CHARACTERIZATION OF PATHOGEN EFFECTORS AND HOST …
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The Pennsylvania State University The Graduate School The Huck Institutes of the Life Sciences CHARACTERIZATION OF PATHOGEN EFFECTORS AND HOST ENDOGENOUS PEPTIDE ELICITORS IN THE RICE-MAGNAPORTHE ORYZAE INTERACTION A Dissertation in Plant Biology by Wenhua Liu 2013 Wenhua Liu Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2013
Transcript of CHARACTERIZATION OF PATHOGEN EFFECTORS AND HOST …
The Pennsylvania State University
The Graduate School
The Huck Institutes of the Life Sciences
CHARACTERIZATION OF PATHOGEN EFFECTORS AND HOST
ENDOGENOUS PEPTIDE ELICITORS IN THE RICE-MAGNAPORTHE
ORYZAE INTERACTION
A Dissertation in
Plant Biology
by
Wenhua Liu
2013 Wenhua Liu
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
August 2013
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The dissertation of Wenhua Liu was reviewed and approved* by the following:
Yinong Yang
Associate Professor of Plant Pathology and Environmental Biology
Dissertation Advisor
Chair of Committee
Teh-hui Kao
Distinguished Professor of Biochemistry and Molecular Biology
Chair of Intercollege Graduate Degree Program in Plant Biology
Timothy McNellis
Associate Professor of Plant Pathology and Environmental Biology
Dawn Luthe
Professor of Crop and Soil Sciences
*Signatures are on file in the Graduate School
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ABSTRACT
The arms race is going on forever between plants and pathogens. Along their ever-
lasting co-evolutionary path, plants have developed multilayered surveillance systems
and defense lines, whereas pathogens have acquired vast capabilities of evading
perception and breaking defense lines. In the interaction of Magnaporthe oryzae and
rice, M. oryzae delivers a phalanx of effectors that could enter rice cells and subvert
rice defenses, and rice cells deploy an array of surveillance receptors that could sense
pathogen attack and initiate defense responses against M. oryzae. This study aims to
shed some new light on the rice-M. oryzae interaction from both the pathogen and the
host perspectives.
A family of one-finger zinc finger proteins was identified as pathogen effectors from
the secretome of M. oryzae isolate 70-15. Rice leaf sheath live-cell imaging showed
that zinc finger effectors were first accumulated in the biotrophic interfacial complex
(BIC) and then translocated into rice cells during M. oryzae infection. The underlying
mechanism for translocation was likely associated with two host-targeting motifs
L/IPXP and L/IXAR, as demonstrated by the analysis of the localizations of BAX-
and GFP-tagged fusion proteins. Yeast two-hybrid screening of a blast-induced rice
cDNA library revealed that the rice nucleus-localized protein HIRA was one of the
interactors of this family of zinc finger effectors. The interaction between zinc finger
effectors and HