Basal resistance of barley to adapted and non-adapted ... · Basal resistance of barley to adapted...

Basal resistance of barley to adapted and

non-adapted forms of Blumeria graminis

Reza Aghnoum

II

Thesis committee

Thesis supervisors

Prof. Dr. Richard G.F. Visser

Professor of Plant Breeding

Wageningen University

Dr.ir. Rients E. Niks

Assistant professor, Laboratory of Plant Breeding

Wageningen University

Other members

Prof. Dr. R.F. Hoekstra, Wageningen University

Prof. Dr. F. Govers, Wageningen University

Prof. Dr. ir. C. Pieterse, Utrecht University

Dr.ir. G.H.J. Kema, Plant Research International, Wageningen

This research was conducted under the auspices of the Graduate school of

Experimental Plant Sciences.

III

Basal resistance of barley to adapted and

non-adapted forms of Blumeria graminis

Reza Aghnoum

Thesis

Submitted in partial fulfillment of the requirements for the degree of doctor

at Wageningen University

by the authority of the Rector Magnificus

Prof. Dr. M.J. Kropff,

in the presence of the

Thesis Committee appointed by the Doctorate Board

to be defended in public on Tuesday 16 June 2009

at 4 PM in the Aula.

IV

Reza Aghnoum

Basal resistance of barley to adapted and non-adapted forms of Blumeria graminis

132 pages.

Thesis, Wageningen University, Wageningen, NL (2009)

With references, with summaries in Dutch and English

ISBN 978-90-8585-419-7

V

Contents

Chapter 1

General introduction

1

Chapter 2

Which candidate genes are responsible for natural variation in

basal resistance of barley to barley powdery mildew?

15

Chapter 3

Transgressive segregation for extreme low and high level of

basal resistance to powdery mildew in barley

47

Chapter 4

New perspectives to identify genes that determine non-host

resistance of barley to non-adapted Blumeria graminis forms

61

Chapter 5

Rust-induced powdery mildew resistance in barley

79

Chapter 6

General discussion

101

Abbreviations 111

Glossary 113

Summary in English 115

Summary in Dutch (Samenvatting) 119

Acknowledgments 123

About the author 127

List of publications 129

Education certificate 131

VI

This thesis is dedicated with deep gratitude to my dear wife Fatemeh and our lovely

children, Mahrokh and Pouria

General Introduction

Chapter 1

2

Introduction Powdery mildews, a large group of Ascomycota fungal species, are obligate biotrophs

that colonize a wide range of plant species. These fungi consist of 16 genera and

about 650 species (Inuma et al., 2007). Nearly 10,000 species of angiosperms have

been reported as host plants for powdery mildews (Braun, 2002). The host range of

these fungi is restricted to angiosperms, and does not include ferns and

gymnosperms (Takamatsu, 2004). They cause economic loss on a wide range of

important agricultural crops, including cereals. Powdery mildew of the grass family is

caused exclusively by members of the species Blumeria graminis. On the basis of

host range, B. graminis is divided into formae speciales (f.sp., plural ff.spp.), each of

which can infect mainly species of one genus of gramineous plants (Wyand & Brown,

2003). Powdery mildew of barley caused by Blumeria gramins (DC.) Speer f.sp.

hordei Em. Marchal (syn. Erysiphe graminis DC. f.sp. hordei Em. Marchal) is one of

the most important diseases of barley in Europe and many major barley cultivation

areas.

Barley- Blumeria interaction as a model system Due to several reasons, powdery mildew of barley has been studied extensively and it

can be considered as an experimental model system for the study of host-pathogen

interactions.

1- The development of the fungus is restricted to the host epidermal surface, so the

successive developmental stages of germlings including germination, appressorial

germ tube growth and haustorium formation can be easily monitored by light

microscopy. The synchronized development of the fungus on the host facilitates

meaningful experiments where physiological and molecular responses of the barley

host can be correlated perfectly with the development of the fungus, using bioimaging

analyses and transcript profiling (Collinge et al., 2008).

2- The mode of infection by mildew exclusively involves the epidermis. Epidermal

cells can be transiently transformed relatively easily and transient-induced gene

silencing (TIGS) and transient over-expression in single epidermal cells can be

applied, allowing reverse genetics to identify genes that influence the barley-Blumeria

interaction (Douchkov et al., 2005).

3-There is a great availability of valuable genetic stock. More than 85 race-specific

resistance genes for powdery mildew have been identified in barley (Jørgensen,

1994). Many near-isogenic lines (see Glossary) have been developed by back-

crossing different resistance gene donors into common genetic background, resulting

in, e.g., the Pallas differential set (Kølster et al., 1984). Such sets of near-isogenic


3

lines form excellent experimental material for comparative studies of defense

responses (Thordal-Christensen et al., 2000). The incompatible phenotype of different

R genes/barley mildew interactions varies from single cell to multi-cell hypersensitive

reaction (HR) or are based on formation of papillae with a subsequent HR. So far six

major resistance genes of barley to powdery mildew (Mla1, Mla6, Mla7, Mla10, Mla12

and Mla13) have been cloned and characterized (Chełkowski et al., 2003, Stein &

Graner, 2004). Furthermore, some genes have been cloned that are required for R-

gene-mediated resistance viz. Rar1 (required for Mla12 resistance-1) and Sgt1

(suppressor of the G2 allele of skp1) (Kitagawa et al., 1999; Azevedo et al., 2002).

4- Barley is the first crop in which an alternative, durable (see Glossary) type of

resistance, mlo resistance, has been found. This resistance is mediated by the

recessive mlo allele of the Mlo (Mildew resistance locus-o) locus. mlo resistance is

effective against all known isolates of the barley powdery mildew fungus (Jørgensen,

1992;Büschges et al., 1997), and has remained so despite extensive cultivation of

mlo-cultivars in Europe since 1979 (Lyngkjær et al., 2000). mlo resistance is

associated with formation of cell wall appositions (papillae) at the site of attempted

fungal penetration. Cloning of the Mlo wild type allele and Ror2, a gene that is

required for mlo-mediated resistance, provided insights into papilla-based penetration

resistance (Büschges et al., 1997; Collins et al., 2003). Non-host resistance of barley

against non-adapted powdery mildews, the basal host resistance and mlo-mediated

resistance are based on similar mechanisms (Trujillo et al., 2004; Humphry et al.,

2006; Schweizer, 2007). Only recently, more than 60 years after the first report of mlo

in barley, effective homologues have been found in tomato, pea and Arabidopsis

thaliana (Collins et al., 2003; Fondevilla et al., 2006; Bai et al., 2008). In Arabidopsis

thaliana these genes are called penetration mutant genes (PEN), and they have been

shown to be required for both non-host resistance and a host resistance that is similar

to the mlo resistance in barley to B. graminis (Consonni et al., 2006).

5. Barley is a diploid autogamous crop, in which DH production is technically

feasible. This is an advantage over an allogamous cereal crop like rye (heterozygous)

and allopolyploid crops like wheat and oat that possess in principle two or three

homologous copies of gene of interest, one on each genome (Elliott et al., 2002).

Barley-powdery mildew: Compatible interaction The powdery mildew pathogen disperses by asexually formed windborne conidia.

Approximately 2 hours after contact with a leaf surface, the conidium germinates with

a primary germ tube (PGT) (Figure 1). This germ tube penetrates through the leaf

cuticle and may have a function in conidial adhesion, water uptake, recognition of leaf

Chapter 1

4

surface structures and perception and transduction of leaf derived signals (Heitefuss,

2001; Eichmann & Hückelhoven, 2008). Within about 4 h, a secondary germ tube,

that is called the appressorial germ tube (AGT), will be formed. AGT differentiates an

appressorium by 8-12 h, from which the fungus tries to penetrate the host cell wall by

a penetration peg. Attacked epidermal cells react to the fungal penetration peg by

papilla deposition. If fungal penetration succeeds, a haustorium will be formed in the

attacked cell. This is an organ by which the fungus obtains its nutrition from the plant

cells and probably organizes reprogramming of the plant cell’s gene expression. After

successful formation of the first haustorium (called establishment) an elongated

secondary hypha (ESH) will start to grow from the primary appressorium. From these

ESH, secondary appressoria are formed that will penetrate epidermal cells to

establish secondary haustoria. Three to four days after formation of the first

haustorium, the pathogen develops conidiophores, on which chains of conidia are

formed. Therefore this development stage is also called conidiation. Released mature

conidia can initiate a new infection cycle (Figure 1).

Penetration resistance in barley-powdery mildew int eraction Penetration thorough the epidermal cell walls is a critical stage during the infection

process of powdery mildews. Epidermal cells actively resist penetration by powdery

mildews in association with papilla formation at the site of attempted fungal

penetration, and hence may prevent haustorium formation (Figure 1). Papillae consist

of a variety of compounds, including callose, phenolic compounds, H2O2, proteins and

glycoproteins with hydrolytic properties (Zeyen et al., 2002). Even in a susceptible

plant some germlings fail to penetrate in association with papilla formation. The

speed of papilla deposition and their chemical composition play an important role in

the success or failure of haustorium formation at that site (Heitefuss, 2001). Upon

failure of attempted penetration from the first appressorial lobe, the appressorium

may form one or two subsequent lobes to try again (Carver, 1986).

In interaction of barley with the powdery mildew pathogen, three types of

resistance, viz. basal host resistance, non-host resistance and mlo-mediated

resistance operate on the basis of similar mechanisms, viz. penetration resistance in

association with papilla formation.

Basal host resistance Even in susceptible plants a proportion of germlings stop at penetration stage in

association with papilla formation due to basal resistance. This resistance is defined

as the plant’s ability to reduce the disease severity caused by an adapted pathogen


5

despite a compatible interaction phenotype. Histological studies on apparently

susceptible barley accessions showed that resistance to primary penetration varies

greatly (e.g. Carver, 1986; Asher & Thomas, 1987). Penetration resistance to

powdery mildew in barley also depends on the type of the epidermal cells. Higher

frequencies of successful penetration occurs on the short cells which directly are in

contact with the stomata (epidermal cells type A) and short cells not directly adjacent

Figure 1. Schematic representation of different outcome of interaction of barley with the powdery mildew

pathogen. hai/dai = hours/days after inoculation, PGT=Primary germ tube AGT=Appressorial germ tube,

C=Conidia, HAU=Haustorium, ESH= Elongated secondary hyphae, Pa=Papilla

to stomata (type B) than on the long epidermal cells covering the vascular tissue (Lin

& Edwards, 1974; Koga et al., 1990). Infection rate in both short and long epidermal

cells also depends on the leaf age. On older leaves, a lower proportion of germlings

succeed to establish than on younger leaves (Lin & Edwards, 1974; Carver, 1986).

Basal resistance is a quantitatively inherited trait, conferred by many, mostly

additively acting genes, each with small effect, so it is also called quantitative

resistance. Advances in molecular marker technology and QTL analysis have led to

identification of many QTLs in different genetic backgrounds of barley, but the genes

underlying the resistance QTLs are still elusive. Using transient-induced gene

silencing and transient over-expression in single epidermal cells, many candidate

genes have been identified that play a role in host, non-host and/or mlo-mediated

resistance (e.g. Douchkov et al., 2005; Zimmermann et al., 2006) but still it is

questionable whether the allelic variation in those candidate genes also determines

natural variation in level of basal resistance between plant accessions.

:::

ESH

Hypersensitive response

R genes, Mla1, Mla6 etc.

Papillae defense

mlo-mediated, Non-host

and basal host resistance

5-8 dai

AGT C

Compatible interaction

HAU

72hai

12hai

:::

PGT PA

C

Chapter 1

6

Non-host resistance The grass powdery mildew pathogen, Blumeria graminis, is classified into several

formae speciales each colonizing an individual genus of the grass family. Blumeria

graminis f.sp. hordei (Bgh), the causal agent of powdery mildew disease on barley,

for example, successfully colonizes cultivated barley Hordeum vulgare and its wild

ancestor H. spontaneum but is probably not able to colonize other related species in

the genus Hordeum nor other species in the Triticeae, such as wheat (Eshed & Wahl,

1970; Menzies & MacNeill, 1989). Conversely, cultivated barley cannot be colonized

by powdery mildew isolates collected on other Hordeum species and other Triticeae.

This resistance is known as non-host resistance, and is mainly associated with papilla

formation at sites of attempted fungal penetration. A proportion of challenged

epidermal cells show, however, a hypersensitive reaction. Although a similar

mechanism of defense is involved in basal resistance of barley to the adapted

pathogen, Bgh, at macroscopic and microscopic level a clear discrimination can be

made between basal host resistance and non-host resistance, since in the latter, the

resistance is complete. Since there is not any barley accession which is susceptible

to the non-adapted mildew forms, like the wheat powdery mildew pathogen (Bgt), it is

not known which genes determine the host status of barley to the non-adapted Bgt.

mlo-mediated resistance Resistance mediated by the recessive loss-of-function mutant at the Mlo locus is

race-non-specific, since it is effective against all known isolates of Bgh (Büschges et

al., 1997). Several resistance alleles of mlo have been derived from mutagenic

treatment of susceptible wildtype Mlo barley cultivars, but the mlo resistance allele

has also been identified from non-mutagenized landraces of barley from Ethiopia

(Jørgensen, 1992). In mlo resistant plants, resistance at the penetration stage in

association with papilla formation prevents the fungus to form haustoria in all kinds of

epidermal cells, except the subsidiary cells of stomata. Papilla formation occurs also

in Mlo susceptible plants when challenged by the penetration peg of the germinated

powdery mildew conidium, but the penetration efficiency of Bgh is considerably higher

in Mlo susceptible plants than in mlo resistant plants (Freialdenhoven et al., 1996).

On the other hand, papilla based responses occur earlier and the size of papillae are

generally larger at the failed penetration sites in mlo resistant lines than at the sites of

failed penetration in Mlo susceptible plants (Jørgensen, 1992).

mlo-mediated resistance shows some level of pathogen specificity, since mlo alleles

are not effective against other fungal pathogens including barley leaf rust (Puccinia

hordei), stripe rust (Puccinia striiformis), stem rust (Puccinia graminis), scald


7

(Rhynchosporium secalis), and the take all fungal disease (Gaeumannomyces

graminis) (Jørgensen, 1977; Collins et al., 2007). However mlo seems to play a role

in non-host resistance of barley to the non-adapted mildew pathogen, Bgt

(Peterhänsel et al., 1997; Trujillo et al., 2004; Humphry et al., 2006). Mlo gene

homologs have now been identified in a wide range of plants, including wheat, rice,

tomato, pea, grapevine and the model plant Arabidopsis thaliana, some of which

have been demonstrated to contribute to penetration resistance to powdery mildew

(Elliott et al., 2002; Consonni et al., 2006; Fondevilla et al., 2006; Bai et al., 2008;

Winterhagen et al., 2008).

mlo and non-host resistance share similar molecular co mponents mlo and non-host resistance share many analogous features (Humphry et al., 2006).

1- Both types of resistance are associated with strong penetration resistance based

on papilla formation at the penetration sites. 2- Both mlo and non-host resistance are

broad spectrum and durable. 3- They do not depend on Salicylic acid (SA), Jasmonic

acid (JA) or Ethylene-mediated defense signaling pathways but both depend on actin

cytoskeleton components. 4- Both types of resistance require PEN genes

(Arabidopsis) or Ror genes (barley). 5- Both depend on the function of the SNAP34

protein.

Cloning of the Mlo wild type allele, identification of Ror genes and characterization

of Arabidopsis mutants with atenuated non-host interaction with Bgh contributed a lot

to our understanding of the molecular basis of penetration resistance to powdery

mildews (see Figure 2; Freialdenhoven et al., 1996; Büschges et al., 1997; Collins et

al., 2003; Consonni et al., 2006). Two genes, Ror1 and Ror2 (required for mlo-

specified resistance) are required for the function of mlo in barley. Mutation either in

Ror1 or Ror2 decrease powdery mildew resistance in mlo genotypes (Freialdenhoven

et al., 1996). Peterhänsel et al., (1997) showed that mlo and Ror genes are active

when barley is challenged with Bgt, a non-adapted powdery mildew. Trujillo et al.

(2004) indicated that mutants of Ror1 and Ror2 showed higher frequencies of fungal

penetration in interaction with Bgt. Screening of mutagenized Arabidopsis populations

for increased frequencies of penetration by the non-adapted powdery mildew fungus

Bgh, resulted in identification of several penetration (pen) mutants allowing higher

frequencies of Bgh entry into epidermal cells of which three, designated pen1, pen2,

and pen3, have been characterized and the corresponding genes were identified

(Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006). Cloning of PEN1 showed

that it is a homologue of barley Ror2 and that this gene encodes SYP121, a syntaxin

which is known to contribute to vesicle fusion events through the formation of SNARE

Chapter 1

8

complexes with a corresponding VAMP (vesicle associated membrane protein) and a

SNAP25 homologue (Collins et al., 2003). PEN2 encodes a glycoside hydrolase that

is required for the enzymatic activation of small molecules and is involved in the

production of an antifungal compound (Lipka et al., 2005). PEN2 protein was found to

be localized to peroxisomes, organelles that accumulate at the sites of attempted

powdery mildew penetration (Koh et al., 2005; Lipka et al., 2005). PEN3 encodes an

ATP-binding cassette (ABC) transporter protein (Stein et al., 2006) possibly involved

in exports of a potentially fungi-toxic compound that is processed by PEN2 in

peroxisomes into the apoplast (Figure 2).

Figure 2. Schematic overview of molecular components involved in penetration resistance to powdery mildew. A

fungal appressorium trying to breach the host cell wall by a penetration peg is stopped in a cell wall apposition

(papilla). PEN1/Ror2 syntaxin and HvSNAP34 protein contribute to SNARE complex formation and secretion of

antimicrobial compounds at sites of attempted fungal penetration. PEN3 is involved in export of potentially toxic

compounds that were released by PEN2 enzyme activity in peroxisomes (reprinted by permission from Collins et

al., 2007; http://www.publish.csiro.au/nid/40/paper/AR06065.htm).

The possible functional link between mlo-mediated and non-host resistance was

recently supported by identification of some candidate genes that emerged from

transient overexpression or transient-gene silencing studies. These candidate genes

were found to be required for mlo-mediated resistance against Bgh as well as for

non-host resistance to Bgt (Douchkov et al., 2005; Miklis et al., 2007; Schweizer,

2007). Furthermore, overexpression of Mlo in epidermal cells of barley resulted in

some degree of susceptibility to the non-adapted Bgt pathogen suggesting that Mlo

may negatively regulate non-host resistance as well (Elliott et al., 2002).


9

Suppression and induction of penetration resistance Adapted pathogens have the ability to overcome preformed defense barriers and

suppress induced defense responses, but non-adapted pathogens do not have such

ability (Thordal-Christensen, 2003). Different fungal pathogens employ different

suppression strategies to infect and colonize their hosts (Staples & Mayer, 2003).

Biotrophic and haustorium forming fungal pathogens, like powdery mildews and rusts,

first should suppress penetration resistance to be able to successfully penetrate

through epidermal or mesophyll cell walls. Successful penetration and haustorium

formation of virulent isolates of Blumeria graminis induces susceptibility to haustorium

formation by subsequent challenge of either adapted or non-adapted powdery mildew

pathogens. On the other hand, failed attempted penetration induces resistance to

haustorium formation by subsequent challenge inoculation with a compatible isolate

(e.g. Lyngkjær & Carver, 1999; Lyngkjær et al., 2001; Kunoh, 2002; Olesen et al.,

2003). This induction of susceptibility and resistance at cell level is often called

accessibility and inaccessibility. Studies on non-host interactions with cowpea rust

fungus and plantain powdery mildew fungus suggests that the adhesion between the

plant cell wall and the plasma membrane plays a role in cell wall-associated defense

responses and powdery mildews and rust fungi employ different strategies to

successfully induce accessibility of plant cells (Mellersh & Heath, 2001; Heath, 2002).

Scope and outline of the thesis In this thesis we investigated the genetic variation in barley for resistance to the

adapted and non-adapted powdery mildew pathogens and we intend to identify the

genes that are responsible for natural variation in host status and non-host resistance

level. We aimed also to determine whether the basal resistance of barley to powdery

mildew is being suppressed by prior attack by a different pathogenic haustorium

forming fungus, Puccinia hordei that differs from the mildew fungus in its strategy to

induce accessibility.

In Chapter 2, a multi-population QTL analysis was conducted for powdery mildew

resistance of barley at seedling and at adult plant stage. Twenty-three EST/cDNA-

clone sequences were mapped that are likely to play a role in the barley-Blumeria

interaction based on transcript profiling, gene-silencing or over-expression data, and

mlo, Ror1 and Ror2. To compare the map position of QTLs over different mapping

populations, we constructed an improved high-density integrated linkage map of

barley, based on the marker data of seven mapping populations of barley.

In Chapter 3, two experimental barley lines were developed with extremely high and

low level of basal resistance to powdery mildew by convergent crossing between the

Chapter 1

10

most resistant and the most susceptible lines from four mapping populations of

barley.

In Chapter 4, a large collection of barley germplasm was screened and some rare

barley accessions were identified with rudimentary susceptibility to the non-adapted

wheat powdery mildew, Bgt. Those accessions were intercrossed in two cycles, and

resulted in two lines, called SusBgt SC and SusBgt DC, with exceptional susceptibility

to Bgt, that were tested with seven non-adapted forms of Blumeria graminis, to

investigate the specificity of the non-host resistance of barley.

In Chapter 5, the rust-induced powdery mildew resistance at the cellular level was

characterized. We also identified differentially expressed genes that may explain the

strong papilla-based penetration resistance in double inoculated barley plants.

In Chapter 6, the results obtained in the previous Chapters, the importance of the

experimental barley lines that were developed in this study for the identification of

genes that determine basal resistance of barley to adapted and non-adapted

Blumeria graminis forms is being discussed.

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Chapter 1

14

Which candidate genes are responsible for natural

variation in basal resistance of barley to barley

powdery mildew?

R. Aghnoum, T.C. Marcel, A. Johrde, N. Pecchioni, P. Schweizer and R.E. Niks

Submitted

Chapter 2

16

Abstract

Basal resistance of barley against powdery mildew is a quantitatively inherited trait

that is based on non-hypersensitive mechanisms of defense. A functional genomic

approach indicated many candidate genes to be involved in defense against fungal

haustorium formation. It is not known for which of those candidate genes allelic

variation may contribute to the natural variation for powdery mildew resistance, since

many of them may be highly conserved within the barley species. Twenty-three

EST/cDNA-clone sequences that are likely to play a role in the barley-Blumeria

interaction based on transcript profiling, gene-silencing or over-expression data, and

mlo, Ror1 and Ror2, were mapped and are considered candidate genes to contribute

to basal resistance. We mapped QTLs for powdery mildew resistance in six mapping

populations of barley at seedling and/or at adult plant stage and developed an

improved high-density genetic integrated map containing 6,990 markers for

comparing QTLs and candidate gene positions over mapping populations. We

mapped 12 QTLs at seedling stage and 13 QTLs at adult plant stage of which four

were in common between the two developmental stages. Six of the candidate genes

showed coincidence in their map positions with the QTLs identified for basal

resistance to powdery mildew. This co-localization justifies to give priority to those six

candidate genes to verify them as being responsible for the phenotypic effects of the

QTLs for basal resistance.

Introduction The barley-powdery mildew pathosystem is one of the best characterized and highly

specialized systems of host-parasite interaction. More than 85 race-specific

resistance genes to powdery mildew have been identified in barley (Jørgensen, 1994;

Chelkowski et al., 2003). They act through a hypersensitivity response (HR), which is

only elicited by particular avirulent pathogen isolates according to the gene-for-gene

concept (Collins et al., 2002). Upon deployment of resistance genes in breeding

programs, virtually all of these genes became ineffective (Czembor, 2000). Six major

resistance genes of barley to powdery mildew (Mla1, Mla6, Mla7, Mla10, Mla12, and

Mla13) have been cloned and characterized (Chelkowski et al., 2003; Stein & Graner,

2004).

Another type of resistance is known as basal (partial) resistance and is conferred by

many, mostly additively acting genes, each with a small effect. Basal resistance has

been used to indicate a low-level penetration resistance expressed in 'susceptible'

plants, which is not due to hypersensitivity (e.g. Collins et al., 2003). Since partial

resistance is defined as a resistance characterized by a susceptible infection type

Which candidate genes are….

17

and a reduced disease severity (Parlevliet, 1975) we presume in this manuscript that

partial and basal resistance in this pathosystem are synonymous terms. Basal

resistance is considered as an important and potentially durable type of resistance.

Nevertheless, it is the least investigated type of resistance. In barley-powdery mildew

interaction, basal resistance is associated with formation of cell wall appositions, so-

called papillae, at the fungal penetration sites and this reinforcement of cell walls

prevents haustorium formation in the attacked epidermal cells. Failure of haustorium

formation is also called “pre-haustorial” resistance and is in general not associated

with a hypersensitive reaction of the challenged plant cells (Niks & Rubiales, 2002).

Even the most susceptible barley genotypes prevent a substantial proportion of

mildew infection units from forming their first haustorium, implying that all genotypes

have at least some degree of basal resistance. Genotypic variation in level of basal

resistance of barley to powdery mildew has been reported by several researchers

(Asher & Thomas, 1984; Jones & Davies, 1985; Carver, 1986; Knudsen et al., 1986;

Mastebroek & Balkema-Boomstra, 1991). Histological studies on partially resistant

genotypes showed that resistance to primary penetration by non-hypersensitive

mechanisms affect a varying proportion (26-75%, dependent on barley accession) of

attempted penetrations to fail (Asher & Thomas, 1987). Carver (1986) reported that in

spring barley lines with various levels of basal resistance, a range from 48 to 90% of

infection units failed at penetration stage.

Development of molecular marker techniques has enabled researchers to dissect

quantitatively inherited traits, like basal resistance, by QTL analysis and to study the

effects of individual QTLs, including their interactions. In contrast to single and

dominant major genes, the genetic and molecular basis of quantitative disease

resistance is less well understood, because of the polygenic inheritance, the relatively

small effect of individual genes and significant genotype × environment interactions

that make such traits experimentally more difficult to assess. However, research is

underway to isolate the genes underlying QTLs for basal resistance of barley to

barley leaf rust Puccinia hordei following map-based cloning approaches (Marcel,

2007b). Such an approach would be feasible to identify the genes underlying the

powdery mildew QTLs.

The recessive allele of the Mlo-locus of barley mediates a race non-specific

resistance to the barley powdery mildew. mlo resistance and basal resistance share

the same mechanism of defense, i.e. both are “papilla-based” and “pre-haustorial”

(Trujillo et al., 2004; Schweizer, 2007). Mutational approaches revealed that two

genes (Ror1 and Ror2) are required for full expression of mlo resistance

(Freialdenhoven et al., 1996). These two genes have also been implicated in basal

Chapter 2

18

penetration resistance of barley expressed in susceptible (Mlo) genotypes (Collins et

al., 2003). So, mlo, Ror1 and Ror2 can be considered as candidate genes to be

involved in basal resistance of barley to powdery mildew.

Candidate gene analysis is based on the hypothesis that genes with known function

could correspond to loci controlling traits of interest (Pflieger et al., 2001). This

approach has been widely applied for genetic dissections of complex and quantitative

traits (Zhu & Zhao, 2007). For plant geneticists, a candidate gene is any gene

putatively involved in trait variation, based on its biological function, its map position

and/or its expression pattern (Pflieger et al., 2001). Ideally, the list of candidate genes

to explain a particular phenotype can be shortened by following a prioritarization

approach, which consist in combining information on the function, position and

expression of the genes involved (Zhu & Zhao, 2007). However, finally the candidate

genes should be validated through physiological analyses or genetic transformation.

To identify genes that influence barley-Blumeria interactions in host and non-host

combinations, a high-throughput RNAi system for transient-induced gene silencing

(TIGS) as well as a transient over-expression in single epidermal cells of barley has

been developed (Schweizer et al., 1999; Douchkov et al., 2005; Ihlow et al., 2008).

Using this system, the function of 930 barley candidate genes including 693 up-

regulated genes and 101 resistance-gene analogues expressed in barley epidermis

have been tested for their effect on non-host resistance of barley to wheat powdery

mildew and on the level of basal host resistance. The role of 43 genes in non-host

resistance, basal resistance and/or mlo-mediated resistance of barley has been

confirmed by using this method (Dong et al., 2006; Zimmermann et al., 2006; Johrde

& Schweizer, 2008). Identification of candidate genes whose expression is correlated

with haustorium formation as shown after overexpression or silencing does not

necessarily mean that they are also responsible for natural variation in levels of basal

resistance. Such candidate genes may simply be conserved within the plant

population.The study of the inheritance of natural variation can be considered as an

alternative approach to elucidate the functional role of candidate genes in a given

phenotype (Mitchell-Olds & Schmitt, 2006).

In the present study we mapped genes that determine natural variation in level of

basal resistance in six barley mapping populations and compared their map positions

with those of 23 markers derived from gene sequences that were found to be

pathogen-induced or that produced a resistance phenotype upon RNAi or over-

expression during the interaction of barley with powdery mildew and also mlo, Ror1

and Ror2 genes. We combined the expression and function of candidate genes with


19

their map position in order to prioritize them. We also determined whether QTLs

tended to map to major gene loci of resistance to powdery mildew.

Materials and Methods

Marker data sets Segregating marker data from seven individual mapping populations (Table 1) were

compiled to prepare a high-density genetic integrated map of barley. The populations

consisted of four doubled haploids (DH) populations, i.e. Igri × Franka (IgFr, Graner et

al., 1991), Steptoe × Morex (StMo, Kleinhofs et al., 1993), the Oregon Wolfe Barleys

(OWBs, Costa et al., 2001) and Nure × Tremois (NuTr, Francia et al., 2004), and of

three recombinant inbred lines (RIL) populations, i.e. L94 × Vada (LnVa, Qi et al.,

1998), Vada × SusPtrit (VaSu, Jafary et al., 2006) and Cebada Capa × SusPtrit

(CCSu, Jafary et al., 2008).

We reused the data sets compiled by Marcel et al., (2007a) after excluding the

AFLP markers mapped on the OWBs. We added the DArT (diversity arrays

technology) and TDM (transcript-derived marker) marker data sets released by Wenzl

et al., (2006) and Potokina et al., (2008) [StMo], respectively, the SSR and EST

marker data sets released by Varshney et al., (2007) and Stein et al., (2007) [IgFr,

StMo, OWBs], respectively, and new marker data posted on the Oregon State

University (OSU) Barley Project web site (http://www.barleyworld.org/) [OWBs]. The

marker data set of NuTr consisted of 542 markers, mostly DArT markers (72%), and

was obtained from Dr. Nicola Pecchioni (Università di Modena e Reggio Emilia, Italy).

Segregating data from most of the BIN markers defined by Kleinhofs & Graner (2001)

[StMo] were obtained from Dr. Andris Kleinhofs (Washington State University). Three

new marker data sets were also included in the present integrated map and consist of

168 “PERO” markers mapped in LnVa and VaSu by peroxidase-profiling (to be

published elsewhere by our laboratory), 26 new EST-SSR markers mapped in NuTr

or CCSu by A/T labelling (Marcel et al., 2007a), and the 23 candidate genes

described in the present study (Table 4).

Construction of the barley integrated map The integrated map was constructed essentially as described in Marcel et al., (2007a)

using the software JoinMap 4TM and MapChart (Voorrips, 2002). All the bridge

markers (i.e. markers common to two or more populations), the 210 BIN markers

defined by Marcel et al., (2007a) and the additional BIN markers provided by Dr. A.

Kleinhofs were used to prepare the framework map. The order of the 210 BIN

markers was initially used as fixed order to run the regression mapping algorithm.

Chapter 2

20

After each mapping, the obtained marker order was compared with the marker order

on the integrated map of Marcel et al., (2007a). Most discrepancies between the two

marker orders were investigated by graphical genotyping in order to reject or to

validate the newly calculated map. In cases where the newly calculated map was

rejected, the fixed order was modified and the mapping repeated until the

discrepancy was corrected. Consequently, in cases where the new map was

validated, it could only represent an improvement from the previous integrated map of

Marcel et al., (2007a). Then, the marker order on the constructed framework map was

used as fixed order to run the maximum likelihood mapping algorithm on the 49

linkage groups (i.e. seven barley chromosomes for seven mapping populations);

default values were increased to five map optimization rounds with 3,000 chains.

Three maps were generated for each linkage group and aligned to compare the order

and distances between markers. From each alignment/linkage group, the map

showing the most consistent marker order and the shortest total length was

integrated into the framework map following the “neighbors” approach. The obtained

integrated map was divided into 217 BINs of about 5 cM each. The BIN-defining

markers of Kleinhofs & Graner (2001) were maintained in their role while the BIN-

defining markers of Marcel et al., (2007a) were sometimes modified to obtain a more

homogeneous distribution of the BINs over the map.

Plant and pathogen materials Six out of seven (IgFr excluded) mapping populations were evaluated for their level of

basal resistance to barley powdery mildew at seedling stage under controlled

conditions and five of them (IgFr and OWBs excluded) at adult plant stage under field

conditions. An isolate of Blumeria graminis f.sp. hordei collected in Wageningen 2004

(Wag.04) was used for evaluation of resistance levels at seedling stage. This isolate

was maintained in a climate cabinet and propagated on young seedlings of the

susceptible barley cultivar ‘Manchuria’.

Table 1. Characteristics of the seven barley mapping populations used to construct the integrated map of

which six were used for QTL analysis.

Name of population Type Lines N umber of markers

L94 × Vada (LnVa) RIL 103 1083

Vada × SusPtrit (VaSu) RIL 152 533

C.Capa × SusPtrit (CCSu) RIL 113 519

OWBs DH 94 882

Steptoe × Morex (StMo) DH 150 3561

Nure × Tremois (NuTr) DH 118 542

Igri × Franka (IgFr) DH 71 737


21

Table 2 . Comparison of several (integrated) linkage maps published for barley Map length

(cM)

Number

of loci

Average locus number per cM Contributing Populations 1

1H

2H

3H

4H

5H

6H

7H

Rostoks et al.,

2005

1,211 1,237 0.9 1.1 1.0 1.1 0.9 1.0 1.2 LiHs, OWBs, StMo

Wenzl et al., 2006 1,161 2,935 2.8 3.1 2.5 1.9 2.0 2.5 3.0 BaCP, ClSa, DaZh, FoCI, FrSt, IgAt, PaTa,

StMo, TXFr, YeFr

Marcel et al., 2007 1,081 3,258 2.6 3.7 3.3 2.4 2.3 2.9 3.8 LnVa, VaSu, StMo, OWBs, IgFr, CCSu

Stein et al., 2007 1,118 1,055 1.0 1.1 0.9 0.8 0.9 0.8 1.2 StMo, OWBs, IgFr

Potokina et al.,

2008

1,010 1,596 1.3 1.8 1.9 1.3 1.4 1.5 1.7 StMo

This report 1,093 6,990 6.4 7.9 6.9 4.5 5.7 5.9 7.3 LnVa, VaSu, StMo, OWBs, IgFr, CCSu,

NuTr

1 LiHs (Lina x Hordeum spontaneum HS92); OWBs (Oregon Wolfe Barleys); StMo (Steptoe x Morex); BaCP (Barque-73 x CPI71284-48); ClSa (Clipper x Sahara);

DaZh (Dayton x Zhepi2); FoCI (Foster x CI4196); FrSt (Fredickson x Stander); IgAt (Igri x Atlas68); PaTa (Patty x Tallon); TXFr (TX9425 X Franklin); YeFr (Yerong x

Franklin); LnVa (L94 x Vada); VaSu (Vada x SusPtrit); IgFr (Igri x Franka); CCSu (Cabada Capa x SusPtrit); NuTr (Nure x Tremois)

Chapter 2

22

Assessment of resistance level at seedling stage Evaluation of powdery mildew resistance at seedling stage was performed on

detached leaves (Schwarzbach, 2004). Barley plants were grown in spore-free

greenhouse compartments. Twelve days after sowing, when the first leaf was fully

expanded, a segment of about 4 cm long was cut from the middle part of the first

leaves, and both cut ends were immersed in 0.6% water agar medium containing 125

ppm benzimidazole in a square (12 × 12 cm) polystyrene petri dish, the adaxial side

of the leaf facing upwards. Each petri dish accommodated about 16 leaf segments.

One leaf segment was taken per RIL/DH line per experiment. Both parents of each

mapping population and the susceptible cultivar ‘Manchuria’ were added to each petri

dish as reference. To use freshly produced conidia for inoculation, young seedlings of

the susceptible cultivar ‘Manchuria’ were densely inoculated with powdery mildew 7

days prior to inoculation of the experiment. A settling tower was used for inoculations

(Schwarzbach, 2004). Per inoculation, four petri dishes containing the leaf segments

were put inside the tower and conidia from heavily infected leaves were blown into

the tower using compressed air and were allowed to settle for 10 minutes. The

density of inoculum was monitored by haemocytometer and was adjusted to 10-15

conidia per mm2. Following inoculation, the closed petri dishes were incubated in a

growth chamber at 20 oC with fluorescent light irradiance (102.8 micromol/m2/s)

supplied for 12 h per day. Four days after incubation, the accessions were scored by

counting the number of young powdery mildew colonies per 2 cm2 of leaf segment,

using a magnifying glass (10x). The experiment was repeated three times and the

average infection frequency used for QTL analysis. Because L94 is a mlo-barley and

fully resistant to powdery mildew, in our inoculation experiment we only included non-

mlo carrying RILs (51 accessions) of the LnVa population (Shtaya et al., 2006).

Histology of infection by powdery mildew To determine the mechanism of resistance at the cellular level, one DH line or RIL

from each mapping population showing high levels of basal resistance at seedling

stage together with the parents were selected for microscopic observation. The

susceptible barley cultivar ‘Manchuria’ was included as reference. From each

accession two seedlings were grown in boxes (37 × 39 cm). Twelve days after

sowing, first formed leaves were fixed in a horizontal position, with the adaxial side

up. The inoculation was performed using the settling tower with freshly produced

spores collected from young susceptible barley plants. The inoculum density was

adjusted to 10-15 conidia per mm2. In each inoculation, one seedling from each

accession was sampled for histological analysis and the other one was kept for


23

checking the efficiency of inoculation. The experiment was performed in two

replications. Forty eight hours after inoculation (hai) a leaf segment from the

inoculated leaves was cut and transferred to a solution of 1 mg.ml−1 3,3'-

Diaminobenzidine (DAB) for 8 h and, subsequently, to a solution of acetic acid-

ethanol (3/1 v/v) to be cleared overnight. The leaf segments were stained with

0.005% Trypan Blue as described by Anker & Niks (2001). To make the preparations,

the leaf segments were embedded adaxial side up in glycerol. Leaf segments were

examined at 400x magnification using an Axiophot microscope (Zeiss, Germany). In

all microscopic observations only interstomatal (type B epidermal cells, following the

terminology proposed by Koga et al., 1990) were examined. Those germlings that

were stopped by papillae at penetration stage were considered as failed primary

penetration (FPP) and germlings that formed a haustorium and elongated secondary

hyphae (ESH) were considered as successful primary penetration (SPP) (Asher &

Thomas, 1983). Those epidermal cells that were challenged by more than one

conidium were not considered.

Phenotypic assessment at adult plant stage Evaluation of basal resistance at adult plant stage was carried out at field conditions

under naturally occurring powdery mildew infections. The parents and lines of

different mapping populations were sown in three replications according to a

randomized complete block design in spring of 2006 and 2007. Each genotype was

sown in three-row plots of one meter long, row distance 25 cm. To minimize the effect

of interplot interference (Parlevliet & Van Ommeren, 1984; Lipps & Madden, 1992),

three rows of oat were sown between two neighbouring genotypes. The plots were

treated following standard agricultural practices. Mildew severity was recorded two or

three times with 8-10 days interval according to the 0-9 scale (0= no infection and 9=

fully covered by mildew) (Saari & Prescott, 1975). The area under the disease-

progress curve (AUDPC) was calculated (Jeger & Viljanen-Rollinson, 2001) and used

as quantitative trait for QTL analysis. For the CCSu population, in the 2006 field test,

disease severity was recorded only once and directly used for QTL analysis. For

NuTr, the data were collected from 2 × 3 meter yield trial plots of 88 spring lines

during the spring of 2005 and 2006, without alternating oat plots. The possible effects

of interplot interference were minimized by sampling only the middle rows of the plots.

QTL analysis The quantitative resistance data were combined with the already available biparental

marker map data and analyzed with the software package MapQTL version 5 (Van

Chapter 2

24

Ooijen, 2004). First an interval mapping (IM) analysis was performed. Markers with

the highest log-likelihood ratios (LOD value) were selected as the initial set of

possible co-factors for multiple-QTL mapping (MQM) analysis (Jansen, 1993; Jansen

& Stam, 1994). The LOD threshold for detecting QTLs was calculated by a

permutation test with 10000 iterations at the significance level α = 0.05. A threshold

LOD value above or equal to the LOD value suggested by the permutation test was

used to declare the presence of a QTL. Detected QTLs were transferred from the

individual maps to the barley integrated map. QTLs were named as ‘Rbgq’

(quantitative genes for Resistance to Blumeria graminis) followed by the QTL number

as suggested by Shtaya et al., (2006). A common name is given to QTLs for which

the LOD-1 interval was overlapping.

Mapping candidate genes and their co-localization w ith QTLs Thirty four EST-derived sequences were provided by Dr. Patrick Schweizer (The

Leibniz Institute of Plant Genetics and Crop Plant Research, IPK, Germany) and 5

sequences by Dr. Ralph Hückelhoven (Technical University of Munich, Germany).

These ESTs represent candidate genes implicated in basal resistance, since they

promoted or decreased haustorium formation by Blumeria graminis upon RNAi

silencing or over-expression. We tried to convert those sequences to polymorphic

PCR markers to map them on one of the barley mapping populations used to

construct the integrated map. Primer pairs were designed using DNASTAR software

package based on the full-coding-sequence available or using databases of genomic

or cDNA sequences (CR-EST: The IPK Crop EST Database; The National Centre for

Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/ and The TIGR Gene

Index at DFCI). EST sequences were converted to Cleaved Amplified Polymorphic

Sequence (CAPS), Derived Cleaved Amplified Polymorphic Sequences (dCAPS) or

Sequence Characterized Amplified Region (SCAR) markers to be mapped in one of

the four mapping populations: CCSu, OWBs, StMo and LnVa.

The candidate gene based markers were placed on the integrated map of barley to

be compared with the position of QTLs for resistance to powdery mildew. We

considered mlo and the two genes that are required for the mlo-mediated resistance,

Ror1 and Ror2, as candidate genes to compare their map positions with QTLs. mlo

gene was already mapped directly in the LnVa population and was present in our

integrated map. We located the Ror genes on the map based on the position of

reported co-segregating markers (Collins et al., 2001; Collins et al., 2003). To

determine the global significance of co-localizations between QTLs and candidate

genes, a chi-square test was performed. The null hypothesis of independent


25

distribution of 5 cM BINs containing a QTL peak marker and BINs containing a

candidate gene was tested.

Comparing the map position of QTLs with known R gen es The Mla6 and MlLa genes were already located on the integrated map based on the

marker data of IgFr and LnVa populations. Other R genes (Mlra, Mlk, Mlnn, Mlga,

Mlg, Mlj, mlt, Mlf) were located on the map based on the reported position of co-

segregating markers (Schonfeld et al., 1996; Kurth et al., 2001; Jahoor et al., 2004).

Results

The new barley integrated map The new barley integrated map contains 6,990 morphological/molecular markers

covering a total genetic distance of 1,093 cM, which is similar to previously reported

barley integrated genetic maps (Table 2). The framework map contained 94 (for

chromosome 1H), 149 (2H), 131 (3H), 85 (4H), 126 (5H), 116 (6H) and 122 (7H) (=

823 in total) bridge markers and covers a genetic distance of 1,051 cM. The most

represented types of molecular markers on this integrated map are RFLP (20%),

AFLP (20%), SSR (9%), DArT (19%) and TDM (23%) (= 91% of all markers). RFLP

and AFLP marker techniques are not used so commonly anymore but the integration

of such markers with more modern types of markers allow to integrate past and

present data collected on many different mapping populations as well as to select

markers or candidate genes linked to a specific trait in any of these populations. More

than 43% of the markers on the present barley integrated map target ESTs or gene

sequences (gene-targeted markers, GTM) and can be used for candidate gene

identification (Rostoks et al., 2005; Marcel et al., 2007a) or to perform synteny

analyses (Rostoks et al., 2005; Stein et al., 2007). The StMo map contributed more

than half of the total number of markers (3,561 markers) on the integrated map, most

of these markers coming from the recently released array-based marker data sets

(Wenzl et al., 2006; Potokina et al., 2008). The very low percentage of errors and

high quality of these marker data greatly increase the reliability of our integrated map.

The present integrated linkage map was aligned with the one of Marcel et al., (2007a)

and with the TDM-based map of Potokina et al., (2008) in order to compare their

locus order. The marker order between the present integrated map and the TDM-

based map was highly consistent, with only a few and very localized inversions

pinpointing small inaccuracies inherent to map integration projects (Jackson et al.,

2008). The marker order was also consistent with the integrated map of Marcel et al.,

(2007a) with the exception of reordering between several AFLP markers mapped at

Chapter 2

26

the telomeres of chromosomes 5HL and 6HL in the CCSu population. The inspection

of graphical genotypes mostly agreed with the marker order on the present integrated

map. The constructed barley integrated map has been posted on the GrainGenes

website (Barley, Integrated 2008, Marcel" at http://wheat.pw.usda.gov/).

QTLs mapped at seedling stage The infection frequency of powdery mildew in detached seedling leaf tests exhibited

continuous variation in all six mapping populations indicating quantitative inheritance.

In all tested mapping populations, transgressive segregation towards resistance and

towards susceptibility was observed (Figure 1), indicating that the resistance and

susceptibility alleles were contributed by both parents.

The number, genomic position on the barley integrated map, and effect of QTLs

associated with basal resistance to powdery mildew at seedling stage are

summarized in Table 3 and Figure 2. In total, 12 QTLs were mapped that affect the

infection frequency at seedling stage in six mapping populations. The mapped QTLs

were found on all chromosomes. One QTL on chromosome 5H (Rbgq15) was in

common between VaSu and LnVa populations. The phenotypic variation explained by

the QTLs ranged from 6.2-24.8%. The LOD scores of QTLs were generally low

(≤6.1). Our results indicate a high level of diversity in QTLs to be involved in powdery

mildew resistance at seedling stage in barley.

QTLs mapped at adult plant stage The RIL and DH populations showed a continuous distribution for AUDPC or disease

severity data collected at field experiments. Transgressive segregation was common

in four out of five (StMo, no transgression) populations tested (Figure 3). In total, 13

QTLs for adult plant resistance, including QTLs mapped in different populations at the

same location, were detected and these were distributed over all barley

chromosomes (Table 3 and Figure 2). Three out of the 13 QTLs (Rbgq7, Rbgq8 and

Rbgq9) were mapped in more than one population. Four QTLs were detected in both

developmental stages (Rbgq2a, Rbgq7, Rbgq9, and Rbgq13) of which two were

detected in the same population at seedling and adult plant stages. The phenotypic

variation explained by the QTLs ranged from 7.3% to 52.1%, indicating great

differences in effect size of QTLs conferring resistance at adult plant stage. Four

QTLs (Rbgq12, Rbgq4, Rbgq7 and Rbgq8) were detected and confirmed in both

years. The most consistent QTLs were also the ones with the highest LOD scores

(Table 3).The inconsistency of the other QTLs may be due to a high level of

environment dependency or due to different race composition of the pathogen


27

population between years. Our results indicate that most QTLs for powdery mildew

resistance in barley are highly plant development stage dependent and that a high

level of genetic diversity exists in barley for basal resistance to powdery mildew.

Figure 1. Histograms of the frequency distribution of powdery mildew resistance phenotypes in six

mapping populations of barley tested at seedling stage. Infection frequency represents the number of

colonies in 2 cm2 of infected leaf segments 4 days after inoculation.

OWBs-seedling stage

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Chapter 2

28

Table 3. Characteristics of QTLs mapped in six barley populations against powdery mildew (Blumeria graminis f.sp. hordei) QTL

Name

Growth

stage

Population Chrom. Peak marker

(PM)

Position

of PM

LOD % exp. a Add. b Donor c

Rbgq4 Field2005 NuTr 1H bPb-4657 23.8 7.2 28.4 10.05 Tremois


Rbgq12 Field2005 NuTr 4H E39M61-181 71.6 6.3 31.2 10.66 Tremois


Rbgq7 Field2006 LnVa 2H E41M32-83 32.3 7.3 52.1 17.08 Vada

Rbgq21 Field2006 VaSu 7H P15M47-184 40.9 3.3 8.6 0.58 SusPtrit


Rbgq18 Field2006 CCSu 6H E33M55-333 58.5 2.8 10.4 0.37 Cebada Capa


Rbgq8 Field2006 VaSu 2H E33M54-182 61.1 7.7 20.0 0.85 Vada

Rbgq12 Field2006 NuTr 4H E39M61-181 71.6 4.9 37.1 6.34 Tremois


Rbgq9 Field2006 VaSu 2H P17M54-152 148.4 3.6 9.9 0.60 Vada


Rbgq7 Field2007 StMo 2H MWG858 37.8 8.7 17.2 2.33 Steptoe

Rbgq5 Field2007 StMo 1H ABG053 43.1 5.7 10.9 1.89 Morex

Rbgq8 Field2007 CCSu 2H E38M54-169 54.3 3.4 14.3 4.00 Cebada Capa

Rbgq8 Field2007 VaSu 2H E38M54-169 54.3 13.3 31.1 4.08 Vada


29

Table 3 continued

QTL

Name

Growth

stage Population Chrom.

Peak marker

(PM)

Position of

PM

LOD % exp. a Add. b Donor c

Rbgq2a Field2007 LnVa 3H E33M61-131 63.8 4.3 24.9 4.27 L94

Rbgq11 Field2007 StMo 3H CDO113B 106.7 3.6 7.3 1.49 Morex

Rbgq13 Field2007 VaSu 4H E41M40-79 138.3 3.9 7.9 2.07 SusPtrit

Rbgq16 Field2007 StMo 5H CDO484 161.1 4.2 7.6 1.55 Morex

Rbgq17 Field2007 VaSu 5H E33M61-144 178.0 4.8 9.5 2.22 SusPtrit

Rbgq20 Seedling OWBs 7H ABG704 7.4 3.5 18.3 11.12 Rec

Rbgq10 Seedling StMo 3H MWG571C 16.4 4.7 15.5 9.90 Morex

Rbgq7 Seedling CCSu 2H E37M32-288 25.5 3.8 14.4 6.24 SusPtrit

Rbgq14 Seedling OWBs 5H Bmac0047e 44.9 4.9 19.9 11.40 Dom

Rbgq2a Seedling NuTr 3H bPb-0312 47.7 6.1 24.8 9.36 Tremois

Rbgq22 Seedling CCSu 7H E38M61-128 48.7 4.2 20.0 8.68 SusPtrit

Rbgq6 Seedling VaSu 1H E38M54-441 61.5 3.4 8.0 6.07 SusPtrit

Rbgq2b Seedling LnVa 3H E35M48-410a 69.5 2.8 19.8 9.98 L94

Rbgq19 Seedling NuTr 6H scssr05599 96.9 5.6 22.4 8.76 Nure


Rbgq15 Seedling LnVa 5H E33M58-306 117.6 3.1 21.5 10.71 Vada


Rbgq9 Seedling VaSu 2H E42M48-405 146.4 5.0 11.5 6.97 Vada

a The proportion of the phenotypic variance explained by the QT b Effects of the resistant allele in reduction of the number of colonies per 2 cm2; c Donor of the resistance allele

Chapter 2

30

*BCD1434

*ABA305

*E42M51-113GBM1447*MWG938Mlra

*cMWG645

*MWG837Mla6Mlk*GBM1007

*ABA004

*GBR1154

*BCD098

*ABG053Mlnn

*Ica1

*E35M61-92

*JS074GBM1412Ror1GBM1487*Pcr2GBM1336GBM1216GBM1480*bPb-1193*Glb1GBM1153*GBM1108WBE223*DAK123BMlgaGBM1481*GBR0643*PSR330*Bmac0144aGBM5162*cMWG649B

*cMWG706A

*ABC257

*BCD193

*GBR1418

*ABG702

*ABC261WBE221GBM1308*GBR0828GBM1358GBM1407*GBR1390GBM1314

Rbgq4(Trem

ois)_7.2R

bgq6(SusPtrit)_3.4

Rbgq4(Trem

ois)_9.6

Rbgq5(M

orex)_5.7

1H*MWG844A

*GBR0613

*ABG703B

*E37M33-160

*MWG878

*GBS0535

GBM5023GBM5018*ABG318*E33M61-135GBM5140*ABG358*GBR1550WBE226*PoxGBM5095*E38M54-169GBM1446*Bgq60GBM1158GBM1172GBM1203GBM1366GBM1129*GBM5230WBE219*ABC468WBE231*GBMS160GBM1513*ABC451*MWG865GBM1468GBM1408*Bmag0125WBE229*MWG503GBM1328GBM1440*MWG882*GBM1208*ksuD22GBM1469*E33M54-307

*GBS0379

GBM1437*ABC252GBM1200

*GBM1047

*ABC165A

WBE220*E35M61-358

*MWG844C

MlLa*MWG2200

GBM5080

Rbgq7(SusP

trit)_3.8R

bgq8(C.C

apa)_3.4R

bgq9(Vada)_5.0

Rbgq7(V

ada)_3.1R

bgq8(Vada)_13.3

Rbgq7(Steptoe)_8.7

Rbgq8(T

remois)_6.0

Rbgq7(V

ada)_7.3R

bgq8(Vada)_3.3

Rbgq9(V

ada)_4.7

Rbgq8(T

remois)_6.0

Rbgq9(V

ada)_3.6

Rbgq8(V

ada)_7.7

2H*BCD907

WBE212*ABG070

*JS195F

*E35M48-250

*E42M55-233

*ABG321

GBM1430*MWG798B

*MWG584GBM1284

*BCD153

*E37M32-471

*ABG396

*GBM1372

*MWG571BGBM1163

*ABC176GBM5020GBM1343*ABG377WBE232Contig7079_atContig7080_atGBM1090GBM5047*MWG555B*ABG453*ABR320*CDO345*bPt-7238

*CDO113B

*GBS0510

*His4B

GBM1226*MWG847

*ABG004

*WG110

*ABC161GBM1118

*MWG902WBE211*ABC174WBE225GBM1288WBE218*Adh10

*ABC166

*GBR1425

*ABC172GBM1420

Rbgq10(M

orex)_4.7R

bgq2a(Trem

ois)_6.1R

bgq2b(L94)_2.8

Rbgq2a(L

94)_4.3R

bgq11(Morex)_3.6

3H0

5

10

15

20

25

30

35

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45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

140

145

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155

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165

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180

Figure 2. Localization of candidate gene markers and QTLs for resistance to powdery mildew on an integrated

map of barley. The candidate genes, including mlo, Ror1 and Ror2 genes, are in bold red. The locations of QTLs

are indicated by vertical bars at the right side of each linkage group. Solid bars represent QTLs identified at

seedling stage under controlled conditions, and shaded bars represent QTLs identified at adult plant stage under

field conditions. The length of a bar approximately represents the LOD-1 support interval and the length of the

exceeding line the LOD-2 support interval of the QTL based on rMQM results. The ruler indicates the distance in

cM. (Continued on next page)


31

*bPt-7602

*bPt-7852

*MWG634

*E35M54-548

*JS103.3

*HVM40

GBM1252*Ole1*LoxA

*BCD402B

*HVKNOX3

*BCD808B

Mlg*ABC303*ABG484GBM1364*PBI25

*bBE54A

*Bmac0310GBM5210*BCD453B

*GBM1350

*ABG319A

*E39M61-367

GBM1249*KFP221

WBE216*bPb-1325mlo*ABG397

*GBM1448

WBE214*ABG319C

*MWG2037BGBM1324GBM1147WBE104*E42M58-312GBM1453*GBM1018

Rbgq12(T

remois)_6.3

Rbgq13(SusP

trit)_4.2

Rbgq12(T

remois)_4.9

Rbgq13(SusP

trit)_3.9

4H*bPb-2330

*DAK133

*ABR313

*MWG920-1A

*bPb-0091

*scssr07106*E45M55-400GBM5028*ABG705WBE227*CDO669BContig5975_atContig5974_s_atWBE100*ABG395GBM1478*E32M62-321GBM1222GBM1325GBM1508*Ltp1*GBMS196GBM1293*WG530*E35M55-181*GBS0042*ABC302GBM1506

*E38M55-128

*BCD926

*MWG522

*ABG473Mlj*GBM1227GBR0180*E42M51-335*MWG2230GBM1295GBM1141*GBS0531WBE228GBM5008*MWG514BGBM5222GBM5212GBM1384GBM5214*GBS0138*WG908GBM1166Ror2*E42M55-75

*ABG496GBM1490

*GBS0800

*ABG391

*ABG390

*DsT-33

*ABG463

*ABC309GBM1474*MWG851B

Rbgq14(D

om)_4.9

Rbgq15(SusP

trit)_2.7R

bgq16(Morex)_4.2

Rbgq15(V

ada)_3.1R

bgq17(SusPtrit)_4.8

5H*ABG062

*ABG466

*ABG378

*E42M54-160

*E33M61-401

*cMWG652A

*E33M55-63

GBM1270*DD1.1C

*ABG387B*E35M61-269GBM5086GBR0504*GBM1053*iLdh1GBM1267WBE215GBM5012*GBS0468*ABG474

*GBMS180

*ABC170B

*GBS0767

*Nar7

*GBR0621

*MWG934

*Tef1

*scssr00103

*Bmac0040a

*P16M51-215GBM1154GBM5064*GBM1274GBM1275GBM1276*MWG2196*cMWG684A

Rbgq19(N

ure)_5.6

Rbgq18(C

.Capa)_2.8

6H*E37M50-401

*ABG704

mlt*bPt-3623

*ABG320

WBE224*P14M61-73

*ABC151AWBE217*GBM1326GBM5060*ABG380

*bPt-6944

*ksuA1A

*GBM1464

GBM5226*Brz

*ABC255

*HVCMA

*E33M54-261*ABG701GBM1237*ABC455WBE101Contig2163_atContig2170_atMlf*Amy2GBM1303WBE210GBM1472GBM1297GBM1396GBM1428*GBMS141*RZ242GBM5225*bPb-9908*ABC310B*bPb-9104*ABC305*GBR1547GBM1362WBE230*ABG461A

*GBR1326

*GBM1456

*GBR1567

*bPb-1858

Rbgq20(R

ec)_3.5R

bgq22(SusPtrit)_4.2

Rbgq21(SusP

trit)_3.3

7H0

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Chapter 2

32

NuTr-Field 2006

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Figure 3. Histograms of the frequency distribution of powdery mildew resistance phenotypes in 5

mapping populations of barley tested at adult plant stage in field conditions.


33

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Dom Rec

OW

B 3

4( R

)

Nur

e

Tre

moi

s

NuT

r 12

2( R

)

Step

toe

Mor

ex

StM

o 39

( R)

L94

Vad

a

LnV

a 26

( R

)

SusP

trit

VaS

u 11

1( R

)

CC

apa

CC

Su 9

2( R

)

Man

chur

ia

Inte

ract

ion

site

s

SPP-HR

SPP+HR

FPP

Figure 4. Defense reaction of the parents and most resistant line of different mapping populations to Bgh at 48

hai. Data of each group represent the mean of two leaf segments; in each leaf 100 infection sites have been

evaluated. Gray-dotted column (FPP) represent failed primary penetration where the fungus was stopped at

penetration stage; Black column (SPP+HR), successful primary penetration resulting in a haustorium but showed

HR; White column (SPP-HR), successful primary penetration with haustorium and without HR. The lines with the

(R) represent the most resistant line from each mapping population.

Histology of resistance at seedling stage The reaction to Bgh at 48 hai are presented for the parental lines and for the most

resistant lines of each of the six studied mapping populations in Figure 4. At

macroscopic level, all parental lines, except L94 that carries the mlo gene, showed a

compatible infection type (infection type 4). This implies that they do not carry an

effective race-specific resistance gene (R gene) to the isolate Wag.04 that was used.

The proportion of failed infection units that were stopped at penetration stage (pre-

haustorial resistance) in parental lines ranged from 20% (Rec) to 63% (SusPtrit) of

attempted penetration sites. Always a higher proportion of infection units were

stopped at penetration stage in the most resistant lines rather than in the parents of

the mapping populations. Apart from the mlo-caused pre-haustorial resistance in L94,

the highest proportion of papilla-based resistance (83 %) was in line CCSu (RIL 92).

The proportion of penetrated infection units that were stopped in association with a

hypersensitivity reaction of the challenged epidermal cells ranged from 1% (Rec) to

11% (Vada) of the infection units. These results indicate that the QTLs that were

mapped at the seedling stage should determine papilla-based, non-hypersensitive,

basal or partial resistance.

Chapter 2

34

Candidate gene mapping Twenty three out of 39 EST-derived sequences representing candidate genes were

mapped in at least one of the available mapping populations. Eighteen candidate

genes were converted to CAPS markers. For one candidate gene, a SNP was used

to design a dCAPS marker using the dCAPS Finder online software

(http://helix.wustl.edu/dcaps/dcaps.html). Two sequences were mapped as SCAR

markers and two other sequences were previously mapped as RFLP markers. One

sequence(ID: HO02M14) was polymorphic in the CCSu population but could not be

assigned to a map position with confidence. Fiftheen candidate gene sequences

remained for which no polymorphism was detected or for which it was not possible to

obtain a clear PCR amplification product in any of the parental combinations (ID:

HO14H18, HO14K08, HO16C19, HO26F22, HO13M13, HZ40O15, HI05K17,

HO15M05, HO13K06, HW03O11, HU02G09, AJ344223, AJ518932, AJ518933,

AM265370). The position of the mlo gene was determined directly in the LnVa map

on chromosome 4H (Qi et al., 1998; Shtaya et al., 2006). Based on Collins et al.,

(2001), Ror1 is located within a 0.1 cM distance of ABG452 marker on chromosome

1H. Ror2 was located on chromosome 5H based on its flanking markers as published

by Collins et al., (2003).

Co-localizations between candidate genes and QTLs The Chi-square test applied did not indicate a significant global co-localization

between QTLs and candidate genes (Chi-square value, X2=0.79). Nevertheless, six

out of 26 mapped candidate genes showed coincidence in their map positions with

the QTLs identified for basal resistance to powdery mildew (Figure 2, Table 4). We

defined coincidence of a QTL and a candidate gene, when the candidate gene

mapped within the LOD-1 interval of the QTL in our integrated map. In this paper we

only discuss those candidate genes which are within 5 cM from QTL peak markers

because they are the most relevant ones.

Despite similarities in resistance mechanism between basal resistance and mlo

mediated resistance, none of the detected QTLs showed coincidence with the map

positions of mlo and Ror2 genes. The peak marker of Rbgq6, a QTL mapped in VaSu

population at seedling stage, is located within a 0.1 cM distance of ABG452, a marker

closely linked to Ror1 on chromosome 1H. Shtaya et al., (2006) detected a QTL in

LnVa population in the same region where Ror2 is present.


35

Co-localizations between QTLs and race-specific res istance genes In most cases, the map positions of powdery mildew resistance QTLs did not coincide

with those of the race-specific resistance genes (R genes). Only five out of 21 QTLs

mapped in the same region where an R gene has been reported. We defined

coincidence of a QTL and an R gene, when the putative map position of the R gene

in our integrated map was located within the LOD-1 support interval of a QTL. Since

the R-genes were mapped mostly in mapping populations that did not contribute to

our integrated map, their map position in our integrated map is more indirectly

obtained, and therefore the cM distance to our QTL peak marker is less precise than

the map positions of the candidate gene-derived markers. A QTL mapped in LnVa

and VaSu populations (Rbgq9) showed coincidence with the known map position of

the MlLa gene, a gene derived from “Hordeum laevigatum” and located on the long

arm of chromosome 2H. This gene causes an intermediate type of hypersensitive

reaction to avirulent isolates (Giese et al., 1993; Marcel et al., 2007b). In both

populations, the resistance QTL allele was contributed by Vada, a cultivar that carries

the MlLa gene. Since the powdery mildew isolate used for evaluation of the

populations at seedling stage was virulent to the MlLa carrying genotypes, it is

plausible that the explained phenotypic variation in this region is due to a QTL and

not to MlLa itself. One QTL detected on chromosome 7H in the OWBs (Rbgq20)

showed coincidence with the map position of mlt. A QTL mapped on chromosome 1H

in NuTr population (Rbgq4) showed coincidence with Mla and Mlk loci. Backes et al.,

(2003) also reported co-localization of QTLs mapped in the cross Vada × 1B-87 on

chromosomes 1H and 7H with the Mla and Mlf loci, respectively. A QTL mapped in

the StMo population (Rbgq5) on chromosome 1H showed coincidence with the map

position of Mlnn gene. Finally, a QTL mapped in the NuTr population (Rbgq12) on

chromosome 4H showed coincidence with the map position of Mlg. But at the end, in

the case of co-localizations between QTLs and R genes, only fine-mapping of the

concerned loci can tell whether the phenotypic variation is due to the R gene or not.

Chapter 2

36

1Regulation in barley leaves inoculated with Blumeria graminis ff.ssp.;

2RNAi effect, the effect of transient-induced gene silencing (TIGS) of test sequence on resistance phenotype

in the single-cell system; OEX effect, the effect of single-cell transient over-expression of test sequence on resistance phenotype in the single-cell system; 3Regulation by Bgh or

stem-rust inoculation in experiments BB2, BB4, BB7 or BB49 of Plant Expression Database (PlexDB) .Corresponding probe-set IDs: HV02H04, Contig12036_at; L37358, HI02E21u_s_at; HY07A14, Contig15618_at; AJ534447, Contig11241_at.;

4 Not significant;

5 Not analyzed

Table 4 . Characterization and map position of ESTs or cDNA-clone representing candidate genes for powdery mildew resistance in barley

Clone

ID/Acc.

Regul. 1 OEX or

RNAi

effect 2

Function References Name of

marker

Position

Chrom./cM

Co-localization

with QTL(s)

HY05J20 up yes Bax-inhibitor 1 Hückelhoven et al., 2003 GBR0504 6H/ 51.6

HO08H12 up yes HvGER4d Zimmermann et al., 2006 WBE104 4H/ 135.8 Rbgq13

HW05P16 up yes HvGER5a Zimmermann et al., 2006 GBR0180 5H/ 97.4

HO06J21 up n.a. WIR1a Schweizer et al., 1999 WBE101 7H/ 83.1

HO09K14 up n.a. WIR1b Zierold et al., 2005 WBE100 5H/ 35.5

DQ647621 up yes HvGER3a Zimmermann et al., 2006 WBE210 7H/ 88.6

HO02P09 up n.a. unknown Zierold et al., 2005 WBE216 4H/ 108.5

HO07K06 up n.a. unknown Zierold et al., 2005 WBE218 3H/ 149.0

HO11O12 up n.a. unknown Zierold et al., 2005 WBE224 7H/ 23.0

HO13M20 up n.a. unknown Zierold et al., 2005 WBE212 3H/ 11.8 Rbgq10

HO14K22 up yes Receptor protein kinase-like Zierold et al., 2005 WBE211 3H/ 142.7

HO14P02 up n.a. Receptor protein kinase-like Zierold et al., 2005 WBE220 2H/ 136.2

HO12E03 up n.a. unknown Zierold et al., 2005 WBE217 7H/ 33.0

HO07H15 up n.a. 4-Coumarate coenzyme A ligase Zierold et al., 2005 WBE215 6H/ 59.8 Rbgq18

HO40A01 up n.a. Hydrolase Zierold et al., 2005 WBE214 4H/ 126.1

HO12F09 up yes SNAP-34 Douchkov et al., 2005; Jensen et al., 2007 WBE219 2H/ 63.6 Rbgq8

HO06D23 up yes P-type ATPase Zierold et al., 2005 WBE223 1H/ 79.8

HvPrx40 up yes Peroxidase Johrde et al., 2008 WBE232 3H/ 81.2

L37358 up 3 n.a.5 Lipoxygenase 2 Bachmann et al., 2002

http://www.plexdb.org/index.php

WBE228 5H/ 117.0 Rbgq15

Hv07A14 up 3 n.a.5 ACC synthase http://www.plexdb.org/index.php WBE229 2H/ 83.9

AJ536667 up yes WRKY1 transcription factor Eckey et al., 2004 WBE230 7H/ 123.3

AJ534447 up 3 n.a.5 Apoplastic invertase http://www.plexdb.org/index.php WBE231 2H/ 69.0


37

Discussion

Diversity of QTLs for powdery mildew resistance in barley Most mapping studies on quantitative, basal resistance, of barley to powdery mildew

have been based on individual populations. Here we studied the genetic variation of

basal resistance using multiple populations to assess also the diversity of the genes

involved in the genetic variation. The QTLs conferring basal resistance were

distributed over all seven chromosomes of barley without obvious clustering. In

different mapping populations different sets of QTLs were involved in basal

resistance. These indicated a great diversity for QTLs (genes) for this trait in barley. A

similar great diversity has been reported in genes conferring basal resistance in some

other barley-pathogen interactions, like barley-Puccinia hordei (Qi et al., 2000),

barley-Rhyncosporium secalis (Zhan et al., 2008), barley-Fusarium (Fusarium head

blight) (Sato et al., 2008) and barley-spot blotch (Bilgic et al., 2005). Qi et al., (2000)

reported, on the basis of two tested mapping populations, that many loci for basal

resistance to leaf rust (Puccinia hordei) should exist on the barley genome. Marcel et

al., (2007a) reported 19 QTLs contributing to the basal resistance of barley to leaf

rust in five mapping populations. They showed that in each mapping population of

barley different sets of QTLs segregate for basal resistance and only few QTLs were

found to be in common in more than two different barley cultivars. QTL analysis for

Fusarium head blight (FHB) resistance in different barley mapping populations

indicate that quantitative resistance of barley to FHB is conditioned by many loci

(Mesfin et al., 2003). QTLs for powdery mildew resistance have been reported in

different mapping populations of barley (e.g. Backes et al., 1995; Backes et al., 1996;

Spaner et al., 1998; Falak et al., 1999; Backes et al., 2003; Von Korff et al., 2005;

Shtaya et al., 2006). We compared the position of the peak marker of the presently

found QTLs with the peak markers of already reported QTLs using our high-density

integrated map of barley. In four cases (Rbgq2a, Rbgq4, Rbgq7 and Rbgq8), the

location of peak marker of QTLs are only about 0-2 cM away from previously reported

QTLs for mildew resistance, and in two cases (Rbgq2b and Rbgq14) the peak

markers are 4-6 cM away. One of these QTLs (Rbgq4) already has been reported in

two different populations of barley, Blenheim × E224, Vada × 1B-87 (Thomas et al.,

1995; Backes et al., 2003). This suggests that some QTLs found in our study might

be the same as already reported QTLs in other genetic backgrounds.

Growth-stage specificity of QTLs for basal resistan ce Growth-stage dependent and independent QTLs both have been reported in many

host-pathogen interactions, including powdery mildew and leaf rust on barley,

Chapter 2

38

septoria blotch on wheat, ascochyta blight on pea, and downy mildew on lettuce (e.g.

Qi et al., 1999; Backes et al., 2003; Eriksen et al., 2003; Prioul et al., 2004; Marcel et

al., 2007a; Zhang et al., 2009). In our study, only 4 out of a total of 21 QTLs were

effective at both growth stages (Rbgq2a, Rbgq7, Rbgq9, and Rbgq13), suggesting

that QTLs for powdery mildew resistance are mainly growth-stage dependent. Similar

results have been reported by Backes et al. (2003). They found that only one out of

five QTLs that mapped in a cross between the barley cultivar ”Vada” and a wild barley

(H. vulgare ssp. spontaneum line ”1B-87”) was growth-stage independent. This is

also in agreement with the report of Mastebroek & Balkema-Boomstra (1991) in which

it is concluded that the phenotypic expression of basal resistance of barley to

powdery mildew is growth-stage dependent. Our histological observations confirm

that the QTLs mapped in the present work contribute mainly to a prehaustorial non-

hypersensitive basal resistance, rather than to an incomplete hypersensitive

resistance.

Co-localization of candidate genes and R genes with QTL regions In field experiments under natural disease infection, R-gene resistance can behave

as QTL resistance, since 1) the R-genes may have an intermediate effect (like MlLa),

and 2) the natural population of pathogen may consist of a mixture of virulent and

avirulent races to a certain R-gene, resulting in a lower epidemic development on

genotypes carrying the R-gene than in genotypes carrying the susceptibility allele

(Parlevliet, 1983; Parlevliet & van Ommeren, 1985). Furthermore, QTLs that co-

localize with R-genes for resistance may be allelic variants of R-genes or residual

effect of defeated R-genes (Kelly & Vallejo, 2005).

The molecular basis of quantitative (basal) resistance, like other genetically

complex traits, is poorly understood. Candidate gene analysis can be considered as

an important approach toward understanding the molecular basis of quantitative

resistance, partly thanks to the availability of huge numbers of ESTs in databases. In

the present study, we found a co-localization of QTLs for resistance with genes that

have been suggested by reverse genetics to be involved in barley-powdery mildew

interaction. Six out of 26 mapped candidate genes showed coincidence with the

position of QTLs for powdery mildew resistance (Figure 2). Transient-induced gene

silencing (TIGS) experiments showed that a t-SNARE protein, HvSNAP34, is involved

in three types of durable, non-race specific resistances, viz. mlo-mediated, non-host

and basal resistance (Douchkov et al., 2005). Map co-localization of HvSNAP34 with

a QTL (Rbgq8) for powdery mildew resistance provides further evidence of the role of

this gene in basal resistance. An other QTL (Rbgq7) co-localized with the known map


39

position of Ror1 on chromosome 1H. None of the QTLs co-localized with mlo and

Ror2 genes. We conclude that, despite the similarity in resistance mechanism, there

is little evidence that basal resistance is determined by allelic variants of the Mlo or

Ror2 genes.

Map co-localization between resistance gene analogs (RGA) (see Glossary) and

defense-related (DR) genes with QTLs for seedling and/or adult plant resistance have

been reported in many other host-pathogen systems (Faris et al.1999; Geffroy et al.,

2000; Wang et al., 2001; Prioul-Gervais et al., 2007; Marcel et al., 2007a). Hu et al.,

(2008) established a candidate gene strategy that integrates linkage map, expression

profile and functional complementation analyses with the purpose of cloning QTLs

that are responsible for quantitative resistance of rice to bacterial blight and fungal

blast. Using more than one mapping population, however, provides an impression on

the diversity of the genes responsible for basal resistance. With a larger number of

mapping populations and QTLs detected, it is possible to test a larger sample of

QTLs for their statistical coincidence with the candidate genes. The use of an

integrated map to compare data obtained in different populations also increases the

chance of discovering relevant co-localizations. Here we prioritized the candidate

genes by accumulating evidence of position, biological function and functional proof

by TIGS. However, map co-localization of candidate genes with the QTLs involved in

the trait variation is not a sufficient proof of causal relationship, and actual

involvement of the candidate genes in the trait variation should be validated by

complementation tests through genetic transformation and/or physiological analyses

of genes at the mRNA and/or the protein level and by ultra-fine-mapping of both the

candidatee gene and the QTL (Pflieger et al., 2001). The integrated candidate gene

strategy has proven to be a fast and efficient approach for isolation of genes with

small effect (Hu et al., 2008). Information gained from this approach may help

breeders to use more efficiently the valuable sources of diversity to improve the level

of basal resistance in crop plants.

Acknowledgements

Reza Aghnoum was supported by the Agricultural Research and Education Organization (AREO) and the Ministry of Science Research and Technology of Iran. The excellent technical assistance of Anton Vels in greenhouse and field experiments is greatly appreciated. We are indebted to Dr. Ralph Hückelhoven (Technical University of Munich, Germany) for sharing five sequences of candidate genes; to Christine Boyd and Dr. Andris Kleinhofs from the Washington State University for making available the segregation data of many BIN markers; to Prof. Fred van Eeuwijk for allowing us to collect powdery mildew data from his field experiments of the NuTr population; to Dr. Rajeev K. Varshney from ICRISAT for providing primer aliquots and markers information on the new set of EST-SSR markers. Those EST-SSR markers have been developed at IPK in Germany. Marc-Antoine

Chapter 2

40

Bonvoisin from ESITPA in France is acknowledged for his hard work on mapping this new set of EST-SSR markers. We gratefully acknowledge the critical reading of the manuscript by Prof. Richard Visser.

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Chapter 3

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Transgressive segregation for extreme low and high

level of basal resistance to powdery mildew in barley

R. Aghnoum, R. Tadayonirad and R.E. Niks

Chapter 3

48

Abstract

Basal resistance of barley to powdery mildew is a quantitatively inherited trait that

limits the growth and sporulation of barley powdery mildew pathogen by a non-

hypersensitive mechanism of defense. Two experimental barley lines were developed

with extremely high (ErBgh) and low (EsBgh) level of basal resistance to powdery

mildew by convergent crossing followed by phenotypic selection between the most

resistant and the most susceptible lines from four mapping populations of barley. We

tried to verify the alleles of the seven identified QTLs in the four mapping populations

from which the most resistant and the most susceptible individuals had been derived.

Only four out of seven QTLs were confirmed to be present in the selected resistant

lines. Surprisingly, none of the expected QTLs could be detected in the resistant line

ErBgh. Our results showed that phenotypic selection in convergent crossing is very

effective and resulted in very contrasting phenotypes for basal resistance. Histological

observations showed that in ErBgh almost 90% of infection units failed to penetrate

the epidermal cells in association with papilla formation. Established infection units

may fail in formation of a substantial proportion of the secondary and subsequent

haustoria. The extremely resistant and susceptible lines developed here are valuable

material to be used in further experiments to characterize the molecular basis of basal

resistance to powdery mildew.

Introduction R-gene triggered resistance is considered easy to select for, because of the large

phenotypic effect. Most R-gene resistance appears to be associated with HR cell

death (Watanabe & Lam, 2005). This resistance is only able to protect the plants

against those races of the pathogen that possess an avirulence (Avr) gene

corresponding to the R-gene. Pathogens like Blumeria graminis, the cereal powdery

mildew pathogen, that possess a mixed (sexual and asexual) reproduction system

and a high potential for gene flow due to the long-range wind dispersal of spores, are

easily able to break down R-gene mediated resistance. Use of quantitative resistance

has been proposed as a breeding strategy for durable protection against such a

pathogens (McDonald & Linde, 2002). The minor gene, quantitative, resistance is

often considered much more difficult to select for, due to the small contribution of

each gene to the total level of resistance. Marker assisted selection has been

proposed as a strategy to accumulate such resistance genes, in order to achieve high

levels of this durable, non-race specific resistance (Castro et al., 2003, Friedt &

Ordon, 2007). Quantitative resistance of barley to powdery mildew is based on

impaired haustorium formation in the attacked epidermal cells, and is not associated

Transgressive segregation for ….

49

with a hypersensitive reaction of challenged cells.This resistance also called basal

resistance (see Glossary). Such a quantitative pre-haustorial resistance also occurs in

various other mildew and rust fungal pathosystems (e.g. Carver, 1986; Niks, 1986;

Asher & Thomas, 1987; Sillero & Rubiales, 2002). It has been reported frequently that

among the progenies of a cross between two plant genotypes with intermediate levels

of resistance or even susceptibility, descendants can be found with a higher or lower

level of resistance compared to that of both parents (e.g. Jones, 1983; Wallwork &

Johnson, 1984; Roderick & Jones, 1991). This phenomenon is known as

transgressive segregation. Transgressive segregation implies that both parents carry

a similar gene dose for the trait of interest, but those genes are located at different

loci, and alleles for resistance from both parents can end up together in some of the

progeny plants.

Four barley mapping populations that had not been developed with the aim to study

quantitative resistance to powdery mildew demonstrated that there is a great genetic

diversity of minor genes for powdery mildew resistance, with very few of such genes

in common between the populations (Chapter 2). Such a high diversity leads to

substantial transgressive segregation for resistance levels. We presume that further

accumulation of minor genes by convergent crossing and rounds of selection should

result in even higher levels of resistance to be achieved.

The objective of the present study was to investigate genetic plasticity of a limited,

medium resistant barley gene pool for obtaining extremely susceptible and extremely

resistant progeny by phenotypic selection for low and high resistance levels,

respectively. On the basis of the knowledge of the contributing QTLs (see Chapter 2)

we intended to test whether the resulting extremely susceptible and resistant progeny

would indeed carry the expected QTL alleles for susceptibility or resistance.

Material and Methods

Crossing and selection program In a previous study (Chapter2) we mapped QTLs for powdery mildew resistance at

seedling stage in four mapping populations of barley, viz. one recombinant inbred line

(RIL) population, L94 × Vada (LnVa) and three doubled haploid (DH) populations,

Nure × Tremois (NuTr), Steptoe × Morex (StMo) and Oregon Wolfe Barleys (OWBs).

Per mapping population, one line was selected with the lowest infection frequency on

the primary leaf and the lowest haustorium formation rate of challenged epidermal

cells. These homozygous most resistant lines were crossed pairwise (F1), and their

most resistant descendants (in F2) were intercrossed again, forming a Double Cross

(DC), to develop, by phenotypic selection, a descendant (in DC-S1) with the highest

Chapter 3

50

level of quantitative (basal) resistance (Figure 1). In parallel, per mapping population,

one line was selected with the highest infection frequency on the primary leaf and the

highest haustorium formation rate of challenged epidermal cells, representing the

most susceptible line of that mapping population. These lines with very low level of

basal resistance were intercrossed in two rounds to form a Double Cross (DC) to

accumulate QTL alleles for susceptibility. For development of DH barley lines with

extremely high level of basal resistance, the DC-S1 seeds were allowed to self-

pollinate and the most resistant DC-S2 plant was selected. A bulk of microspores of

six plants (DC-S3) derived from the self-pollination of this DC-S2 plant was used for

DHs production. For development of a DH barley line with extremely low level of basal

resistance, three DC-S1 plants were used to make DH lines. From each DH plant

three seedlings (12 days-old) were inoculated with powdery mildew and based on the

infection frequency, four days after inoculation, two DH lines, one with the highest and

one with the lowest infection frequency, were selected. The susceptible barley cv.

Manchuria was used as reference.

Evaluation of basal resistance at seedling stage For evaluation of the mapping populations, selection of the most resistant and the

most susceptible progenies and DHs, the following method was used. Barley plants

were sown in boxes (37 × 39 cm) and were grown in spore free greenhouse

compartments at 18-20 °C with 70% relative humidity and a photoperiod of 16 h. After

12 days, the fully expanded primary leaf was fixed in a horizontal position, with the

adaxial side up. A settling tower was used for inoculation. Freshly produced conidia

on young seedlings of the susceptible cultivar ‘Manchuria’ were blown into the tower.

The density of inoculum was monitored by haemocytometer and was adjusted to 10-

15 conidia per mm2. The inoculated plants were incubated in a growth chamber at 18-

20 oC with fluorescent light irradiance (102.8 micromol/m2/s) supplied for 12 h per

days. Four days after incubation, the barley accessions were scored by counting the

number of young powdery mildew colonies per 2 cm2 of leaf area using a frame with a

0.5 × 4 cm window and a magnifying glass (10x).

Histological assessment of infection Samples were collected from the middle part of inoculated leaves at 48 hai (hours

after inoculation) and transferred to a solution of acetic acid-ethanol (3/1 v/v) to be

cleared overnight. Then the leaf segments were stained with 0.1 mg ml-1 Trypan Blue

in alcoholic lactophenol as described by Peterhänsel et al. (1997). To make the

prepreations, the leaf segments were embedded adaxial side up in glycerol and


51

examined at 400× magnification using a transmitted white light Axiophot microscope

(Zeiss, Germany). Only stomatal and interstomatal cells (type A and type B of

epidermal cells following the terminology of Koga et al., 1990) were examined. Long

epidermal cells over-lying vascular bundles (type C) and subsidiary cells were not

considered. Only those epidermal cells that were attacked by a single germinated

conidium were evaluated.

Verification of QTLs via putative flanking markers One DH line was selected from each double cross DH population. For the resistant

selection (DC-R) there were 132 DHs, for the susceptible selection (DC-S) 129 DHs.

From DC-R and DC-S the most resistant and the most susceptible DH lines,

respectively, were selected based on the phenotypic reaction of DHs to powdery

mildew. The inoculation with powdery mildew and evaluation was performed in the

same way as described earlier. The selected lines were genotyped for flanking

markers of the seven identified QTLs (Table1).

Results

Development of barley lines with extremely low and high level of basal

resistance to powdery mildew Based on both macroscopic and histological data, DC-S1 families showed

transgressive segregation both towards resistance and susceptibility (data not

shown). One DH line was selected from each double cross (DC-R and DC-S)

combination out of 132 and 129 total DH lines produced, respectively. The extremely

resistant and the extremely susceptible barley lines to Bgh were called ErBgh and

EsBgh, respectively. ErBgh and EsBgh showed a remarkable difference in infection

frequency of powdery mildew at 6 dai (Figures 2 and 3). Histological evaluations

revealed that in the most resistant lines of the mapping populations always a higher

proportion of germlings was stopped in association with papilla formation at the sites

of attempted penetration than in the most susceptible lines (Figure 4). The ErBgh line

allowed only about 12 % and the EsBgh line 67% of the mildew germlings to form a

primary haustorium (Figure 4). In ErBgh a considerably lower proportion of secondary

penetration attempts along the elongated secondary hyphae (ESH) resulted in a

haustorium than in EsBgh at 48 hai (19% and 60% respectively). Interestingly, in

ErBgh, most of the successful secondary penetrations were observed in the same cell

as where the first haustorium was formed (Figure 5). On ErBgh no substantial

hypersensitivity reaction was observed.

Chapter 3

52

Our results demonstrated that just by visual selection QTLs from different sources

can be combined to reach an extreme level of basal resistance.

QTL verification We tried to verify the alleles of the seven identified QTLs in the four mapping

populations from which the most resistant and the most susceptible individuals had

been derived. It seemed reasonable to presume that line ErBgh would combine for all

or most of the QTLs the resistance allele, EsBgh for all or most of the QTLs the

susceptibility allele. We checked the genotype of the most resistant and the most

susceptible genotypes of each mapping population that were used for making the first

round of crossings (Table 1). Only four QTLs (Rbgq14, Rbgq2a, Rbgq19 and

Rbgq10) were confirmed to have the resistance allele in those four selected resistant

lines (Table 1). Surprisingly, none of the QTLs for which we can have a conclusion

(Rbgq14, Rbgq2a, Rphq19 and Rphq10) could be detected in the resistant line

ErBgh. Only three QTLs (Rbgq2a, Rbgq19 and Rbgq15) were confirmed to have the

susceptibility allele in the selected susceptible lines. Surprisingly, none of the QTLs

for which we can have a conclusion (Rbgq2a, Rbgq19 and Rbgq15) could be

detected in the susceptible line EsBgh.

The verification of the QTL alleles present in ErBgh and EsBgh was hampered by at

least two technical points.

1-Lack of multiple allelic variations makes a QTL peak or flanking marker-allele not

unique for selection if this allele also occurs in at least one of the parents of the other

mapping populations. This is especially true for markers like AFLP, where each

marker has only two variants (“0” or “1”).

2 -We searched on the latest integrated barley linkage map (Chapter 2) for multiple

allelic SSR markers close to the target QTLs, to increase the chance that the allele

that was indicative for a particular QTL allele, did not occur in any of the other

ancestral lines. However, we found such SSR markers often at some distance from

the peak marker of the QTL. Therefore, recombination between the marker locus and

the QTL might occur during the crossing procedure. In case of such a distance,

absence of the indicative marker allele does not prove absence of the QTL allele.


53

Mapping QTLs for powdery mildew resistance at seedling stage in four mapping populations

Selection of the most resistant line of each population

Two cycles of recurrent selection to accumulate QTLs for resistance

Figure 1. A schematic representation of a crossing programs to develop a barley line with extreme high level of basal resistance to powdery mildew. A parallel crossing program followed to develop a line with extreme low level of resistance.

R1 × R2 R3 × R4

F1 Selfing F1 F2 Selection F2 F2 R1 × R2 × F2 R3 × R4

DC

DC-S1 DC-S2 Selection DC-S3 Making DH lines Selection of the extremely resistant line

Figure 2. Phenotypeof the extremely resistant (ErBgh, left) and the extremely susceptible (EsBgh) barley lines six days after inoculation with Bgh with the same inoculom density and incubation conditions.

Chapter 3

54

Table1: Characteristics of QTLs mapped at seedling stage for powdery mildew resistance and

the genotype of the most resistance and the susceptible lines used for crossing Population QTLs Peak marker LOD Exp% a Donor b R parent c S parent d

OWBs

Rbgq20

Rbgq14

ABG704

Bmac0047e

3.5

4.9

18.3

19.9

Rec

Dom

-

+

+

+

NuTr

Rbgq2a

Rbgq19

bPb-0312

scssr05599

6.1

5.55

24.8

22.4

Tremois

Nure

+

+

-

-

LnVa

Rbgq2b

Rbgq15

E35M48-410a

E33M58-306

2.8

3.1

19.8

21.5

L94

Vada

-

-

+

-

StMo Rbgq10

MWG571C 4.7

15.5

Morex

+ +

a The proportion of the phenotypic variance explained by the QTL b Donor of the resistance allele

c The genotype of the most resistant (R ) line of each population regarding the expected QTL; + indicates that the

line contained a molecular marker that suggested the expected resistance allele of the QTL, - that the line

contained a molecular marker that suggested the unexpected susceptibility allele. d The genotype of the most susceptible (S) line of each population regarding the expected QTL; + indicates that

the line contained a molecular marker that suggested the expected susceptibility allele of the QTL, - that the line

contained a molecular marker that suggested the unexpected resistance allele.


55

0

10

20

30

40

50

60

70

80

90

StMo-

r

LnVa-r

OWBs-r

NuTr-r

StMo-

s

LnVa-s

OWBs-s

NuTr-s

ErBgh

EsBgh

Man

churia

Infe

ctio

n fr

eque

ncy

Figure 3. Infection frequency [colonies per 2 cm2] of the most resistant (r) and

the most susceptible (s) genotypes of different mapping populations. LnVa (L94

× Vada), StMo (Steptoe × Morex), OWBs (Oregon Wolf Barleys) and NuTr

(Nure × Tremois). ErBgh represents the extremely resistant and EsBgh the

extremely susceptible DH barley line to Bgh derived from the double crosses.

Data were collected at 4 dai.

0

10

20

30

40

50

60

70

80

StMo-

r

LnVa-r

OWBs-r

NuTr-r

StMo-

s

LnVa-s

OWBs-s

NuTr-s

ErBgh

EsBgh

Man

churia

Pen

etra

tion

eff

icie

ncy

%

Figure 4. Penetration efficiency (% successfully penetrating germlings) of Bgh

on the most resistant (r) and the most susceptible (s) genotypes of different

mapping populations. ErBgh represents the extremely resistant and EsBgh the

extremely susceptible DH barley line to Bgh derived from the double crosses.

Chapter 3

56

Discussion

Accumulation of genes with minor effect by phenotyp ic selection Our data support the proposed strategy that genotypes with much higher (and lower)

level of resistance than the parents can be selected from progenies derived from

convergent crosses between several parents with intermediate levels of resistance,

due to high transgression of the genetic components from both parents (Wallwork &

Johnson, 1984). This high success of accumulation of quantitative genes for basal

resistance can be ascribed to the great diversity of such genes in the barley

germplasm, as shown in Chapter 2, involving different loci in each parental pair. Such

an accumulation was, for the same reason, also easily achieved by Parlevliet &

Kuiper (1985), in barley to barley leaf rust (Puccinia hordei). Also to that pathogen

loci for basal resistance occur all over the barley genome (Qi et al., 2000; Marcel et

al., 2007), making transgression for basal resistance a very common phenomenon.

Efficiency of phenotypic selection to achieve a hig h level of basal

resistance Our results showed that phenotypic selection in convergent crossing is very effective

and resulted in very contrasting phenotypes for basal resistance. MAS has some

problems, like the lack of unique markers close to the peak marker of the QTLs of

Figure 5. Develpoment of Bgh (48 hai) on ErBgh. A, a successful primary penetration resulted in a haustorium and a failed secondary attempted penetration (SAP). B, a closer view of failed SAP showed in fig. A. C, a successful primary penetration resulted in formation of a secondary haustorium in the same cell. The scale bar represents 100 µm.


57

interest, especially if more than two parents will be used in a convergent crossing

programme. With strong enough linkage disequilibrium (see Glossary) between

markers and QTLs (Dekkers & Hospital, 2002), MAS should in theory be easier,

faster, cheaper, or more efficient than classical (phenotypic) selection but in practice

the MAS is not always as efficient as expected (Hospital, 2008). It should be pointed

out here that the efficiency of MAS also depends on the type of the trait and to the

population. Phenotypic selection is more effective than MAS in some cases or at least

both are of the same value (Romagosa et al., 1999; Flint-Garcia et al., 2003). Flint-

Garcia et al. (2003) compared the efficiency of phenotypic selection versus Marker-

assisted selection for rind penetrometer resistance (RPR) and European corn borer

(ECB). They reported that MAS for high and low RPR was effective in the three

populations studied. Phenotypic selection for both high and low RPR was more

effective than MAS in two of the populations. However, in a third population, MAS for

high RPR using QTL effects from the same population was more effective than

phenotypic selection. MAS is probably not needed for a rather simple trait like

quantitative powdery mildew resistance. Selection for powdery mildew resistance is

quite straightforward, plenty of genes in the ancestors are available that can be

accumulated and non-genetic variation is relatively low if inoculum is applied

homogeneously over the plants to be compared. MAS may be more useful to

introgress or accumulate QTLs for such traits like drought and salt tolerance that are

difficult to select for phenotypically. However, also in such cases unique marker

alleles are required if a convergent crossing scheme is used.

It is remarkable that our initially selected most resistant and most susceptible lines

from the mapping populations did not contain (nearly) all the expected QTL alleles

(Table 1). One possible explanation may be that the effect of QTLs depends strongly

on the genetic background. Marcel et al. (2008) showed that some QTLs for basal

resistance to P. hordei had a particularly high effect if combined with other QTLs, that

alone had hardly any effect. Such interactions would make phenotypic selection

probably more successful than MAS, in case the partner QTLs of such interactions

are still unknown.

High level of basal penetration resistance in ErBgh line Basal resistance of barley to powdery mildew is mainly based on impairment of

haustorium formation. The observation that established infection units in ErBgh may

fail in formation of a substantial proportion of the secondary and subsequent

haustoria (Figures 5A and B) is unexpected. In other barley and oat genotypes

(Carver & Carr, 1978; Gustafsson & Claesson, 1988), lower secondary haustorium

Failed SAP

Chapter 3

58

formation is reported but it is not mentioned whether this is due to failure of

attempted secondary penetration or to slow growth resulting in fewer penetration

attempts. The percentage of successful primary penetration in ErBgh is nearly as low

(12%) as the lowest (10%) reported ever (Carver, 1986), excluding non-host and mlo

resistance. It seems plausable that further rounds of convergent crossing and

selection, involving parents with high quantitative resistance might easily increase

resistance levels even further. It should be further investigated whether the failed

secondary and attempted penetration in ErBgh is due to papilla formation or that

other mechanisms are involved.

The present observation that in ErBgh at 48 hai the second and next haustoria are

often formed in the same cell as the first haustorium (Figure 5C) is intriguing. A

similar phenomenon was observed in SusBgt lines that show a high level of

susceptibility to the non-adapted wheat powdery mildew, B. graminis f.sp.tritici

(Chapter 4). This is a further indication that basal host resistance has similarities with

non-host resistance.

We consider the extremely susceptible and resistant lines developed here as

valuable material to be used in further experiments to characterize the molecular

basis of basal resistance to powdery mildew. Additional work on this material could

also lead to a critical comparison of phenotypic versus marker assisted selection for a

quantitative trait like basal resistance.

Acknowledgements

The excellent technical assistance of Anton Vels in greenhouse experiments is greatly appreciated. Dr.

TC. Marcel is acknowledged for his help in QTL verification using SSR markers.

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Friedt W, Ordon F. 2007. Molecular Markers for Gene Pyramiding and Disease Resistance Breeding

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Hospital F. 2008. Challenges for effective marker-assisted selection in plants. Genetica

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Jones IT. 1983. Transgressive segregation for enhanced level of adult plant resistance to mildew in

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trait loci for partial resistance of barley to Puccinia hordei confirmed in mapping populations and

near-isogenic lines. New Phytologist 177: 743–755.

Marcel TC, Varshney RK, Barbieri M, Jafary H, de Ko ck MJD, Graner A, Niks RE. 2007. A high-

density consensus map of barley to compare the distribution of QTLs for partial resistance to

Puccinia hordei and of defence gene homologues. Theoretical and Applied Genetics 114: 487–

500.

McDonald BA, Linde C. 2002. The population genetics of plant pathogens and breeding strategies for

durable resistance. Euphytica 124: 163-180.

Niks RE. 1986. Failure of haustorial development as a factor in slow growth and development of

Puccinia hordei in partially resistant barley seedlings. Physiological and Molecular Plant

Pathology 28: 309–322.

Parlevliet JE, Kuiper HJ. 1985. Accumulating polygenes for partial resistance to barley leaf rust,

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Qi X, Fufa F, Sijtsma D, Niks RE, Lindhout P, Stam P. 2000. The evidence for abundance of QTLs

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Roderick HW, Jones IT. 1991. The evaluation of adult plant resistance to powdery mildew (Erysiphe

graminis f.sp. avenae) in transgressive lines of oats. Euphytica 53: 143-149.

Romagosa I, Han F, Ullrich SE, Hayes PM, Wesenberg DM. 1999. Verification of yield QTL through

realized molecular marker-assisted selection responses in a barley cross. Molecular Breeding 5:

143–152.

Sillero JC, Rubiales D. 2002. Histological characterization of the resistance of faba bean to faba bean

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Chapter 3

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New perspectives to identify genes that…

54

New perspectives to identify genes that determine

non-host resistance of barley to non-adapted

Blumeria graminis forms

R. Aghnoum and R.E. Niks

Submitted

Chapter 4

62

Abstract The genetic basis of non-host resistance of barley to non-adapted formae speciales of

Blumeria graminis is not known, since there is no barley line that is susceptible to, for

example, the wheat powdery mildew pathogen, Blumeria graminis f.sp. tritici (Bgt).

We identified barley accessions with rudimentary susceptibility to the non-adapted

Bgt. Those accessions were intercrossed in two cycles and selected two lines, called

SusBgt SC and SusBgt DC, with exceptional susceptibility to Bgt at seedling stage.The

quantitative variation among barley accessions and in the progenies after convergent

crossing suggests a polygenic basis for this non-host resistance. Both lines show a

remarkable level of haustorium formation and colony development by the non-

adapted target mildew. The SusBgt lines and their ancestor lines also allowed

haustorium formation and conidiation by four out of seven tested non-adapted

Blumeria forms. Component analysis of the infection process suggested that non-host

resistance factors are specific to the form and developmental stage of Blumeria.

Resistance to establishment (first haustorium), colonization (subsequent haustoria)

and conidiation are not clearly associated.

The lines developed will serve to elucidate the genetic basis of non-host resistance

in barley to Bgt, and they are useful tools in gene expression and complementation

studies on non-host resistance.

Introduction The majority of plant species are immune to the majority of potentially pathogenic

microbial invaders. This is the most common and broadest spectrum resistance,

termed non-host resistance. It is defined as resistance shown by all members of a

plant species against all members of a given pathogen species (Heath, 2000;

Thordal-Christensen, 2003). Non-host resistance relies on a variety of preformed and

inducible mechanisms of defense (Thordal-Christensen, 2003; Mysore & Ryu, 2004).

Pathogens that are able to overcome the preformed defense mechanisms encounter

extracellular surface receptors that recognize general elicitors, the so-called

pathogen-associated molecular patterns (PAMP) (see Glossary) (Nürnberger et al.,

2004; Chisholm et al., 2006). The pathogen should suppress the PAMP-triggered

immunity (PTI) in order to establish a compatible interaction and successfully colonize

the plant (Chisholm et al., 2006). Those pathogen species that are not able to

suppress the PTI are often called non-adapted, inappropriate or heterologous

pathogens. It is presumed that would-be pathogens deliver effectors (see Glossary) in

the attacked plant cells that may suppress PTI if there is sufficient correspondence

between effectors and their respective operative targets (Block et al., 2008; van der


63

Hoorn & Kamoun, 2008). The degree of correspondence between effectors and

targets may determine the host status of a plant to a would-be pathogen (Niks &

Marcel, 2009). Two interesting questions are whether different stages of development

of a pathogen in a plant may require different sets of effectors and whether different

strains of a pathogen species need to address the same operative targets in a

particular plant species.

Powdery mildew in cereals is caused by the biotrophic Ascomycete fungus Blumeria

graminis (Bg) formerly known as Erysiphe graminis. Bg has a high level of biological

specialization for its cereal and grass hosts. Barley is considered a strict non-host

plant species to the wheat forma specialis, Blumeria graminis f.sp. tritici (Bgt), and

also to the forms of Blumeria graminis that are pathogenic to oats and rye (Hardison,

1944; Eshed & Wahl, 1970; Menzies & MacNeill, 1989). At the macroscopic level, no

barley accession has been identified that allowed Bgt colonies to develop (Menzies &

MacNeill, 1989; Olesen et al., 2003; Atienza et al., 2004). At the microscopic level,

however, some barley accessions have been reported to permit some development of

Bgt (Tosa & Shishiyama, 1984; Trujillo et al., 2004). Penetration of the plant

epidermal cell walls and subsequent haustorium formation, hereafter called

establishment, is a critical stage in the infection process. In compatible interactions of

barley with the adapted powdery mildew pathogen, Blumeria graminis f.sp. hordei

(Bgh), the fungus can successfully penetrate through epidermal cell walls and form

haustoria to obtain nutrients from the host cells. In non-host interactions of barley with

non-adapted formae speciales such as Bgt, establishment typically fails in association

with formation of cell wall appositions (papillae), in some cases supplemented with

the hypersensitive cell death reaction (HR) of single attacked cells. In the interaction

of barley with the adapted powdery mildew, Bgh, a proportion of attempted germlings

are also stopped by such defense mechanisms (Tosa & Shishiyama, 1984;

Hückelhoven et al., 2001; Trujillo et al., 2004).

It is unknown which genetic factors determine that barley is a host to Bgh but a non-

host to non-adapted formae speciales of Bg. Since there is no barley line that is

susceptible to, for example, Bgt, inheritance studies are not possible. Some candidate

genes have been implicated to play a role in resistance of barley to the wheat forma

specialis (Bgt) by transient overexpression or transient-induced gene silencing (TIGS)

in single epidermal cells (Miklis et al., 2007; Douchkov et al., 2005; Eichmann et al.,

2004; Schweizer, 2007), but it is not known whether allelic variation between these

genes explains the host status difference between wheat and barley to Bgt. Barley is

indeed a model plant in which to study the molecular basis of basal and non-host

resistance to powdery mildews (Hückelhoven, 2007; Schweizer, 2007; Collinge et al.,

Chapter 4

64

2008). We set out to identify the genes that are responsible for natural variation in

host status and non-host resistance level.

In the present paper, we identified barley accessions with rudimentary susceptibility

to the non-adapted wheat powdery mildew (Bgt) and crossed them to develop

experimental barley lines with exceptional susceptibility to Bgt. We quantified

components of infection development of Bgh, Bgt and several other non-adapted

powdery mildew formae speciales and species on these experimental lines to

determine the specificity of resistance factors.


Plant and Pathogen Materials A collection of 439 accessions of barley was evaluated at the seedling stage to the

non-adapted wheat powdery mildew fungus, Bgt. The collection consisted of 136

accessions of Hordeum spontaneum and 303 accessions of Hordeum vulgare: viz.,

227 accessions (landraces and wild H. spontaneum barley from the ICARDA barley

germplasm collection) provided by Dr. Rajeev K. Varshney, 54 accessions received

from the Centre for Genetic Resources (CGN), Wageningen, the Netherlands and 22

parental lines of available mapping populations of barley at the Laboratory of Plant

Breeding, Wageningen University. The seeds of barley cv. Turkey 290 were kindly

provided by Dr. H.E. Bockelman (National Small Grains Collection, USDA, USA). An

isolate of Bgt (Swiss field isolate FAL92315), kindly provided by Dr. Patrick Schweizer

(IPK, Germany), was propagated on the susceptible wheat cv. Vivant and used for

inoculation. The lines with some level of susceptibility to Bgt were intercrossed in two

cycles and resulted in two lines with exceptional susceptibility to Bgt (see Results

section). These lines were named SusBgt SC and SusBgt DC. SusBgt lines and the

parental lines were tested to several non-adapted powdery mildew formae speciales

(ff. spp.) and species. The isolates will be referred to according to their source host.

The following non-adapted ff. spp. of Bg were tested: Blumeria graminis f.sp. avenae

(Bga), the powdery mildew of oat (Avena sativa); Blumeria graminis f.sp. secalis

(Bgs), the powdery mildew of rye (Secale cereale); and four isolates collected from

wild grasses, Hordeum murinum, Blumeria graminis f.sp. hordei-murini (Bghm);

Agropyron repens, Blumeria graminis f.sp. agropyri (Bgar); Bromus mollis, Blumeria

graminis f.sp. bromi (Bgb); and Dactylis glomerata, Blumeria graminis f.sp. dactylidis

(Bgd). All of these isolates were collected in the Wageningen region and maintained

in isolation on their respective host plants. We also tested two non-adapted species,

viz. Oidium neolycopersici, the powdery mildew of tomato, and Sphaerotheca

fuliginea, the powdery mildew of cucumber.


65

Inoculation

Three seedlings of each barley accession were grown in boxes 37 × 39 cm in size.

The susceptible wheat cv. Vivant was sown in each box to monitor the effectiveness

of inoculation. Turkey 290, SusPmur and SusPtrit which have been reported to allow

relatively high haustorium formation by Bgt (Tosa & Shishiyama, 1984; Trujillo et al.,

2004) were included as references. The fully expanded primary leaves of 12-day-old

seedlings were stapled horizontally, adaxial side up. To make a dense inoculation,

freshly produced conidia of a Bgt isolate formed on the susceptible wheat cv. Vivant

were blown onto the leaves to reach a density of approximately 50 conidia per mm2.

The inoculated plants were incubated in a growth chamber at 18-20 °C with 70%

relative humidity and a photoperiod of 16 h (200 micromol/m2/s).

Macroscopic and microscopic evaluation of barley- Bgt interaction First, macroscopic evaluations of all tested accessions were carried out using a

magnifying glass (10x) eight days after inoculation (dai). Those accessions that

macroscopically showed some degree of susceptibility were selected. These

accessions were sown and inoculated again for quantification of haustorium formation

and conidiation under the microscope. At two time points, 72 hours after inoculation

(hai) and 8 dai, a leaf segment approximately 3 cm long was collected from the

middle part of the infected primary leaves. Leaf segments were transferred to a

solution of acetic acid-ethanol (3:1 v:v) to be cleared for at least 3 hours. Then, the

leaf segments were stained in Coomassie Brilliant Blue according to the method of

Wolf & Fric (1981). The experiment was carried out in two consecutive replications.

Per barley accession, 200 observed germlings were scored for the result of the first

penetration attempt to establish a haustorium. Because different types of host

epidermal cells show different degrees of resistance to penetration (Lin & Edwards,

1974; Koga et al., 1990) and penetration attempts by different germlings on the same

cell may induce resistance or susceptibility (Carver et al., 1999; Olesen et al., 2003),

we considered only cells that were attacked by a single conidium and only epidermal

cells adjacent to stomata and interstomatal epidermal cells (type A and type B of

epidermal cells following the terminology of Koga et al., 1990).

Evaluation of susceptibility of SusBgt lines SusBgt lines and the parental lines were tested to non-adapted powdery mildew

isolates in order to quantify the degrees of haustorium formation, hypersensitive and

non-hypersensitive mechanism of defense and conidiation. At 72 hai, leaf samples

were cut and placed in a solution of 1 mg ml−1 3,3'-Diaminobenzidine (DAB)-HCL, pH

Chapter 4

66

3.8, which allows whole-cell accumulation of H2O2, for 8 h and subsequently

transferred to a solution of acetic acid-ethanol (3:1 v:v) to be cleared overnight. Then

the leaf segments were stained with 0.1 mg ml-1 Trypan Blue in alcoholic lactophenol

as described by Peterhänsel et al., (1997). H2O2 accumulation appears as a reddish-

brown coloration of epidermal cells and indicates the degree of HR (Thordal-

Christensen et al., 1997).

Results

Genotypic variation in interaction of barley with t he wheat powdery

mildew pathogen The majority of the 439 barley accessions were (nearly) immune, showing no

macroscopic symptoms or signs of infection whatsoever with the non-adapted

pathogen (Bgt) even 10 days after inoculation. However, a few barley accessions

were identified that showed a low degree of susceptibility to Bgt. On these accessions

very small lesions, or even in a few cases tiny colonies, were visible by 10x

magnifying glass or by naked eye, indicating some haustorium formation by the non-

adapted pathogen. Six such accessions were examined under the microscope to

quantify the amount of haustorium formation in comparison with lines Turkey 290,

SusPtrit and SusPmur and with the immune barley cv. Vada. The cellular reactions of

these selected lines to Bgt is shown in Figure 1. On barley cv. Vada, all germlings

were stopped at the penetration stage in association with the deposition of cell wall

appositions (papillae), and the fungus could not develop further to establish any

haustoria. On the other accessions, the large majority of germlings were also stopped

at penetration stage, but 12 to 25 % of the germlings succeeded in penetrating

through the epidermal cell wall and established a haustorium and elongated

secondary hyphae (ESH). Accessions with similar rates of establishment by Bgt, such

as Hsp17 and SusPtrit, could differ greatly in the rate of conidium formation (Figure

1). The results confirm earlier reports (Tosa & Shishiyama, 1984; Trujillo et. al., 2004)

that there are differences among barley accessions in the degree to which they allow

the non-adapted wheat powdery mildew pathogen to establish haustoria. Such rare

barley accessions with some level of susceptibility to this non-adapted mildew were

used as parental lines to accumulate genes for susceptibility to Bgt.


67

0

5

10

15

20

25

30

35

Hiproly

Trisuli B

azar 9

Chame 2

Bimtak

othi 7

HSP14

HSP17

Turkey

290

SusPtri

t

SusPmur

Vada

Inte

ract

ion

site

s[%

]

Figure 1. Penetration efficiency and conidiation of Blumeria graminis f.sp. tritici (Bgt) on different barley

accessions that showed some level of susceptibility to Bgt. The immune barley cv. Vada was used as a reference.

White columns represent the successful primary penetration with a haustorium, collected from two leaf segments

(each from independent inoculation experiments) fixed at 72 hai. In each leaf segment, 100 attempted

penetrations were recorded. Gray columns represent the percentage of all successful primary penetrations that

resulted in formation of conidia at 8 dai. Data represent the means of two leaf segments collected from two

independent inoculation experiments.

Development of SusBgt lines, susceptible to the non -adapted wheat

powdery mildew Based on macroscopic and microscopic observations, including the degree of

conidiation, four accessions of barley, viz. Hiproly (CGN00588; Egypt), Trisuli Bazar 9

(CGN00931; Nepal), Chame 2 (CGN01042; Nepal) and SusPtrit (Atienza et al., 2004)

were selected as the most promising lines for crossing to accumulate genes for

susceptibility to Bgt. We made pairwise crosses between these four lines (Trisuli

Bazar 9 × Hiproly) and (SusPtrit × Chame 2) (Figure 2). The second generation

offspring (F2) derived from each of the two crossing combinations were evaluated first

macroscopically for susceptibility to the Bgt isolate, and then a few F2 plants from

each crossing combination that seemed to be more susceptible than the parents were

tested microscopically for penetration efficiency and conidiation. A double cross (DC)

was made between a selected individual F4 plant derived from the cross Trisuli Bazar

9 × Hiproly and a selected individual F3 plant derived from the cross SusPtrit ×

Chame 2. Four DC-S1 plants coming from the same single DC plant were used for

making doubled haploid (DH) lines by microspore culture. We had expected additional

transgression in the DC (because it had four susceptible ancestors) compared to the

single cross (SC) with only two ancestors. However, during the procedure, the

progeny from the cross SusPtrit × Chame 2 seemed so susceptible that we produced

also a DH from that lineage. Two selected F4 individuals derived from one selected F3

plant from the single cross (SC) SusPtrit × Chame 2 were also used for making DH

Chapter 4

68

lines. Progeny from selfed DH plants were first evaluated macroscopically, and those

lines that showed a clear susceptibility were analyzed microscopically. Since the

selected DC and SC lines looked similarly susceptible, we decided to test them both

extensively. The most Bgt-susceptible lines derived from the single cross and double

cross were named SusBgt SC and SusBgt DC.

Susceptibility of SusBgt lines to the adapted ( Bgh) and non-adapted

wheat powdery mildew pathogen ( Bgt) SusBgt lines and all the parental lines showed a compatible interaction (infection type

4 according to the scale of Mains & Dietz (1930)) with the barley powdery mildew.

Interestingly, SusPtrit and both SusBgt lines arrested at least 50% of the infection

units of the adapted pathogen, Bgh, from developing haustoria. This indicates a rather

high level of basal resistance to Bgh (Figure 3).

SusBgt lines allowed the non-adapted Bgt to form many colonies that were clearly

visible by the naked eye at 8 dai and even earlier (Figure 4A). In the barley

accessions, a proportion (depending on genotype, 12 to 27%) of challenged

epidermal cells without detectable haustoria responded by HR (Figure 5A).

Figure 2. Two cycles of convergent crossing in barley to accumulate genes for susceptibility

to the wheat powdery mildew (Blumeria graminis f.sp. tritici).

Crossing Selfing Selection of the Selfing Selfing Double cross

most susceptible (selection) (selection)

F2 plants

P1 × P2 F1 F2 F3 F4

F4 P1 × P2 × F3 P3 × P4

P3 × P4 F1 F2 F3

DC-S1

Making DH lines

Making DH lines

P1 = SusPtrit Selection o f the most

susceptible DH line

P2 = Chame 2 SusBgt SC

Selection of the most

P3 = Trisuli Bazar 9 susceptible DH line

SusBgt DC

P4 = Hiproly


69

There were great differences between the accessions in the rate of cells in which a

haustorium was formed. Vada was fully resistant to penetration by Bgt. Both SusBgt

SC and SusBgt DC lines showed a higher level of haustorium formation by Bgt than the

parental lines (Figure 5A). In SusBgt SC and SusBgt DC, 51% and 59%, respectively, of

germlings had succeeded in penetration and haustorium formation, while in Chame 2,

the most susceptible parental line, only 35% of germlings had successfully formed a

first haustorium. In SusBgt DC, which showed the highest frequency of successfully

penetrated cells, a larger proportion of penetrated cells with a haustorium (59%) were

stopped by single-cell HR than in SusBgt SC (42%) and the parental lines. The highest

frequency of successfully penetrated epidermal cells without HR was detected in

SusBgt SC (29%).

0%10%20%30%40%50%60%70%80%90%

100%

Hipro

ly

Trisuli

Bazar

9

Chame 2

SusPtri

t

SusBgt

SC

SusBgt

DC

Inte

ract

ion

site

s

Figure 3. Defense reactions of SusBgt lines and their parental lines to the barley powdery mildew (Bgh) at 48 hai.

White columns represent failed primary penetration. Black columns represent the successful primary penetration

with a haustorium and with hypersensitivity (HR). Cross-hatched columns represent the successful primary

penetration with a haustorium but without HR. Data represent the means of four leaf segments collected from two

independent inoculation experiments. In each leaf segment, 100 infection sites were recorded.

Figure 4. Macroscopic phenotypes of interaction of SusBgt lines with Blumeria graminis f.sp. tritici (Bgt) and B.

graminis f.sp. hordei-murini (Bghm). (A) Development of colonies of Bgt on SusBgt SC and SusBgt DC at 8 dai. (B)

Hypersensitive reaction of SusBgt SC to Bghm at 8 dai. Vada is immune to both ff.spp.

Chapter 4

70

The majority of those germlings that had formed a primary haustorium were stopped

at the secondary penetration attempts. In both SusBgt SC and SusBgt DC, a

considerable proportion of successfully penetrated germlings succeeded in

establishment of a secondary haustorium (44 and 42%, respectively, Figure 5D).

Interestingly, the secondary and subsequent haustoria were almost always detected

in the same cell in which the primary haustorium had been established, typically

resulting in two to five haustoria in one epidermal cell (Figure 6E). Subsequent

penetration attempts in epidermal cells located away from the first infected cell usually

failed (Figure 6F). At 8 dai, lines differed in the proportion of established germlings

that had proceeded to the formation of at least some conidiophores. This conidiation

was most conspicuous (34% of those germlings that had formed a haustorium) in

SusBgt SC, and much less so in SusBgt DC (6%) (Figure 5G). In the parental line

Trisuli Bazar 9, a similar proportion of established germlings reached conidiation as in

SusBgt SC (Figure 5G), but the rate of establishment was lower than in SusBgt SC

(Figure 5A). In summary, the results showed that SusBgt lines show an exceptional

susceptibility to the non-adapted Bgt, and the amenability to formation of haustoria is

not necessarily associated with amenability to conidiation. Both lines are valuable to

study the inheritance of non-host resistance in barley to powdery mildews.

The level of susceptibility of SusBgt lines to othe r non-adapted powdery

mildew pathogens To determine whether the SusBgt lines are susceptible to other non-adapted powdery

mildew pathogens, the four parental lines, SusBgt lines and the reference barley cv.

Vada were inoculated with one isolate each of other non-adapted formae speciales

and species of powdery mildew pathogens. SusBgt lines and their parental lines showed relatively high haustorium formation

and conidiation by the powdery mildew fungus of Hordeum murinum, Bghm (Figure

5B, 5E and 7B). At the macroscopic level, SusBgt SC showed a remarkably strong HR

reaction at 8 dai (Figure 4B). All tested accessions allowed at least some haustorium

formation and conidiation by the rye powdery mildew pathogen (Bgs) (Figure 5C and

5F), but the SusBgt lines were not more susceptible to this mildew form than the other

lines were. SusBgt SC and SusPtrit that allowed relatively high establishment by Bgt

(>50 %) allowed considerably less establishment by Bgs (< 26 %). Surprisingly, Vada,

which did not allow any haustorium formation by Bgt, allowed relatively high

haustorium formation by Bgs (31 %). Relatively few germlings of Bgs that had formed

a first haustorium proceeded to form a secondary haustorium (< 22%) and conidia

(<14%) compared to Bgt (< 45 and < 35%, respectively) (compare Figure 5D with


71

Figure 5F, and note the difference in the scale of the ordinates). The tested barley

lines were all nearly immune to the mildew fungi of oat, Bromus mollis and Dactylis

glomerata. At most, 10% of the germlings formed a haustorium, and none of the

germlings formed conidia. Both SusBgt lines allowed some haustorium formation (35-

51%) by the Agropyron powdery mildew (Bgar), but the rate of established germlings

reaching conidiation was considerably higher in SusBgt SC than in SusBgt DC (80%

and 21%, respectively) (data not shown). As with the Bgt, during infections by the

other non-adapted mildew strains, except Bghm (Figure 7A), second and subsequent

haustoria were almost always formed in the same cell as the first haustorium, and

failed penetration attempts along the spreading hyphae were commonly observed.

The two additional non-adapted mildew species, O. neolycopersici and Sphaerotheca

fuliginea, were not observed to form any haustoria on any of the barley accessions.

Discussion Although we have not yet started to perform formal inheritance studies, several

interesting conclusions on the genetic basis of the basal resistance of barley to non-

adapted Bg mildews have emerged.

1-The resistance has a quantitative inheritance, as suggested by the quantitative

differences between accessions and by the transgressive segregation when

combining the parental lines. Our results suggest that crossing and selection among

some barley accessions with some level of susceptibility resulted in the accumulation

of susceptibility factors (or depletion of resistance factors) to the non-adapted wheat

powdery mildew, Bgt. These lines showed an unprecedented level of haustorium

formation and colony development by this non-adapted target mildew. We conclude

that non-host resistance of barley to Bgt is based on several minor genes, each with a

small effect.

2-Genes for basal resistance have pathogen developmental stage-specific effects. If

quantitative trait loci (QTLs) indeed represent operative targets for pathogen-delivered

effectors to silence aspects of the defense (Jafary et al., 2006; Niks & Marcel, 2009),

silencing of the defense at the first penetration stage may require different effector-

target interactions than ESH development or conidiation. Also, it seems that making

the first haustorium (= silencing the defense of the first plant cell) does not imply that

the defenses of the next cells are silenced easily as well. Arabidopsis penetration

mutants (pen) allow higher frequencies of penetration by the non-adapted powdery

mildew pathogen (Bgh), but other genes (Sag101, Pad4) compromise subsequent

stages in the infection process (Lipka et al., 2005). Consonni et al. (2006) reported

Chapter 4

72

Bgt

0%10%20%30%40%50%60%70%80%90%

100%

Hiproly

Tris

uli Baz

ar 9

Cham

e 2

SusPtri

tSusB

gt SC

SusBgt

DC

Vada

Vivant

Inte

ract

ion

site

s

A Bghm

0%10%20%30%40%50%60%70%80%90%

100%

Hiproly

Tris

uli B

azar

9

Chame 2

SusPtri

tSusB

gt SC

SusBgt

DC

Vada

Inte

ract

ion

site

s

B

Bgs

0%10%20%30%40%50%60%70%80%90%

100%

Hipro

ly

Trisuli B

azar

9

Chame 2

SusP

trit

SusB

gt SC

SusBgt

DC

Vada

Rogo

Inte

ract

ion

site

s

C

Bgt

0

10

20

30

40

50

Hipro

ly

Trisuli B

azar 9

Chame 2

SusPtri

t

SusBgt

SC

SusBgt

DC

Inte

ract

ion

site

s [%

]

D

Bghm

05

10152025303540

Hiproly

Tris

uli Baz

ar 9

Chame 2

SusP

trit

SusBgt

SC SusB

gt D

C

Vada

Inte

ract

ion

site

s [%

]

E

Bgs

0

5

10

15

20

25

30

Hiproly

Trisuli B

azar 9

Chame 2

SusPtri

t

SusBgt S

C

SusBgt D

CVad

a

Inte

ract

ion

site

s [%

]

F

Bgt

0

10

20

30

40

Hiproly

Tris

uli Baz

ar 9

Chame 2

SusPtri

t

SusBgt

SC SusB

gt DC

Vada

Inte

ract

ion

site

s [%

]

G

Bghm

0102030405060708090

100

Hipro

ly Tris

uli Baz

ar 9

Chame 2

SusP

trit

SusBgt

SC SusB

gt DC

Vada

Inte

ract

ion

site

s [%

]

H

Bgs

0

5

10

15

20

25

30

Hiproly

Trisuli B

azar 9

Chame 2

SusPtri

t

SusBgt S

C

SusBgt D

CVada

Inte

ract

ion

site

s [%

]

I

Figure 5 . Interaction of SusBgt lines and the parental lines with three non-adapted Bg forms. Three upper figures (A-C): defense reactions at 72 hai. White-dotted

columns (FPP+HR) represent failed primary penetration with HR. White columns (FPP-HR) represent failed primary penetration without HR. Black columns (SPP+HR)

represent the successful primary penetration with a haustorium and with HR. Cross-hatched columns (SPP-HR) represent the successful primary penetration with a

haustorium but without HR. Data represent the means of four leaf segments collected from two independent inoculation experiments. In each leaf segment, 100 infection

sites were recorded. Vivant is a wheat cultivar, and Rogo is a rye cultivar (Each is susceptible to its own mildew forms). Three middle figures (D-F): development of

secondary haustoria in the SPP infection units of A-C. Black-dotted columns represent the percentage of infection units that developed at least one secondary haustorium

at 72 hai. Three lower figures (G-I): development of conidia. The gray columns represent the percentage of established infection units that formed at least one

conidiophore at 8 dai. Data represent the means of four or two leaf segments collected from two independent inoculation experiments.

SPP-HR

SPP+HR

FPP-HR

FPP+HR


73

Figure 6. Interaction of SusBgt lines with non-adapted Bgt mildew fungus. (A) A germling that stopped at the penetration stage in an epidermal cell, which shows an H2O2 reaction in the whole cell, indicating HR. (B) A failed germling that stopped at the penetration stage in association with papilla formation. (C) A successfully penetrating germling that elicited HR. (D) A successfully established germling that formed elongated secondary hyphae (ESH). (E) Multiple haustorium formation (arrows) in an epidermal cell where the primary haustorium was formed. (F) Failed secondary penetrations (arrows) along an ESH. (G) An established colony of Bgt at 8 dai on SusBgt DC that did not result in conidiation. (H) Abundant conidiation by an established colony of Bgt at 8 dai on SusBgt SC. The scale bar represents 100 µm.

Chapter 4

74

Figure 7. Haustorium formation and conidiation of non-adapted Bghm and Bgar mildews on SusBgt

lines at 8 dai. (A) A successfully established germling of Bghm on SusBgt SC that resulted in formation

of a hyphal network and several secondary haustoria (arrows). (B) Abundant conidiation by an

established colony of Bghm at 8 dai on SusBgt SC. (C) An established colony of Bgar at 8 dai on

SusBgt DC that did not result in conidiation. (D) Abundant conidiation by an established colony of Bgar

at 8 dai on SusBgt SC. The scale bar represents 100 µm.

that the Arabidopsis Atmlo2 pen1 double mutant allows more cell entry by

Golovinomyces cichoracearum, but no significant increase in conidiophore

production. For those later stages of development, apparently EDS1, SAG101 and

PAD4 need to be silenced.

3-Genes for basal resistance are mildew forma specialis-specific. SusBgt lines and

SusPtrit are remarkably accessible to Bgt and allow relatively high frequencies of

fungal establishment. Yet, they show a relatively high level of basal resistance to the

adapted powdery mildew pathogen, Bgh. Vada had a high level of resistance to Bgt,

but was not equally resistant to the rye mildew Bgs. The factors in SusBgt DC that

hampered conidiation by Bgt compared to SusBgt SC (Figure 5G) seem to also be

effective against Bgar (Figure 7C and D), but not against Bghm (Figure 5H and 7B) or

Bgs (Figure 5I). Selection for high susceptibility to Bgt has not affected the level of

resistance to Bga and other non-adapted mildews.

Our results in the present study show many parallels with the barley–rust interaction

(Atienza et al., 2004; Jafary et al., 2006; Marcel et al., 2007; Jafary et al., 2008). In


75

both pathosystems, accumulation of genes for susceptibility to non-adapted pathogen

forms and species appears feasible. Non-host resistance to rust species and to non-

adapted formae speciales of powdery mildew seems similar to quantitative, basal host

resistance, and the resistance mechanism in both pathosystems is mainly based on

non-hypersensitive mechanisms.

Powdery mildew pathogens should suppress PAMP-triggered immunity (PTI) to

establish basic compatibility. This suppression of PTI requires compatibility between

the effectors and the operative targets in the plant. Effectors (O’Connell & Panstruga,

2006) and their operative targets in the plant (van der Hoorn & Kamoun, 2008) both

seem to be under strong diversifying selection. For each plant species, a different

bouquet of effectors may be required to suppress PTI (Almeida et al., 2009; Niks &

Marcel, 2009), implying that each mildew form probably has a different set of

effectors, each tuned to the operative targets in their host plant species. Depending

on the fungal development stage, different aspects of defense should be

reprogrammed, again requiring different effectors. By common evolutionary ancestry,

however, some compatible variants of operative target motifs may occur in some non-

host plant accessions. Non-adapted mildews cannot be expected to suppress PTI of

barley effectively, but, in the SusBgt lines, we may have accumulated variants of the

targets that can be suppressed relatively easily by Bgt.

New evidence has emerged on the involvement of some candidate genes in the

basal host and non-host resistance of barley against mildew pathogens (Douchkov et

al., 2005; Miklis et al., 2007). An example of such a candidate gene is HvSNAP34,

which plays a role in basal host and non-host resistance of barley to Bg. Transient-

induced gene silencing (TIGS) of this candidate gene strongly increased the rate of

successful haustorium formation of both adapted (Bgh) and non-adapted powdery

mildew fungi (Bgt) (Douchkov et al., 2005). HvSNAP34 could be one of the operative

targets of effectors to enhance successful establishment of haustoria by invading

mildew fungi. We are now developing mapping populations of SusBgt SC × Vada and

SusBgt DC × Vada to map the QTLs (genes) that determine the level of basal

resistance to the various mildew forms used in this study, similar to what was done for

rusts in Jafary et al. (2006). This will enable us to identify the targets involved in non-

host immunity in barley against mildew pathogens. Finally, the existence of relatively

high levels of susceptibility of SusBgt lines to more than one forma specialis of Bg,

may facilitate the cross-hybridization between different ff. spp. of Bg, as done on the

shared host Triticum urartu for crossing f.sp. tritici with f.sp. agropyri by Tosa (1989).

Such pathogen and plant inheritance studies can contribute considerably to our

Chapter 4

76

understanding of the evolutionary and genetic basis of the host-pathogen interactions

in Gramineous powdery mildew pathosystems. Acknowledgements

Anton Vels is gratefully acknowledged for his excellent technical assistance in greenhouse experiments. We thank Dr. Patrick Schweizer (Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Germany) for providing the wheat isolate of mildew. Bert Essenstam from the Unifarm of Wageningen University is acknowledged for providing the cucumber powdery mildew and multiplication of wheat powdery mildew. We thank Dr. Harold E. Bockelman (National Small Grains Collection, USDA, USA) for providing the seeds of barley cv. Turkey 290, the Centre for Genetic Resources, the Netherlands (CGN) for proving the seeds of 54 accessions of barley, and Dr. Rajeev K. Varshney from IPK, Germany, for providing the seeds of 227 accessions of barley (ICARDA collection) used in this study. ICARDA (Syria) is acknowledged for its indirect contribution by preparing the seeds. We thank Dr. Viktor Korzun and Clemens Springmann (PLANTA Angewandte Pflanzengenetik und Biotechnologie, Germany) for making the DHs. We thank Prof. Richard G.F. Visser for critically reading the manuscript and for his continuous support.

References

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73

Rust)induced powdery mildew resistance

79

Rust-induced powdery mildew resistance in barley

R. Aghnoum, R.G.F. Visser, P. Schweizer and R.E. Niks

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Abstract Different pathogens employ different strategies to suppress their host defense

mechanisms. We observed that a compatible interaction of barley-Puccinia hordei

induced increased papilla-based resistance to a challenge infection by a compatible

powdery mildew isolate despite the different strategies of suppression of defense by

the two organisms. The level of rust-induced mildew resistance varies among barley

accessions and is not determined by the virulent/avirulent spectra of the challenger

isolate. Our histological data suggest that the inducer effect is due to rust-plant

communication during stoma penetration by the rust fungus, or to the presence of the

rust hyphae in the apoplast, or to some degree of pre-haustorial resistance mounted

by the mesophyll cells to the invading rust fungus. Macroarray gene expression

analysis showed that several genes related to metabolism and photosynthesis were

highly down regulated and a few genes involved in plant defense including a

pathogenesis related protein (PR-1), and Cysteine synthase were highly up regulated

in the double inoculated treatment. Both our histological and gene expression

analysis suggest that the rust “primes” the basal mildew resistance genes prior to the

challenge mildew infection.

Introduction Infection by a pathogen or parasite may condition plants to activate or suppress

defense reactions against subsequent infection attempts by other pathogen strains or

species. Induction of plant defense systems as a result of prior exposure to

pathogens (Ryals et al., 1996), non-pathogenic root-colonizing bacteria (van Loon et

al., 1998), feeding by herbivores (Kessler & Baldwin, 2002) or treatment with

chemicals (Oostendorp et al., 2001) is termed induced resistance. Three types of

induced resistance have been described, viz. systemic acquired resistance (SAR)

(see Glossary) (Durrant & Dong, 2004), induced systemic resistance (ISR) (see

Glossary) (van Loon et al., 1998) and induced localized resistance (Kunoh, 2002).

Suppression of plant defense systems against a non-adapted or incompatible race of

a pathogen as a result of prior exposure to a compatible pathogen is called induced

susceptibility.

Induced localized resistance and induced susceptibility have been reported in many

plant-pathogen combinations (Table 1). In cereals induction of localized susceptibility

or resistance has been reported as a result of a prior attack by both necrotrophic and

biotrophic fungi (e.g. Jørgensen et al., 1998; Kunoh et al., 1988; Lyngkjær & Carver,

1999; Niks, 1989). In cereals, most research on localized induction of resistance and

susceptibility has concentrated on interaction of compatible and incompatible isolates


81

of the powdery mildew pathogen, Blumeria graminis (hereafter Bg). Failed

establishment (see Glossary) by an adapted or non-adapted powdery mildew

pathogen induces penetration resistance to subsequent challenge inoculation with a

compatible isolate (e.g. Hwang & Heitefuss, 1982; Cho & Smedegaard-Petersen,

1986; Carver et al., 1999; Lyngkjær & Carver, 1999; Kunoh, 2002). Conversely,

successful penetration and haustorium formation by an adapted, virulent isolate of Bg

induces susceptibility at cell level to haustorium formation by subsequent challenge

with adapted virulent or avirulent isolates and also non-adapted formae speciales and

species (e.g. Tsuchiya & Hirata, 1973; Carver et al., 1999; Lyngkjær et al., 2001;

Olesen et al., 2003). “Susceptibility” at cell level is often called accessibility, because

the avirulent or non-adapted challenger pathogen typically will not complete its life

cycle, due to defense mechanisms that back-up the penetration resistance. For

similar reasons “resistance” at cell level is often called inaccessibility. Most of the

reports indicate that this induction of accessibillity and inaccessibility is localized and

limited to the challenged cell and its adjacent cells.

Induced resistance to rust pathogens as a result of prior inoculation with the same

or different rust pathogens has been reported by several workers (e.g. Kochman &

Brown, 1975; Sebesta et al., 1996; Calonnec, et al. 1996; Pei et al. 2003). In the

barley leaf rust (Puccinia hordei) system, failed haustorium formation by an non-

adapted rust fungus, however, does not induce reduced haustorium formation by a

compatible challenger rust (Niks, 1989) but a compatible interaction suppresses the

defense system of the same cell at challenge with a non-adapted rust species.

Powdery mildew and rust fungi both have biotrophic lifestyles and require living

plant tissues to survive and reproduce. Successful establishment of specialized

feeding structures of the pathogen, the so-called haustoria, is a prerequisite for a

compatible interaction. In barley, non-host resistance against non-adapted powdery

mildews, the quantitatively inherited basal resistance and mlo-mediated resistance to

the adapted powdery mildew all act on the basis of papilla formation at the point of

attempted ingress into the cell, which results in impaired haustorium formation. Papilla

formation is involved in induced inaccessibility in the barley-powdery mildew (Sahashi

& Shishiyama, 1986). A similar papilla formation is involved in basal and non-host

resistance against rust fungi (Niks, 1986, 1989).

Mellersh & Heath (2001) reported evidence that mildews and rust fungi follow

different strategies to successfully suppress plant defense, and hence induce

accessibility of plant cells. Rust fungi should decrease adhesion between the cell wall

and the plasma membrane at the cell wall penetration point, but for successful

infection by mildew no such decreased adhesion is required. We set out to determine

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whether a compatible rust interaction would induce increased accessibility to mildew,

despite the different strategies of suppression of defense by the two organisms. We

found, however, in exploring experiments, that compatible infections by Puccinia

hordei induced increased papilla based resistance rather than decreased papilla

based resistance to a challenge infection by a compatible powdery mildew isolate. In

this paper we characterize this rust-induced mildew resistance at the cellular level.

We also identified differentially expressed genes that can explain the strong papilla-

based penetration resistance in double inoculated barley plants.


Plant material Three barley near isogonics lines (Pallas, Pallas-Mla3, Pallas-MlLa), the mildew

susceptible barley cv. ‘Manchuria’, two barley lines that possess high levels of basal

resistance to leaf rust (Vada and 17-5-16), and three near-isogenic barley lines with

different genotypes of MlLa and Rphq2, the latter being a QTL for basal resistance to

leaf rust (Marcel et al., 2007) (Vada rphq2 mlLa, Vada rphq2 MlLa, and Vada Rphq2

mlLa), were used to study the effect of pre-inoculation with leaf rust on powdery

mildew development. Three seeds of each genotype were sown in trays of 37×39 cm

in two rows on the right and left side of each tray. Plants were grown in a greenhouse

compartment where the temperature was set at 18-20°C with 16 hours of light and a

relative humidity of about 70%.

Pathogen material The isolate 1.2.1 of Puccinia hordei was used as the ‘inducer’. This isolate is able to

establish a compatible interaction with all tested barley lines. Two isolates of Blumeria

graminis f.sp. hordei, isolate C15 and Wag.04, were used as ‘challenger’. Isolate C15

is avirulent to Pallas-MlLa but virulent to Pallas-Mla3. Wag.04 isolate is the reverse,

virulent to Pallas-MlLa but avirulent to Pallas-Mla3. Leaf rust isolate 1.2.1 was

maintained on line L94, and the powdery mildew isolates on Manchuria. An isolate of

Puccinia recondita, the rye leaf rust pathogen, freshly propagated on a susceptible

rye genotype and an isolate of Puccinia antirrhini, the snapdragon rust, collected from

naturally infected common snapdragon (Antirrhinum majus) in Wageningen were

used in this study.

Inducer inoculation Ten days after sowing, the fully expanded first seedling-leaves were positioned

horizontally, adaxial side up. In the preliminary experiments, prior to the inoculation


83

with rust, the middle part of each leaf was covered with a 3 cm strip of aluminum foil

to keep that middle section free of rust infection, in order to determine whether the

effect of inducer inoculation on the challenger is localized or systemic. In each tray

only the plants of one row were inoculated by the inducer and the plants of the other

row were covered to keep them free of rust. For each box, 6 mg of fresh

urediospores of P. hordei isolate 1.2.1 were diluted ten times with Lycopodium

powder and dusted over the plants in a settling tower. This inoculum dose results in

about 350 urediospores per cm2. Inoculated plants were incubated overnight in a

dark chamber with 100% humidity and then transferred to a greenhouse

compartment with the above mentioned conditions.

Challenger inoculation Five days after the inoculation with the inducer, plants were subjected to inoculation

by the challenger mildew. A settling tower was used for inoculation. Freshly produced

conidia of powdery mildew were blown into the tower over the horizontally fixed

adaxial sides of the first leaves. The density of inoculum was adjusted to 100-150

conidia per cm2. The inoculated plants were incubated in a growth chamber at 18-20 oC with fluorescent light irradiance (100 micromol/m2/s) supplied for 16 h per day.

Histological assessment of challenger infection Forty eight hour after inoculation (hai) of the challenger, a leaf segment of about 3 cm

long was collected from the basal side of the rust-mildew inoculated portion and the

whole rust-free middle portion. From the control plants a leaf segment of about 3 cm

long was collected from the middle part of the leaves. From each genotype, two

seedlings were sampled and one seedling was kept to monitor the efficiency of

inoculation and infection type of the powdery mildew at macroscopic level. Infection

types (IT) of powdery mildew were scored based on the 0–4 scale proposed by

Mains & Dietz (1930) on those plants that were only inoculated with the challenger.

The leaf segments were submerged in a solution of acetic acid-ethanol (3/1 v/v) at

least for 3 hours to be cleared. They were stained by boiling for 8 min in a solution of

alcoholic lactophenol (96% ethanol-lactophenol 1/1 v/v) containing 0.1 mg ml-1

Trypan Blue and then cleared in a Chloral Hydrate solution (2.5 mg ml-1) overnight

(Peterhänsel et al., 1997). Those infection units that were stopped at penetration

stage in association with papilla deposition were considered failed primary

penetration (FPP) and infection units that succeeded to penetrate the epidermal cells

and established a haustorium and an elongated secondary hypha (ESH) were


122

1 Induced resistance (IR) and induced susceptibility (IS) have been used for the effect of inducer on challenger development on entire leaves, whole plants or at cell level 2 ISR, Induced systemic resistance; 3nhR, non-host resistance; 4Bga, Bgh and Bgt stand for Blumeria graminis f.sp. avenea, hordei and tritici respectively

Table 1: A summary of published literature on pathogen-induced susceptibility and resistance

Crop Inducer Challenger Plant-inducer interaction

Plant-challenger interaction

Outcome of interaction 1 Reference(s)

Bean Uromyces phaseoli Uromyces phaseoli S S IR Yarwood 1954 Melon Sphaerotheca fuliginea Bgh4, Bgt4 S nhR3 IS Oku & Ouchi 1976

Rice Pyricularia oryzae Bipolaris sorokiniana

Pyricularia oryzae Pyricularia oryzae

R nhR

S S

IR IR

Manandhar et al., 1998

Oat Puccinia triticina P. graminis tritici

P. coronata avenae P. graminis avenae

nhR nhR

S S

IR IR

Kochman & Brown 1975

Oat Oat

Bga4 Bga Bga

Bga Bga Bgh,Bgt

S R S

S S nhR

IS IR IS

Carver et al., 1999; Lyngkjær & Carver 2000 Olesen et al., 2003

Wheat Puccinia striiformis P. striiformis R S IR

Calonnec et al., 1996

Wheat Septoria tritici Septoria tritici R S IR Halperin et al., 1996

Wheat Bgt Bgh

Bgh,Bga Bgt

S R

nhR S

IS IR

Olesen et al., 2003 Schweizer et al., 1989

Barley F. oxysporum f.sp. radicis-lycopersici Bgh nhR S ISR2 Nelson 2005 Barley Cladosporium macrocarpum Bgh Saprophyte S IR Gregersen & Smedegaard, 1989

Barley Puccinia hordei P. recondita f.sp. recondita(Prr)

Prr P. hordei

S nhR

nhR S

IS No IR

Niks, 1989

Barley Bipolaris maydis Septoria nodorum

Drechslera teres Drechslera teres

nhR nhR

S S

IR IR

Jørgensen et al., 1998

Barley Bgh Sph. fuliginea S nhR IS Oku & Ouchi, 1976 Barley Bgh Erysiphe pisi S nhR IS Kunoh et al., 1988; Kunoh, 2002 Barley Bgh Various PM fungi S nhR IS Tsuchiya & Hirata, 1973 Barley Bgh Bgt,Bga S nhR IS Ouchi et al., 1976a; Olesen et al., 2003 Barley Erysiphe pisi Bgh nhR S IR Kunoh et al., 1988; Kunoh, 2002

Barley Bgt Bgh nhR S IR Ouchi et al., 1976a; Ouchi et al., 1976b; Gregersen and Smedegaard 1989

Barley Bgh Bgh R S IR Cho and Smedegaard-Petersen, 1986; Lyngkjær &

Carver, 2000; Lyngkjær et al., 2001

Barley

Bgh Bgh S S /R IS Chin et al., 1984; Lyngkjær et al., 2001; Lyngkjær &

Carver, 2000

84


85

considered as successful primary penetration (SPP) (Asher & Thomas, 1983).

Because different types of epidermal cells show different reactions to powdery

mildew infection (Lin & Edwards, 1974) only stomatal and interstomatal cells (type A

and type B epidermal cells following the terminology of Koga et al., 1990) were

examined, and long epidermal cells over-lying vascular tissues (type C) were not

considered. In each leaf segment 150 powdery mildew germlings were scored. Only

those epidermal cells attacked by a single mildew fungal germling were considered.

Preparations were examined at a magnification of 400× using a Zeiss Axiophot

microscope, using transmitted white light.

Transcriptome analysis of rust-induced mildew resis tance In order to study defense-related gene expression in barley that was double

inoculated with rust and powdery mildew, a macroarray expression analysis was

carried out. Barley line 17-5-16 (Parlevliet & Kuiper, 1985) which showed in our

histological experiments a high level of rust-induced penetration resistance to

powdery mildew was selected for this gene expression study. Treatments were as

follows: treatment 1, inducer (P. hordei, isolate 1.2.1.) + challenger (Bgh, isolate

C15); treatment 2, only inducer; treatment 3, only challenger; treatment 4, mock, no

inoculation, only Lycopodium powder applied as for the other treatments. About 60

plants were grown in each box (37 × 39 cm). For each type of treatment one box was

used. Ten days after sowing, first formed leaves were fixed in a horizontal position,

with the abaxial side up. The inoculation procedure, the density of inducer and

challenger and the incubation conditions were as in the previous experiments.

Twelve hai, 15 leaves were sampled per treatment. Leaf samples were shock-frozen

in liquid nitrogen immediately and stored at –80 oC. The experiment was carried out

in three independent inoculation experiments in a randomized block design.

The barley PGRC2 cDNA array We used the barley PGRC2 cDNA macroarray, which is an extended version of the

PGRC1 array described by Schweizer (2008). These cDNA arrays have been

developed at IPK (Leibniz Institute of Plant Genetics and Crop Plant Research,

Gatersleben, Germany) and contain approximately 13,000 and 10,000 unigenes

respectively, spotted on two membranes.

RNA isolation Total RNA was extracted from whole-leaf material sampled at 12 hai. About 10 leaf

samples were ground in liquid nitrogen and about 100 mg of the homogenized tissue

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samples was used for RNA extraction with TRIZOL® Reagent according to the

manufacturer’s protocol with some modification of the procedure, viz. the aqueous

phase was extracted twice with chloroform.

Synthesis of 33P-labelled cDNA and hybridization procedures mRNA was extracted from 37.5 µg of the total RNA using Dynal magnetic beads

according to the manufacturer’s protocol. 33P-labelled second-strand cDNA probes

were prepared as described by Sreenivasulu et al. (2002). cDNA arrays were first

pre-hybridized and then hybridized with cDNA probes as described by Sreenivasulu

et al. (2002). The hybridization period was prolonged to 18 h. as suggested by

Zierold et al. (2005).

Spot detection and data normalization The hybridized membranes were scanned by the phosphorimager (Fuji BAS3000

reader). Spots were detected and spot intensities were quantified by using the

ArrayVision 8.0 software package (Imaging Research Inc., St. Catharines, Ontario,

Canada). Signal intensities were normalized after background subtraction as

described by Sreenivasulu et al. (2002). Only unigenes with a signal/background

ratio of more than 2.0-fold in at least two hybridizations were considered.

Differentially regulated genes were identified by calculating the Q value (false

discovery rate) by using the EDGE (Extraction of Differential Gene Expression)

software (Leek et al. 2006). Transcripts that showed a Q-value of less than 0.2

(FDR<20%) and a p-value of less than 0.05 between sample pairs were selected for

further analysis.

Results

Effect of prior infection with leaf rust on powdery mildew development Induction of localized inaccessibility to powdery m ildew by inducer

The leaf rust fungus (inducer) was able to establish a compatible interaction with all

tested barley genotypes. At five days after inoculation (dai) with the inducer, when

the plants were subjected to the attack by challenger, the leaf rust had formed pale

green flecks and at 8 dai mature rust pustules had formed.

On the control plants that were only inoculated with challenger mildew, depending

on the barley genotype, 23 to 41% of the infection units were stopped at penetration

stage in association with papilla formation. When the challenger isolate was avirulent

to the accession that carry corresponding R gene, the successfully penetrated host

cells died in response to the attack. The inducer inoculation caused a significant


87

reduction in successful haustorium formation by the challenger mildew in all tested

barley accessions, but the level of induced resistance differed between the

accessions (Figure 2). The strongest effect of induced resistance was observed in

the interaction of Pallas-Mla3 with the Wag.04 mildew isolate. On Pallas-Mla3 plants

pre-conditioned by the inducer, only 9% of germlings succeeded to penetrate and

form a haustorium, versus 58 % on the control plants. In the very mildew susceptible

accession Manchuria, the lowest effect of induced resistance was observed. Data

obtained from the middle parts of the leaves that were covered during inducer

inoculation, indicated that this induction of penetration resistance hardly extends to

tissue away from the rust infections, and therefore is hardly or not systemic (Figures

1 and 2A).

Figure 1. Rust-induced mildew resistance phenotype on barley line Pallas inoculated with leaf rust and powdery

mildew (wag.04 isolate). Colonies of powdery mildew are visible on the right part of the leaf that only was

inoculated with powdery mildew; on the left side, that was inoculated with leaf rust 5 days before mildew

inoculation, hardly any colony is visible.

Induced inaccessibly is not determined by the virul ent/avirulent spectra

of the challenger isolate Because of the different levels of induced resistance on the three Pallas near-

isogenic lines (Figure 2A), an obvious question to be answered was whether the

virulence/avirulence of the challenger isolate (powdery mildew) to the Pallas near-

isogenic lines that carrying the corresponding R gene, can influence the level of

induced resistance. Similar results were obtained when we used another powdery

mildew isolate (C15) that reacted differentially with the two near-isogenic lines

(Pallas-Mla3 and Pallas-MlLa) in comparison with Wag.04 isolate (Figure 2B). This

indicates that induced resistance is not determined by the virulent/avirulent spectra of

the challenger isolate.

We tested Vada recombinants with different genotypes regarding Rphq2 and MlLa.

Rphq2 is an allele for higher basal resistance to P. hordei, rphq2 for lower basal

resistance to this rust fungus. MlLa is an allele for race-specific incomplete

Chapter 5

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hypersensitive resistance to powdery mildew, mlLa is the susceptibility allele. The

Rphq2 and MlLa loci are linked, and recombinants between these loci have been

developed in Vada background (Marcel et al., 2007). The four combinantions of

alleles on these two loci showed similar and rather low levels of induced resistance

(Figure 3). This is preliminary evidence that the level of basal resistance to P. hordei

does not determine the level of induced resistance to powdery mildew, and that

mildew R-gene MlLa is not a determining factor either. We also tested barley

experimental line 17-5-16, a barley line that possesses a very high level of basal

resistance to leaf rust (Parlevliet & Kuiper, 1985). In this line the inducer caused a

high level of penetration resistance to the mildew challenger (Figure 3).

0

10

20

30

40

50

60

70

80

90

100

Pallas Pallas-Mla3 Pallas-MlLa Manchuria

Pen

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[%]

aa

b

b

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a

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aa b

a a

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Pallas Pallas-Mla3 Pallas-MlLa Manchuria

Pen

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[%]

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a

a

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a

b

a

b

B

Figure 2. Penetration efficiency of powdery mildew germlings on different barley lines at 48 hai. A, Wag.04 isolate, avirulent to Pallas-Mla3 but virulent to Pallas-MlLa. B, C15 isolate, avirulent to Pallas-MlLa but virulent to Pallas-Mla3. Gray-dotted columns represent the penetration efficiency of powdery mildew when barley leaves were inoculated with the inducer (Puccinia hordei isolate 1.2.1) five days before inoculation with the challenger. White columns represent penetration efficiency in a 3 cm strip of leaves that was covered during inoculation with the inducer and were inoculated only with the challenger. Gray columns represent penetration efficiency on control plants that were inoculated only by powdery mildew at the same time as inoculation of other treatments with the challenger. In Figure 2A data of each column are based on an average of 600 mildew infection units collected from 4 leaf segments (two leaves in two experiments per accession/treatment combination). In Figure 2B data of each column are based on an average of 300 mildew infection units collected from 2 leaf segments each from an independent inoculation experiment. Means followed by the same letter within a genotype are not significantly different (p= 0.05).


89

Stoma penetration and intracellular hyphae may cont ribute to induced

resistance We determined which aspect of rust infection may contribute to induced resistance.

We applied a non-adapted rust, viz. the snapdragon rust fungus P. antirrhini. We

checked the barley leaves inoculated with this rust at microscopic level. This rust

produces only germ tubes on the barley leaf surface, and hardly or not penetrates

through stomata. Although the snapdragon rust was applied at about two times

higher inoculum dose than the leaf rust, we observed that it did not induce resistance

against powdery mildew (Figure 4). This indicates that induced resistance is not due

to communication between the rust germ tube and the barley epidermal cells. Next

we applied the non-adapted rust P. recondita as inducer. P. recondita is able to find

the stomata and penetrate through them, but hardly forms any haustorium in barley

(Niks, 1989). We found that application of P. recondita reduced the haustorium

formation by the mildew fungus from 47% in the control to 25% (Figure 4). There is a

tendency that haustorium formation is not required for the induction of resistance to

the mildew fungus, more replications are required to test whether this difference is

significant. Application of the inducer inoculum P. hordei and challenger mildew on

opposite leaf sides resulted in a similar level of induced penetration resistance to

subsequent attack by powdery mildew as when both inoculations were applied to the

adaxial leaf surface (Figure 4). This induction was not observed when P. recondita

was applied on the opposite side of the leaf. This suggests that the rust intracellular

hyphae and stoma penetration both may contribute to induced resistance.

Transcript profiling Out of approximately 13,000 unigenes spotted on the array, 6,053 unigenes showed

hybridization signals at least 2.0 fold above the local background and they were

selected for further statistical analysis. Only those unigenes that showed a Q-value

less than 0.2 between sample pairs and P value < 0.05 were considered differentially

regulated genes. In total, 285 gene/treatment combinations showed differential

expression compared with the mock inoculation (Figure 5). A considerable set (67%)

of differentially regulated genes in LR+PM was also differentially regulated in LR.

Higher numbers of genes were found to be differentially regulated in LR+PM

treatment (128) than in single inoculation treatments. On the other hand, direct

comparison of gene expression between LR (Leaf Rust) and LR+PM (Leaf Rust +

Powdery mildew) inoculations did not reveal any transcripts that were significantly

Chapter 5

90

Figure 3. Penetration efficiency of powdery mildew germlings (C15 isolate) on different near isogenic barley

lines on the background of Vada and a line with high level of basal resistant to leaf rust (17-5-16). Gray-

dotted columns represent the penetration efficiency of powdery mildew when barley leaves were inoculated

with the inducer (Puccinia hordei isolate 1.2.1) five days before inoculation with the challenger. Gray

columns represent penetration efficiency on control plants that were inoculated only by powdery mildew at

the same time as inoculation of other treatments with the challenger. Data of each column are based on an

average of 300 infection units collected from 2 leaf segments (one leaf in two independent inoculation

experiments per treatment). C15 isolate is avirulent on genotypes carrying the MlLa allele.

Figure 4. Penetration efficiency of powdery mildew germlings (Wag.04 isolate) on barley cv. Pallas

inoculated with different inducer rusts. The inducers were inoculated five days before inoculation with the

challenger. Data of each column are based on an average of 600 infection units collected from 4 leaf

segments (two leaves in two experiments per treatment). * The mean difference is significant at 0.05 level

(one sided test).

Basal resistance

to P. hordei high low low high very high

Incomplete HR

to Bgh yes no yes no no

0

10

20

30

40

50

60

70

(Rphq2 MlLa) (rphq2 mlLa) (rphq2 MlLa) (Rphq2 mlLa) 17-5-16

Pen

etra

tion

eff

icie

ncy

[%]

0

10

20

30

40

50

60

Only mildew P. hordei P. hordeidownside

P. recondita P. recondita downside

Snapdragonrust

Pen

etra

tion

eff

icie

ncy

[%] *

*


91

different in their abundance, indicating the existence of quantitative rather than

qualitative differences between induced but not challenged and induced-challenged

leaves. Such differential expression patterns may have escaped detection due to the

limited number of samples used for the analysis. To assess the quantitative

difference in normalized signal intensities (Int) of differentially regulated genes

between double inoculation treatment (LR+PM) and single mildew inoculation (PM) in

a more robust manner, a differential index (DI; Zierold et al., 2005) was calculated for

each gene as follows:

DI = (IntLR+PM − IntPM)/ (IntLR+PM + IntPM)

DI > 0 indicates genes that on average show higher transcript abundance in LR+PM

compared to PM. DI < 0 indicates genes that on average show higher transcript

abundance in PM than in LR+PM. The genes with the greatest difference in

expression level between LR+PM and PM showing a DI > 0.33 or DI < -0.33, which

corresponds to a two-fold signal difference on average, are listed in Table 2. The

results indicate that there are great differences in transcript abundance among the

genes with different functions. The majority of the differentially regulated genes found

to be involved in metabolism and photosynthesis and a few genes in disease

resistance (Table 2 and Figure 6). Up-regulation of a pathogenesis related protein

(PR-1), a marker for Systemic Acquired Resistance (SAR) and Cysteine synthase

which is involved in balancing the production of ROS (reactive oxygen species) was

observed in double inoculated treatment and in single rust treatment (Table 2, Figure

6).

Figure 5. Venn diagram representation of overlapping and non-overlapping sets of differentially

regulated genes (versus mock) between different treatments in barley line 17-5-16.

14 23

30

1

55 3

40

Powdery mildew (48) Leaf rust (109)

Leaf rust + powdery mildew (128)

Chapter 5

92

Table 2. Differentially regulated genes in double inoculated ( leaf rust + powdery mildew)

barley plants versus powdery mildew inoculated plants Clone ID Blast X description 1 Regulation DI 2

HD01N05 Pathogenesis-related protein up 0.97

HO16H10 PR-1, a pathogenesis related protein up 0.97

HO06C23 Protease inhibitor up 0.93

HW03J02 Hypothetical protein up 0.66

HO08E02 Cysteine synthase up 0.66

HO05I23 Ribulose-1,5-bisphosphate carboxylase down -0.33

HO03F06 (TPR)-containing protein down -0.34

HO05F14 Ribulose-1,5-bisphosphate carboxylase down -0.34

HB27P08 Phosphoethanolamine methyltransferase down -0.34

HO28H23 Fructose-bisphosphate aldolase down -0.34

HE01K15 Hypothetical protein down -0.36

GCW002K24 Unknown down -0.37

HO19N07 Chlorophyll a/b binding protein down -0.37

HO04E03 Ribulose-1,5-bisphosphate carboxylase down -0.38

HO02G01 Ribulose-1,5-bisphosphate carboxylase down -0.40

HZ48I22 Photosystem I antenna protein down -0.41

HM11H23 Photosystem I antenna protein down -0.42

HO01J24 Ribulose-1,5-bisphosphate carboxylase down -0.45

HB21N18 Chlorophyll a/b binding protein down -0.48

HO01D09 Chlorophyll a-b binding protein down -0.49

HE01E12 Chlorophyll a/b binding protein 2 down -0.50

HZ53A20 Metallothioneine type2 down -0.50

HO36J03 Carbonic anhydrase down -0.51

HA06N06 Fructose-bisphosphate aldolase down -0.51

HO38I13 Carbonic anhydrase down -0.52

HO02J18 Putative GTP-binding protein down -0.53

HG01D09 Carbonic anhydrase down -0.53

HA12L17 Phosphoglycerate kinase down -0.56

HV04H14 Putative thiamine biosynthesis protein down -0.57

HO28I13 Carbonic anhydrase down -0.57

HDP10O13 Photosystem II 10 kDa polypeptide down -0.76

HG01P09 Unknown down -0.80

HV03C20 Metallothionein-like protein type 3 down -0.80

HD08N06 Metallothionein-like protein down -0.81 1Description of the most significant Blast X hit (the lowest E value). 2Differential index of transcript abundance between double inoculated and single mildew inoculated plants. Only genes that showed at least 2-fold difference in transcript abundance were presented.


93

Discussion Our histological data indicated that pre-inoculation of barley with leaf rust increases

the efficiency of the barley defense system to arrest the establishment of the

challenger mildew fungus. This induction of resistance is localized rather than

systemic. The lack of induction by the germinating snapdragon rust suggests that the

effect is not caused by the rust germ tubes growing over the epidermis. The inducing

effect by the rye leaf rust in barley suggests that rust haustoria are not essential

either. We presume that the inducer effect is due to rust-plant communication during

stoma penetration by the rust fungus, or to the presence of the rust hyphae in the

apoplast, or to some degree of pre-haustorial resistance mounted by the mesophyll

cells to the invading rust fungus.

The level of induced mildew resistance varies among barley accessions. Although

great differences in level were found among Pallas near-isogenic lines (Figure 2),

suggesting a role of the Ml genes in these lines, the virulence or avirulence of the

challenging mildew apparently did not matter (Figure 2A versus 2B). The high level of

induced resistance observed in Pallas-Mla3 and Pallas-MlLa compared to Pallas may

be due to some other genetic difference between these NILs either linked to the Ml

gene, or elsewhere on the genome. Also we did not find sufficient evidence that level

of basal resistance to P. hordei determines the level of induced resistance. It is

intriguing that the experimental line 17-5-16 that shows a high level of basal

resistance to P. hordei, shows also a high level of induced resistance. However, this

may be a co-incidental association, since the two Vada NILs with a decreased level

of basal resistance (rphq2), did not induce a convincingly lower level of induced

resistance than the two lines carrying the alleles Rphq2 for higher level of basal

resistance (Figure 3). The control treatment inoculated only with the PM suggests that the lower the basal

resistance to powdery mildew (Manchuria, Figure 2), the lower the induction of

penetration resistance by the rust. This possible association should be verified in

larger sets of barley accessions, for which we might use the material characterized in

Chapter 2 and 3, showing a wide range of levels of such basal resistance. If indeed

the level of rust induced resistance depends on the level of basal resistance to

mildew, an interesting hypothesis would be that the rust “primes” the basal mildew

resistance genes prior to the challenge mildew infection. Such a “priming” of defense

genes (ISR) has been reported to occur in plants that were colonized by

Pseudomonas fluorescence (Verhagen et al., 2004).

Chapter 5

94

0

1

2

3

4

PR-1

Pathogen

esis-

relat

ed p.

Protea

se in

h ibitor

Cystein

e synth

ase

Hypo th

etical

p rote

in

Log

sign

al in

tens

ity

Mildew LR LR+PM

A

0

1

2

3

4

Pho t. II 1

0 kDa

Carbon

i c anhydra

se

Phosp

hoglyc

erate

kinas

e

Chloro

phyll a/b

b. p

.

Ribulose-1

,5-b. c

.

Log

sign

al in

tens

ity

B

Figure 6 Examples of highly up-regulated and down-regulated host genes in PM, LR and LR+PM treatments. A,

five genes with the high DI and B, five genes with the low DI. Data represent mean of signal intensities of three

inoculation experiments.

Macroarray transcriptome analysis To get insight into the molecular basis of the rust-induced mildew resistance

phenomenon, we compared the global gene expression profile of double inoculated

barley plants with that of single inoculated ones.

We found a considerable level of overlap in differentially regulated genes in double

inoculated plants with that of single powdery mildew or rust inoculated ones (25%

and 67% respectively) although these two pathogens are adapted to live in different


95

host tissues. P. Schweizer (IPK, Germany, unpublished data) found a considerable

overlap in transcriptional response between entire leaf samples challenged with

powdery mildew or rust, suggesting that rust and powdery mildew affect the barley

transcriptome in a similar way. However, since powdery mildew interacts exclusively

with the epidermis of the host, a considerable proportion of powdery mildew induced

transcripts that are epidermis-specific may escape from detection in whole-leaf

samples due to dilution of the signal by the extract of the mesophyll (Zierold et al.,

2005). Accordingly, this experiment was initially set up to compare gene expression

using peeled epidermis. However, this turned out to be technically not feasible

because rust pustules damaged the epidermis and thereby, prevented efficient

peeling by 6 dai. Therefore only whole leaf samples were collected.

By using DI as criterion for differential gene regulation we identified 46 genes

belonging to different functional categories, predominantly activated in the double

inoculated plants versus single powdery mildew inoculated plants. In double

inoculated and also single rust inoculated plants, high up-regulation of PR-1 protein,

a marker for induced resistance was observed. Pathogenesis-related proteins (PRs)

are usually defined as host-specific proteins that are induced in several plant species

in response to attack by pathogens or related situations and have antimicrobial

activity (van Loon et al., 2006). Transient silencing of PR-1 expression decreased the

penetration resistance to the powdery mildew fungus in barley, indicating a role for

PR-1 in basal resistance to mildew (Schultheiss et al., 2003). We observed high up-

regulation of Cysteine synthase (Table 2) in the double inoculated treatment but not

in single rust or powdery mildew inoculated treatments. Fofana et al., (2007) reported

that in interaction of wheat with the wheat leaf rust pathogen (Puccinia triticina) the

expression of Cysteine synthase is up-regulated in incompatible (Lr gene) but down-

regulated in compatible interactions. Cysteine synthase is involved in reducing the

concentration of reactive oxygen species (ROS), signaling molecules that are

involved in various processes including pathogen defense (Apel & Hirt, 2004).

However, to confirm that PRs and ROS are indeed involved in rust-induced mildew

resistance, further investigations are necessary.

Biotrophic fungal pathogens including rust and powdery mildews were reported to

decrease the rate of photosynthesis in their host plants (e.g. Scholes & Farrar, 1986;

Scholes et al., 1994; Scholes & Rolfe, 1996). Our results showed that many genes

that were strongly down-regulated in double inoculated plants are involved in

photosynthesis (Table 2). Similar down-regulation of genes involved in

photosynthesis reported in wheat attacked by leaf rust pathogen including the 1,5-

Ribulose bisphosphate carboxylase which is the main source of energy production

Chapter 5

96

for the plant cell, generating ATP and reductive potential (NADPH) through

photosynthesis (Fofana et al., 2007). The role of this high down-regulation of genes

involved in photosynthesis in rust-mildew induced resistance is unknown, but it is

tempting to speculate that the strong suppression of photosynthesis in the mesophyll

might prevent proper powdery mildew development in the epidermis due to the lack

of energy.

This study presents the detection and histological and molecular characterization of

rust induced mildew resistance. Important questions that need to be addressed in

subsequent studies are the genes that determine the level of induced resistance, and

the mechanisms by which the rust infections apparently reprogramme the epidermal

cells to become more resistant to the mildew pathogen. An entirely different type of

question is, whether this interaction may have epidemiological consequences in case

both pathogens occur side-by-side in the field.

Acknowledgement

Reza Tadayonirad is acknowledged for his great help during the inoculations and samplings. The

macroarray analysis was done at the Leibniz Institute of Plant Genetics and Crop Plant Research

(IPK). The excellent technical assistance of Ines Walde is acknowledged. We thank Dr. Axel

Himmelbach from IPK and Dr. Chris Maliepaard form Plant Breeding for thier help in data analysis.

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General discussion

101

General discussion

Chapter 6

102

Introduction In the barley-Blumeria interaction attacked plant epidermal cells defend themselves

against fungal penetration by localized responses leading to papilla formation and

preventing the fungus from haustorium formation. This mechanism of defense

conveys a race non-specific form of resistance and is common to basal host

resistance and mlo-mediated resistance to adapted powdery mildew (Bgh) and non-

host resistance to non-adapted forms of Bg. Identification of molecular components

underlying mlo resistance in barley and non-host resistance in Arabidopsis revealed

that these two types of resistance share many similarites (Humphry et al., 2006).

However, it is not known which genes make some barley accessions less susceptible

to the adapted form of Bg than others, and which genes determine the host status of

barley to non-adapted Bg forms. We undertook an integrated approach combining

QTL mapping and candidate genes analysis in order to identify genes that are

involved in basal resistance to adapted Bgh. Two experimental barley lines were

developed with exceptional susceptibility to a non-adapted form of Bg, the wheat

powdery mildew fungus. In this Chapter we discuss the implications of our findings for

understanding the genetics and mechanisms of basal resistance of barley to adapted

and non-adapted forms of powdery mildew.

Abundance and diversity of genes for basal resistan ce to powdery

mildew Basal resistance of barley to powdery mildew is a quantitatively inherited trait that

limits the growth and sporulation of powdery mildew colonies by a non-hypersensitive

mechanism of defense. This resistance is valuable for the breeders since it is

assumed to be race non-specific and durable. However, due to the polygenic

inheritance, interaction with environmental factors and subjectivity of disease

assessment, this type of resistance has always been considered difficult to breed for.

New molecular tools provided the opportunity to dissect this type of resistance and

understand its molecular basis, and might lead to more efficient breeding methods.

QTL mapping has broaden our understanding of the number and location of the

genes that are involved in basal resistance in different genetic backgrounds of barley

(Chapter 2). Most mapping studies on basal resistance of barley to powdery mildew

have been based on single biparental mapping populations, without verfication of the

mechanism of resistance at the cellular level. QTL mapping in biparental populations

reveals only a slice of the genetic architecture for a trait because only alleles that

differ between the two parental lines will segregate (Holland, 2007). In this thesis, the

genetics and mechanism of basal resistance of barley to powdery mildew have been

General discussion

103

studied using six mapping populations, involving 10 parents, to assess the diversity of

the genes involved in the genetic variation and the mechanism underlying the

resistance conferred by those QTLs. In different mapping populations different sets of

QTLs were responsible for the level of basal resistance and only a few QTLs were

found to be in common between populations. Even when a same parent participated

in two mapping populations (Vada and SusPtrit, see Chapter 2), we found that QTLs

discovered to be contributed by a particular parent in one mapping population, almost

never were detected in a mapping population of that same parent with a different

barley accession. These findings indicate a tremendous wealth of minor genes for

basal resistance to mildew. In addition, the results suggested that QTLs for powdery

mildew resistance in barley are mainly growth-stage dependent. This high diversity of

the genes for the basal resistance and their growth-stage dependency explain why

this type of resistance is race non-specific and probably durable, since multiple genes

with partial and inconsistent effects are involved, pathogen variants that overcome

basal resistance gain only a marginal advantage (Poland, 2008).

The results showing diversity of QTLs for basal resistance to powdery mildew within

the mapping populations of barley suggest that at each development stage there are

sufficient numbers of QTLs available that can be used for plant selection and

breeding.

Co-localization of QTLs and candidate genes Gene expression studies, motif directed profiling and high throughput gene silencing,

transient transformation and large scale mutation assays have been very productive

to indicate candidate genes for traits of interest, including disease resistance. In

Chapter 2, we prioritized many candidate genes that showed an interaction

phenotype with powdery mildew after RNAi silencing or overexpression studies, by

acquiring evidence of their mapping position. Six out of 26 mapped candidate genes

co-localized with the QTLs for powdery mildew resistance. This co-localization

suggests that those candidate genes are likely to contribute to the variation observed

in the mapping population in which the QTL at that locus had been detected. The

information obtained from the co-localization and the knowledge of the sequences of

those candidate genes can speed up the isolation of the genes at those QTLs.

However, for final proof that the QTLs that co-localize with the candidate genes are

indeed caused by allelic variation in those candidate genes, a mapping at much

higher genetic resolution is required. HvSNAP34 is one of the prioritized candidate

genes. This gene encodes a t-SNARE protein that is involved in mlo-mediated, non-

host and host basal resistance (Douchkov et al., 2005). This is an interesting gene to

Chapter 6

104

be cloned to compare its allelic variation between Dom and Rec, that had different

QTL alleles at that locus. Both alleles could be used in transient expression assays to

investigate whether one confers more resistance than the other.

Accumulation of genes for basal resistance The multi-population QTL analysis showed that basal resistance of barley to powdery

mildew is conferred by many genes, most of which are growth stage dependent. QTL

analysis followed by Marker Assisted Selection (MAS) has been suggested to be an

efficient strategy in developing new lines with levels of resistance superior to that of

their parents. This process is known as QTL pyramiding. For pyramiding, after the

discovery of interesting QTLs in the mapping population, QTLs can be combined

through crossing of the genotypes carrying the QTL alleles to be followed by MAS

(Ashikari & Matsuoka, 2006). However in practical breeding, accumulation of genes

using MAS has to be repeated in each crossing cycle since the combined resistance

genes will segregate in the progeny. For traits that are difficult to measure and show a

low heritability, the MAS strategy should be considered as a potential alternative for

crop improvement. However, there are not always marker alleles close to the desired

QTL allele to monitor reliably the introgression of that QTL in new progeny. Other

parents may carry the same marker allele, or the marker allele may be too far away

from the target QTL allele. In that case haplotypes around the target QTL allele

should be determined, which is a laborious effort. Another aspect that may reduce the

efficiency of MAS is the possible occurrence of QTL interactions. That may lead to

disappointing phenotypic performance conferred by designed QTL combinations. We

showed here that accumulation of unknown QTLs just by visual selection for higher

level of basal resistance among the progenies is feasible and easy.

Non-host immunity of barley to non-adapted powdery mildews Complexity of the mechanism of non-host resistance Many hypotheses about the mechanisms of non-host resistance have been

suggested, but two of them are currently in the focus of interest.

1-The non-adapted pathogens produces effectors to suppress PAMP-triggered

immunity in the plant, but these effectors correspond insufficiently with the operative

targets.

2- Plants contain multiple “R genes” acting together to recognize the pathogen

avirulence (Avr) gene products (functionally these are presumed to be effectors).

Since the multiple R-gene combination requires several simultaneous Avr � avr

mutations, it is impossible for the pathogen to break this defense.

General discussion

105

Both of these two models can explain the durability of non-host resistance (Jones &

Dangl, 2006). Genetic studies of interaction of Arabidopsis with non-adapted powdery

mildew forms have shown that both pre- and post-penetration defense mechanisms

are involved in non-host resistance (Lipka et al., 2005). Our histological studies

revealed that resistance to penetration in association with papilla formation is the

main mechanism involved in non-host resistance to non-adapted forms of Bg.

However, a considerable proportion of successfully penetrated germlings (30 to 60%

depending on the barley-non-adapted mildew form combination) were stopped by a

hypersensitive reaction. One forma specialis, from Hordeum murinum, induced

extensive hypersensitivity upon haustorium formation on some tested lines. However,

Christopher-Kozjan & Heath, (2003) presented evidence that hypersensitivity in non-

host combinations not necessarily imply the activity of R/Avr interaction. The

combination of non-hypersensitive and hypersensitive defense mechanisms involved

suggest that non-adapted powdery mildew forms should evolve strategies to

overcome different defense mechanisms in different developmental stages to acuire a

plant species as suitable host.

Genetics of non-host resistance to non-adapted powd ery mildews Most of the genes that have been implicated in non-host resistance have been

discovered by mutagenesis or gene expression studies (Niks & Marcel, 2009; Table

1). Only a few studies reported genes that play a role in natural variation for non-host

resistance (Shafiei et al., 2007; Rodrigues et al., 2004; Jafary et al., 2006; Jafary et

al., 2008; Zhang et al., 2009). Barley is a full non-host to the non-adapted Bgt. We

tested here a large collection of barley accessions against Bgt, and only a few

accessions were found to possess susceptibility to Bgt at seedling stage, and at most

at a very low level. Crossings among some barley accessions with some level of

susceptibility to Bgt resulted in the accumulation of susceptibility factors in two barley

lines called SusBgt SC and SusBgt DC. The accumulation of susceptibility genes in

barley to Bgt by successive crosses suggest that the resistance has a quantitative

inheritance. However we intend to follow a mapping approach to determine the

genetics of non-host resistance in barley against non-adapted forms of Bg.

Furthermore both SusBgt lines are useful tools in gene expression and

complementation studies on non-host resistance. The two lines show surprisingly a

differential level of susceptibility in the conidiation stage of non-adapted powdery

mildews. Therefore, mapping populations based on both lines may lead to the

identification of QTLs that are effective in particular stages of fungal development.

Chapter 6

106

Enhancement of basal resistance to powdery mildew Our results showed that basal resistance of barley against powdery mildew is

enhanced by pre-inoculation with leaf rust. We hypothesized that this rust-induced

mildew resistance might be due to rust-plant communication during stoma

penetration, or to the presence of the rust hyphae in the apoplast, or to some degree

of pre-haustorial resistance mounted by the mesophyll cells to the invading rust

fungus.

Using a barley cDNA macroarray, we compared the genome-wide transcriptome

changes upon double inoculation (LR+PM) with single inoculation leaf rust (LR) and

powdery mildew (PM) treatments. The majority of genes up-regulated by LR are also

up-regulated in the LR+PM, and hence, not silenced by PM. Analysis of quantitative

transcriptome abundance of differentially regulated genes in double inoculation

treatment versus single inoculations (PM and LR) revealed that most of the down-

regulated genes are involved in plant metabolism and photosynthesis. This suggests

that the rust-induced powdery mildew resistance state might be due to a lack of

energy in the plant cells. In double inoculated and also single rust inoculated plants, a

high up-regulation of PR-1 protein, a Pathogenesis-related protein (PR) was

observed. Accumulation of PRs is a hallmark of pathogen-induced systemic acquired

resistance (van Loon et al., 2006). Accumulation of PR proteins after infection by

pathogens has been reported to be associated with increased effectiveness of plant

defense responses upon subsequent pathogen infection, a state that has been called

‘’Priming’’. Primed plants are capable of activating defense responses either faster,

stronger, or both upon attack by pathogens, insects or in response to abiotic stress

(Conrath et al., 2006). Both our histological and expression studies suggest that pre-

inoculation of barley with leaf rust prime the basal defense mechanism of plants

against subsequent challenge inoculation with powdery mildew.

Concluding remarks

In this thesis we found that the barley genome is a rich source of QTLs (genes)

conferring basal resistance to powdery mildew. The great diversity of such genes in

the barley germplasm making transgression for basal resistance a very common

phenomenon. Identification of the genes underlying the powdery mildew seems

feasible considering the prioritarized candidate genes that were found to co-localize

with resistance QTLs. Information gained from the prioritization of candidate genes

can help in the isolation of the genes that underlie resistance QTLs. These candidate

genes have been shown already to affect the level of basal resistance, and now they

have been mapped to the QTL positions found in our mapping studies. In qualitative

General discussion

107

Table 1. Genes with known sequences that were implicated to play a role in basal host and/or non-

host resistance of barley to powdery mildews ( adapted from Niks & Marcel, 2009)

Gene name Description BHR a NHRb References *

Genes involved in transport and secretion systems

ROR2 Plasma membrane-anchored

syntaxin

Bgh Bgt 1,2,3,8,10

SNAP34

ADF3

Synaptosome-associated protein

Actin depolymerizing factor 3

Bgh

Bgh

Bgt

Bgt

11,3,10

11

Genes involved in the positive or negative regulati on of basal defenses

BI-1 BAX inhibitor 1 Bgh Bgt 3,9,11

CDPKs Calcium-dependent protein

kinases

Bgh Bgt 8

MLO Seven-transmembrane domain

protein

Bgh 2,3,4,7,10,11

Genes involved in redox signaling

GLPs Germin-like proteins Bgh 2,3

RBOHs NADPH oxidase Bgh 3

PRXs: Prx07/Prx40 Class III peroxidase proteins Bgh Bgt 6

Transcription factors and signaling cascades

NACs: NAC6 NAC transcription factors Bgh 5

WRKYs: WRKY1/2 Group II WRKYs transcription

factors

Bgh 12

a, basal host resistance

b, non-host resistance

* references:

1: Collins et al. (2003)

2: Collins et al. (2007)

3: Hückelhoven (2007)

4: Jarosch et al. (1999)

5 : Jensen et al., (2007)

6: Johrde & Schweizer (2008)

7: Kumar et al. (2001)

8: Lipka et al. (2008)

9: Nürnberger & Lipka (2005)

10: O’Connell & Panstruga (2006)

11: Schweizer (2007)

12: Shen et al.(2007)

genes this would be sufficient proof to consider these sequences as the “responsible

gene”. In QTLs it is, however, too early to consider these candidates as “confirmed”.

We propose that either complementation tests with allelic forms or ultra-fine physical

QTL mapping are carried out to confirm the candidate genes that emerge from our

study as the most likely. The developed barley lines with exceptional susceptibility to

Bgt will serve to determine the genetic basis of non-host resistance in barley to wheat

powdery mildew by both forward and reverse genetic approaches. This material will

also be useful to quantify subsequent lines of basal defense that the mildew fungus

encounters during its development, such as primary haustorium and secondary

haustorium formation and conidiation. Finally, the rust-induced mildew resistance

Chapter 6

108

phenomenon that was reported here for the first time can be further investigated to

improve our knowledge about the molecular mechanism of penetration resistance at

cell level and the mechanisms that plants deploy to protect themselves to different

biotic stress agents.

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Abbreviations

111

Abbreviations

AUDPC Area Under the Disease-Progress Curve

Bg Blumeria graminis

Bga Blumeria graminis f.sp. avenea

Bgar Blumeria graminis f.sp. agropyri

Bgb Blumeria graminis f.sp. bromi

Bgd Blumeria graminis f.sp. dactylidis

Bgh Blumeria graminis f.sp. hordei

Bghm Blumeria graminis f.sp hordei-murini

Bgt Blumeria graminis f.sp. tritici

CAPS Cleaved Amplified Polymorphic Sequences

dai day after inoculation

DArT Diversity Arrays Technology

DC Double Cross

dCAPS derived Cleaved Amplified Polymorphic Sequences

DHs Doubled Haploids

DI Differential Index

ESH Elongated Secondary Hypha

EST Expressed Sequence Tag

f.sp. forma specialis

ff.spp. formae speciales

FPP Failed Primary Penetration

FPP+HR Failed Primary Penetration with HR

FPP-HR Failed Primary Penetration without HR

hai hour after inoculation

HR Hypersensitive Response

IR Induced Resistance

IS Induced Susceptibility

ISR Induced Systemic Resistance

LOD Logarithm of odds

LR Leaf Rust

Abbreviations

112

MAS Marker-Assisted Selection

Mla Mildew resistance locus a

Mlo Mildew resistance locus o

nhR non-host resistance

NILs Near Isogenic Lines

PAMP Pathogen-Associated Molecular Patterns

PEN Penetration (mutant genes)

PM Powdery mildew

PRs Pathogenesis-Related proteins

PTI PAMP-Triggered Immunity

QTL Quantitative Trait Locus

Rbgq quantitative genes for Resistance to Blumeria graminis

RILs Recombinant Inbred Lines

RNAi RNA interference

Ror Required for mlo-specified resistance

Rphq2 An allele for quantitative resistance to Puccinia hordei

SAP Secondary Attempted Penetration

SAR Systemic Acquired Resistance

SC Single Cross

SCAR Sequence Characterized Amplified Region

SPP Successful Primary Penetration

SPP+HR Successful Primary Penetration with a haustorium and with HR

SPP-HR Successful Primary Penetration with a haustorium but without HR

TDM Transcript-Derived Marker

TIGS Transient-Induced Gene Silencing

Glossary

113

Glossary Avirulent: carrying an avirulence (Avr) gene that hampers development and

reproduction of pathogen in a host plant accession that carries a corresponding

resistance gene.

Basal defense/resistance:

qualitative: defense that plant species mount against non-adapted microbial

intruders.

quantitative: defense that inhibits pathogen spread after successful infection and

onset of disease.

Basic compatibility: state that results from the capacity of the microbe to deal

effectively with the defense that plant species mount against non-adapted microbial

intruders.

Challenger: in induced-resistance terminology, it refers to an adapted or non-

adapted pathogen which is inoculated to a plant subsequently after inoculation with

inducer pathogen (see there).

Colonization: the development stage after establishment of the infection (see

there). In mildews the stage of development of secondary and subsequent haustoria.

Compatible interaction: a host–pathogen interaction that results in production of

the pathogen without elicitation of gross defense responses (the host is susceptible).

Durable resistance: resistance that has remained effective for a long period in

which it has been applied at large scale in an environment conducive to the

pathogen.

Effector: secreted pathogen protein that manipulates host cell functions to suppress

defense and promote infection. As a secondary effect, an effector may elicit in

particular plant genotypes a hypersensitive response, and be an avirulence factor.

Establishment: the development of a primary haustorium in attacked plant cells by

a pathogenic fungus, especially rust and powdery mildew fungi.

Haustorium: specialized organ formed by most biotrophic (pathogenic) fungi and

oomycetes in a living plant cell for nutrient absorption and molecular communication

with the plant cell.

Host specificity: the property of a pathogen species to be able to establish basic

compatibility with only one or a few plant species.

Host status: status of a plant species being susceptible to a particular pathogen

species. If not, the plant is a marginal host or a non-host.

Incompatible interaction: a host–pathogen interaction that does not result in

disease (the host is resistant).

Glossary

114

Induced Systemic Resistance (ISR): the phenomenon that plants acquire an

enhanced defensive capacity against subsequent pathogen attack as a result of root

colonization by selected strains of non-pathogenic bacteria.

Inducer: In induced-resistance terminology, it refers to a pathogen, non-pathogenic

microorganism or other factors that induce resistance or susceptibility to a

subsequent challenger.

Linkage disequilibrium (LD). The condition in which the frequency of a particular

haplotype for two loci is significantly different from that expected under random

mating.

Near isogenic lines (NILs): Inbred lines that differ at only a small genomic region.

Non-adapted pathogen: pathogen species that cannot infect a particular (non-host)

plant species (syn. heterologous, inappropriate).

Non-host resistance: resistance occurring in all genotypes of a plant species to all

genotypes of a potential pathogen species.

Operative target: host target that, when manipulated by a pathogen effector, results

in enhanced pathogen fitness.

PAMP: pathogen-associated molecular pattern; a pathogen-specific molecular

sequence or structure, often indispensable for the microbial lifestyle that, elicits an

innate immune response on receptor-mediated perception.

PAMP-triggered immunity (PTI): first line of active plant defense that relies on the

recognition of pathogen-associated (or microbe-associated) molecular patterns

(PAMPs/MAMPs) by pattern-recognition receptors (PRRs).

Pattern-recognition receptors: plant proteins that identify PAMP (see above)

molecules, such as flagellin or chitin components, that are associated with microbial

pathogens.

Quantitative trait locus (QTL): a locus with an effect on a quantitative trait (i.e. a

trait showing continuous variation) that can only be mapped by the application of

QTL mapping software.

Recombinant inbred line (RIL): an inbred line produced from an initial cross

followed by continuous inbreeding; populations of RILs are often used for QTL

mapping studies.

Resistance gene analogs (RGA): a sequence identified in whole genome analyses

based on their structural homology (presence of NBS and/or LRR domains) with

previously identified R-genes.

Systemic Acquired Resistance (SAR): the phenomenon that plants acquire an

enhanced defensive capacity against subsequent pathogen attack as a result of pre-

inoculation with pathogens that causes necrotic lesions.

Summary

115

Summary In the barley-Blumeria interaction, resistance at penetration stage in association with

papilla formation is a commonly occurring mechanism. This mechanism of defense

reduces the infection severity by adapted powdery mildew pathogen (basal

resistance to Blumeria graminis f.sp. hordei, Bgh) and fully protects the plant against

non-adapted powdery mildew pathogens (non-host resistance to non-adapted forms

of B. graminis). In this thesis we followed an integrated approach based on QTL

mapping and candidate gene analysis. It was our objective to determine which of the

known candidate genes have a map position that coincides with one of the QTLs

detected in our mapping study. Such candidate genes might determine the natural

variation of basal resistance against powdery mildew in barley. We also investigated

whether the genotypic variation in level of resistance of barley to the non-adapted

wheat powdery mildew, B. graminis f.sp. tritici (Bgt) could be used to develop an

experimental line to determine the inheritance of non-host resistance of barley to this

pathogen. Finally we aimed to determine whether the basal resistance of barley to

powdery mildew is being suppressed by prior attack by a different pathogenic

haustorium forming fungus, Puccinia hordei.

Chapter 1 presents an introduction about basal resistance of barley to powdery

mildew and briefly lists evidence on molecular associations between basal, mlo-

mediated and non-host resistance. In Chapter 2 , we performed QTL analysis for

powdery mildew resistance at seedling and at adult plant stage in six mapping

populations of barley. In that analysis quantitative resistance of barley to powdery

mildew was found to be based on a large number of genes, with only few detected in

more than one mapping population. These QTLs for powdery mildew resistance in

barley are mainly plant growth stage dependent. From gene-expression, gene

silencing and transient transformation assays 39 genes emerged that increased or

decreased basal resistance of barley to the powdery mildew fungus. They are

considered candidate genes that might be responsible for natural variation in level of

basal resistance. By looking for polymorphism in those genes, we could determine

the map position of 23 of these candidate genes. Mapping positions of three more

genes, mlo, Ror1 and Ror2, were already available, and these were also considered

as candidate genes in our study.To compare the map position of QTLs in different

mapping populations with the map positions of the 26 candidate genes, we

constructed an improved high-density integrated linkage map of barley. This

improved map was based on the marker data of seven mapping populations of

barley. More than 43% of the markers on our improved integrated map target ESTs

or gene sequences (gene-targeted markers, GTM) and can be used for candidate

Sammary

116

gene identification. Six out of the 26 candidate genes co-localized with QTLs for

basal powdery mildew resistance. They are interesting targets for further studies,

since their allelic forms may be responsible for part of the differences in basal

resistance between the parents in our mapping study. Chapter 3 reports the

development of two experimental barley lines with extremely high and low level of

basal resistance to barley powdery mildew. These lines were obtained by convergent

crossing between the most resistant and the most susceptible lines, respectively,

from four mapping populations of barley studied in Chapter 2. We consider the

extremely susceptible and resistant lines developed here as valuable material to be

used in further experiments to characterize the molecular basis of basal resistance to

powdery mildew. The results suggest that phenotypic selection is sufficiently efficient

to achieve high levels of basal resistance. We report some difficulties in the

application of marker assisted selection that might make that approach unnecessary

and less efficient to achieve a similar high level of basal resistance. In Chapter 4 the

development of two more experimental lines is described. These lines have, at the

seedling stage an unprecedented level of susceptibility to the non-adapted wheat

powdery mildew. A large collection of barley germplasm was screened and some

rare barley accessions were identified with rudimentary susceptibility to the wheat

powdery mildew, Bgt. Those accessions were intercrossed in two cycles, and

resulted in the two exceptional research lines, called SusBgt SC and SusBgt DC. The

quantitative variation among barley accessions and in the progenies after convergent

crossing suggest a polygenic basis of this non-host resistance. Component analysis

of the infection process suggested that non-host resistance factors are Blumeria-form

specific. The developed lines will serve to elucidate the genetic basis of non-host

resistance in barley to wheat powdery mildew, and are useful tools in gene

expression and complementation studies on non-host resistance. In Chapter 5 we

reported that a compatible interaction of barley-Puccinia hordei induces increased

papilla based resistance to a challenge infection by a compatible powdery mildew

isolate. These pathogens differ in strategies to suppress plant cell defense. We

showed that the level of rust-induced mildew resistance varies among barley

accessions and is not determined by the virulent/avirulent spectra of the challenger

isolate. Macroarray gene expression analysis showed that several genes involved in

metabolism and photosynthesis were highly down-regulated and a few genes

involved in plant defense including a pathogenesis related protein (PR-1) and

cysteine synthase were highly up-regulated in the double inoculated treatment. Our

histological and gene expression analyses are compatible with the hypothesis that

the rust “primes” the basal mildew resistance genes prior to the challenge mildew

Summary

117

infection. In Chapter 6 , the results obtained in the previous Chapters are being

discussed. The perspectives of the experimental barley lines that were developed in

this study for the identification of genes that determine basal resistance of barley to

adapted and non-adapted Blumeria graminis forms are highlighted.

Sammary

118

Samenvatting

119

Samenvatting

In de gerst-meeldauw (Blumeria) interactie is resistentie tegen celwandpenetratie

door de vorming van papillen een algemeen voorkomend verschijnsel. Dit

resistentiemechanisme reduceert de aantastingsgraad van gerst door

gerstmeeldauw (basisresistentie tegen Blumeria graminis f.sp. hordei, Bgh), en

beschermt de plant volledig tegen niet op gerst gespecialiseerde vormen van

meeldauw (niet-waardresistentie). In dit proefschrift volgden we een geïntegreerde

benadering door de kartering van QTLs te combineren met gegevens uit een

kandidaatgenbenadering. Doel was om te bepalen welke van de bekende

kandidaatgenen karteren op een van de door ons in kaart gebrachte QTLs.

Dergelijke kandidaatgenen kunnen verantwoordelijk zijn voor de gevonden

verschillen in basisresistentie van gerst tegen de gerstmeeldauw. We hebben ook

onderzocht of de genetische variatie in resistentieniveau tegen de

tarwemeeldauwschimmel (B. graminis f.sp. tritici) zou kunnen worden benut om een

experimentele gerstlijn te ontwikkelen die zou kunnen dienen om de overerving te

bepalen van de niet-waard resistentie van gerst tegen dat pathogeen. Tenslotte

bepaalden we of de basisresistentie van gerst tegen meeldauw zou worden

onderdrukt na eerdere infectie door een ander op gerst gespecialiseerd

haustoriumvormend pathogeen, nl. de dwergroestschimmel Puccinia hordei.

Hoofdstuk 1 introduceert het onderwerp, basisresistentie van gerst tegen

meeldauw, en geeft een korte beschrijving van de argumenten die suggereren dat

basisresistentie, mlo-resistentie en niet-waard resistentie op moleculair niveau

geassocieerd zijn. In Hoofdstuk 2 beschrijven we een QTL analyse die we

uitvoerden om de genetische loci te vinden die bijdragen aan variatie in

basisresistentie van gerst tegen meeldauw in het zaailingstadium en in het

volwassenplantstadium. We deden deze analyse in zes karteringspopulaties van

gerst. Die studie leidde tot de conclusie dat deze kwantitatieve resistentie gebaseerd

is op een groot aantal genen. Slechts enkele daarvan werden gevonden in meer dan

een populatie. De meeste van deze QTLs voor meeldauwresistentie zijn

plantontwikkelingsstadium-specifiek. Uit genexpressie-, genexpressie

onderdrukkings- en transformatie-experimenten kwamen 39 genen naar voren die

bijdroegen aan de kans dat een meeldauwschimmel met succes een haustorium in

een plantencel vormde. Deze genen kunnen worden beschouwd als kandidaatgenen

die verantwoordelijk zijn voor natuurlijke verschillen in niveau van basisresistentie.

We zochten sequentieverschillen in deze kandidaatgenen, zodat we hun kaartpositie

konden bepalen. Dit lukte voor 23 van deze kandidaatgenen. Kaartposities van drie

andere bij meeldauwresistentie betrokken genen, mlo, Ror1 en Ror2, waren al

Samenvatting

120

bekend, en ook deze werden voor onze studie beschouwd als kandidaatgenen voor

basisresistentie. Vervolgens vergeleken we de kaartpositie van deze 26

kandidaatgenen met die van de gevonden QTLs voor basisresistentie. Daarvoor

ontwikkelden we een verbeterde en zeer dichte, geïntegreerde koppelingskaart van

gerst. Deze verbeterde kaart was gebaseerd op zeven karteringspopulaties van

gerst. Meer dan 43% van de merkers die op deze kaart zijn samengebracht

vertegenwoordigen gensequenties, en zijn van nut om in de toekomst

kandidaatgenen te identificeren. Zes van de 26 kandidaatgenen hadden een

kaartpositie die samenviel met die van een QTL voor basisresistentie tegen

meeldauw. Deze zes kandidaatgenen zijn interessant voor vervolgstudies, omdat

variatie in deze genen wel eens verantwoordelijk zouden kunnen zijn voor een deel

van de verschillen in basisresistentie die gevonden werden in de karteringsstudies.

Hoofdstuk 3 beschrijft de ontwikkeling van twee experimentele gerstlijnen met een

extreem hoge en een extreem lage basisresistentie tegen gerstmeeldauw. Deze

lijnen werden verkregen door toepassing van convergente kruisingen tussen

respectievelijk de meest resistentie en tussen de meest vatbare lijnen in de

karteringspopulaties die beschreven zijn in Hoofdstuk 2. We beschouwen de extreem

vatbare en extreem resistente lijnen als waardevol materiaal voor verder onderzoek

naar de moleculaire basis van basisresistentie tegen meeldauw. De resultaten wijzen

erop dat fenotypische selectie voldoende effectief is om zeer hoge niveaus van

basisresistentie te verkrijgen. We geven enkele argumenten dat merkergestuurde

selectie wel eens onnodig en minder efficiënt zou kunnen zijn om een dergelijk hoog

niveau van resistentie te bereiken. In Hoofdstuk 4 beschrijven we de ontwikkeling

van twee andere experimentele gerstlijnen. Deze lijnen hebben, in het

zaailingstadium, een niet eerder gemeld hoog niveau van vatbaarheid voor de op

tarwe gespecialiseerde tarwemeeldauwschimmel. In een grote collectie gerstlijnen

werd gezocht naar zeldzame gerstlijnen met een rudimentaire vatbaarheid voor dit

tarwepathogeen. Die gerstlijnen werden in twee cycli onderling gekruist, wat

resulteerde in twee buitengewone onderzoekslijnen, SusBgt SC en SusBgt DC. De

kwantitatieve variatie tussen gerstlijnen en binnen de hier ontwikkelde

nakomelingschappen wijst op een kwantitatieve overerving van deze niet-waard

resistentie. Componentenanalyse van het infectieproces suggereerde dat factoren

die de niet-waard resistentie van gerst tegen meeldauwvormen bepalen Blumeria-

vorm-specifiek zijn. De ontwikkelde lijnen zullen dienen om de overerving van niet-

waard resistentie in gerst tegen de tarwemeeldauw te bepalen. De lijnen zijn voorts

een nuttig instrument in genexpressie- en complementatiestudies naar niet-waard

resistentie. In Hoofdstuk 5 vermelden we dat een compatibele interactie tussen

Samenvatting

121

gerst en Puccinia hordei (de veroorzaker van dwergroest) de basisresistentie tegen

een daarna aangebrachte meeldauw verhoogt. Deze pathogenen verschillen in

manier waarop ze de afweer van plantencellen onderdrukken. We toonden aan dat

het niveau van deze roest-geïnduceerde resistentie verschilt tussen gerstlijnen, en

niet afhangt van het virulentiespectrum van het gebruikte meeldauwisolaat. Een

macroarray genexpressie-experiment liet zien dat in de dubbel-geïnoculeerde

behandeling verscheidene genen die een rol spelen in plant metabolisme en

fotosynthese, verminderd tot expressie kwamen. Dit, terwijl enkele genen die een rol

spelen in de afweer van planten versterkt tot expressie kwamen. Onder de

laatstgenoemde zijn pathogenese gerelateerde eiwitten (PR-1) en cysteine synthase

het vermelden waard. Onze histologische en genexpressiebevindingen zouden erop

kunnen wijzen dat de eerdere infectie door de roest tot gevolg heeft dat genen voor

basisresistentie tegen meeldauw worden “geprimed”, d.w.z. klaargemaakt om snel

en sterk te reageren zodra een meeldauwschimmel de cel aanvalt. In Hoofdstuk 6

bespreken we de in de voorgaande hoofdstukken behaalde resultaten. Met name

wijzen we op de perspectieven die de verkregen experimentele lijnen bieden voor de

identificatie van genen die verantwoordelijk zijn voor basisresistentie van gerst tegen

aangepaste en niet-aangepaste vormen van Blumeria graminis.


122

Acknowledgments

123

Acknowledgments

In the name of God, most gracious, most merciful. Looking back, I feel so fortunate of

having had the opportunity to do my PhD at the Laboratory of Plant Breeding of

Wageningen University. I did my PhD at a very friendly atmosphere and received

continuous technical and emotional support during the last four years. It is a pleasure

to convey my gratitude to all people who helped me in achieving this momentous

task.

First of all, I would like to express my deep sense of gratitude to my supervisor Dr.

Rients Niks, for his patient guidance, encouragement and excellent advice

throughout this study. Dear Rients, you were always ready to help. Your energetic

personality and your enthusiasm for seeing new results have been stimulating me to

improve my scientific background and also to enjoy my research outputs. You were

so quick in commenting and correcting my written materials and your positive words

gave me new confidence in myself and my ability. I will never forget your efforts to

find a good living place for my family before our arrival and how happily you informed

me that you could find a beautiful family house, a case of too-nice-to-be-true! You

and your respectful wife, Marlien was very kind and supportive to me and my family.

Thank you for all your help and support.

I acknowledge my gratitude to Prof. Richard Visser for his valuable advices

throughout my study. Dear Richard, thank you for stimulating discussions and for

your comments and suggestions for the editing of my thesis.

My sincere thanks to Thierry Marcel for his fruitful collaboration and his kind

assistance in the research as well as for his valuable friendship. Dear Thierry, I have

really enjoyed working in the same group with you and have learned a lot from you. I

count you and your respectful wife, Hoa, among my best family friends.

Many thanks go to Hossein Jafary and his respectful wife Shirin. Dear Hossein, I

and my family will never forget your kind help and your warm and generous

hospitality during the first days of our stay in the Netherlands. You kindly helped me

to find my way in the Lab and easily shared your experience. Thank you for your

support, help and friendship.

My cordial thanks to Anton Vels for his kind assistance in setting up the field and

the greenhouse trials. Dear Anton, I was so pleased to hear that you are recovering

from your illness. You are indeed a great colleague.

I am thankful to Annie Marchal, Letty Dijker and Mariame Gada at the secretary of

Plant Breeding for dealing with administration and being always kind and helpful.

I had the great fortune of sharing ideas with every member of resistance group, but

more important, I have been honored with their friendship. It is a pleasure to mention

Acknowledgments

124

Sjaak van Heusden, Ben Vosman, Yuling Bai, Marieke Jeuken, Ningwen Zhang,

Stefano Pavan, Luigi Faino, Zheng Zheng, Erik den Boer and Freddy Yeo.

Furthermore, I would like to give special thanks to our Lab technicians, Petra vanden

Berg, Fien Meijer-Dekens, Koen Pelgrom and Martijn van Kaauwen for creating such

a nice atmosphere in the Lab.

I am thankful to my friendly officemates Paul Dijkhuis, Awang Maharijaya, Firdaus

Syarifin and Heungryul Lee. Extra thanks go to Awang for his kind help to design the

cover of my thesis. I am grateful to Nadeem Khan, Arnold Kuzniar, Anitha Kumari,

Sameer Joshi, Estelle Verzaux, Anoma Lokossou, Xingfeng Huang, Miqia Wang and

Shital Dixit for their friendship. Dear Shital thank you also for your kind acceptance to

be one of my paranimphs.

My special thanks also go to Dr. Patrick Schweizer from IPK, Germany for his

cooperation during the last two years and his great hospitality during my stay at IPK.

I had a great opportunity to meet and establish friendship with many Iranian

scientists in Wageningen. It is a pleasure to mention Dr. Mahmoud Otreshy, Rahim

Mehrabi, Ramin Roohparvar, Mostafa Karbasioun, Benyamin Houshyani, Farhad

Nazarian and their families. Dear Rahim and Farhad we shared various thoughts

during our tea breaks and sport activities in the late afternoon together with Hossein.

Thank you for your friendship. Furthermore I am grateful to Mahdi Arzanlou, Ali

Haghnazari, Reza Talebi, Sara Dezhsetan, Reza Tadayonirad, Leila Samiei, Nasim

Mansouri, Kambiz Baghalian and Alireza Babaei for their friendship. Dear Alireza, we

shared many funny moments during our trips together, I value our friendship very

much.

It is a pleasure to convey my gratitude to Alireza Seifi. Dear Alireza, thank you for

your encouragement and your kind help in the Lab as well as for creating such a

great friendship.

My special thanks go to Hedayat Bagheri, Mohammad Balali, Afshin Mehraban,

Mahmoud Tabib Ghaffary and their families. Dear Hedayat and Mohammad, we have

many good memories from our family trips and gatherings. I have found both of you

trustworthy and honest friends. Dear Afshin and Mahmoud thank you for being

always supportive to me and my family. You are very good friends. Furthermore, I

want to thank Mr. and Mrs. Raei for their support and their kindness to me and my

family.

I would like to take this opportunity to thank my colleagues at Agricultural Research

and Education Organization (AREO) and the Ministry of Science Research and

Technology (MSRT) of my home country, IRAN, for financial support and also dealing

Acknowledgments

125

with a lot of official tasks during the last four years. Many thanks go in particular to

the Staff Training Office of AREO and the scientific counselor of MSRT in Paris.

I would like to express my special thanks to my brother-in-law Reza Tajy and my

sister-in-law Maryam for their support and kindness.

My PhD study could never be fulfilled without the full support from my family. I

would like to express my profound gratitude to my beloved parents for their

continuous emotional support and prayers during my study abroad. You made a long

journey to visit us, indeed those were our best days in the Netherlands. To my lovely

daughter, Mahrokh and my lovely son, Pouria, I feel the joy and happiness as watch

you grow everyday. I am really proud of you! Finally I would like to express my

deepest sense of gratitude to my wife, Fatemeh for her moral support and patience

during my study. Dear Fatemeh, thank you for your generous love and persistent

confidence in me. I love you so much.

Reza Aghnoum

Wageningen, June 2009

Acknowledgments

123

About the author

127

About the author Reza Aghnoum was born on June 22, 1972 in

Tehran, Iran but he grew up in a small city in the

north east of Iran, where his parents had originally

come from. After finishing his high- school, Reza

started his academic studies in Plant Protection at

Ferdowsi University of Mashhad in 1991. He

followed a Master degree at the same university

in Plant Pathology in 1995.

Upon earning his Master degree in 1997, he was employed by the Agricultural

Research and Education Organization (AREO) as a cereal pathologist-breeder at

Mashhad Agricultural Research Center, where he worked for 7 years with a group of

cereal breeders aiming to breed high yielding and disease resistant wheat and barley

cultivars. In 2001 he passed the national examination and received a scholarship to

do a PhD abroad. In February 2005 he started his PhD program at the Laboratory of

Plant Breeding of Wageningen University under the supervision of Dr. Rients E. Niks

and worked on basal resistance of barley to powdery mildew as his PhD thesis. Reza

will join the scientific community of his home country, Iran, after finishing his PhD

studies in the Netherlands.

Reza Aghnoum

Khosaran Agricultural and Natural Resources Research Center

P.O.Box: 91735-488

Mashhad,

IRAN

Email: [email protected]

[email protected]

Acknowledgments

128

List of publications

129

List of publications

Marcel TC, Aghnoum R, Durand J, Varshney RK, Niks RE. 2007. Dissection of the

barley 2L1.0 region carrying the ‘Laevigatum’ quantitative resistance gene to leaf rust

using near isogenic lines (NIL) and subNIL. Molecular Plant–Microbe Interactions 20:

1604-1615.

Aghnoum R , Marcel TC, Johrde A, Pecchioni N, Schweizer P, Ni ks RE. Which

candidate genes are responsible for natural variation in basal resistance of barley to

barley powdery mildew? Submitted.

Aghnoum R , Niks RE. New perspectives to identify genes that determine non-host

resistance of barley to non-adapted Blumeria graminis forms. Submitted.

Abstracts

Aghnoum R, Marcel TC, Jafary H, Niks RE. Genetic diversity for quantitative

resistance of barley against powdery mildew. Plant & Animal Genomes XV

Conference. San Diego, California, USA. January 13-17, 2007.

Aghnoum R, Niks RE. Development of barley lines with extreme low and high level

of basal resistance to powdery mildew.The 10th International Barley Genetics

Symposium. Alexandria, Egypt, April 5-10, 2008.

Acknowledgments

130

Education certificate

131

Education certificate Education Statement of the Graduate School Experimental Plant Sciences Issued to: Reza Aghnoum Date: 16 June 2009 Group: Plant Breeding, Wageningen University and Re search Centre 1) Start-up phase date ► First presentation of your project Genetics of non-hypersensitive resistance of barley to powdery mildew Nov 1, 2005 ► Writing,or rewriting a project proposal Polygenic, non-hypersensitive resistance of barley to barley powdery mildew Feb-Aug 2005 ► Writing a review or book chapter ► MSc courses Genetic Analysis, Tools and Concepts (GEN-30306) Nov-Dec 2005 Breeding for Resistance (PBR 30306) Mar-Apr 2005 ► Laboratory use of isotopes Subtotal Start-up Phase 13.5 credits* 2) Scientific Exposure date ► EPS PhD student days EPS PhD student day 2005 in Radboud University Nijmegen Jun 2, 2005 SDV PhD student day 2006 in Paris Jun 9, 2006 EPS PhD student day 2006 in wageningen Sep 19, 2006 EPS PhD student day 2007 in wageningen Sep 13, 2007

1st Joint Retreat of the PhD Students in EPS

Oct 2-3, 2008

► EPS theme symposia EPS Theme 2 'Interactions between Plants and Biotic Agents, Utrecht University Jun 23, 2005 ► NWO Lunteren days and other National Platforms EPS - ALW Lunteren Apr 3-4, 2006 EPS - Lunteren meeting Apr 6-7, 2009 ► Seminars (series), workshops and symposia Molecular markers in yellow rust and powdery mildew: Dr. Xia-Yu Duan Nov 21, 2007 Plant Breeding Research day 2008, wageningen university, Jun 17, 2008 DNA demethylation in Arabidopsis:Prof.Jian-Kang Zhu Nov 3, 2008 Seminar: Prof. Nürnberger:Receptors in plant immunity Dec 17, 2008 ► Seminar plus ► International symposia and congresses Plant and Animal Genome XV Conference,USA Jan 13-17, 2007 Bioexploit meeting, Wageningen Mar 31-Apr 1, 2009 ► Presentations Poster: Plant and Animal Genome XV Conference,USA Jan 13-17, 2007 Poster:Plant Breeding Research day 2007, wageningen university, Sep 27, 2007 Poster:The 10th International Barley Genetics Symposium, Alexandria, Egypt Apr 5-10, 2008 Poster:Work shop "Natural Variation in plants", Aug 26-29, 2008 Oral presentation:Summerschool "On the Evolution of Plant Pathogen Interactions Jun 20, 2008

Poster:1st Joint Retreat of the PhD Students in EPS Oral presentation:EPS-Lunteren meeting

Oct 2-3, 2008 Apr 7, 2009

► IAB interview Sep 14, 2007 ► Excursions Excursion to Barenburg grass breeding company, Wolfheze, Oct 2,2007 Excursion to Bejo seeds and Royal van Zanten companies, Jun 11, 2008 Excursion to Syngenta company in Einkhuzen Sep 25, 2008

Subtotal Scientific Exposure 10.4 credits* 3) In-Depth Studies date ► EPS courses or other PhD courses Summerschool "Signalling in Plant Development and Plant Defense" Jun 21-23, 2006 Summerschool "On the Evolution of Plant Pathogen Interactions Jun 18-20, 2008 EPS PhD Work shop "Natural Variation in plants", Aug 26-29, 2008 ► Journal club Litterature discussion in Department of Plant Breeding 2005-2008 ► Individual research training Two weeks Lab training on gene expression analysis, IPK Germany Jan 25-Feb 06, 2009

Subtotal In-Depth Studies 9.0 credits* 4) Personal development date ► Skill training courses Working with EndNote 8, Wageningen UR Library Mar 8, 2005 Techniques for writing and presenting a scientific paper Oct 16-19, 2007 ► Organisation of PhD students day, course or confere nce ► Membership of Board, Committee or PhD council

Subtotal Personal Development 1.4 credits* TOTAL NUMBER OF CREDIT POINTS* 34.3

Herewith the Graduate School declares that the PhD candidate has complied with the educational requirements set by the Educational Committee of EPS which comprises of a minimum total of 30 credits * A credit represents a normative study load of 28 hours of study

Acknowledgments

132

This research has been carried out at the Laboratory of Plant Breeding of

Wageningen University and finantially was supported by the Agricultural Research

and Education Organization (AREO) and the Ministry of Science Research and

Technology of I. R. of Iran.

Thesis layout: by the author

Cover design: by Awang Maharijaya and the author

Front page: schematically represents the complexity of the papilla-based defense

Back page: abundant conidiation by the non-adapted wheat powdery mildew on the

barley line SusBgt SC

Printed by: Wöhrmann Print Service, Postbus 92, 7200 AB, Zutphen, the

Netherlands

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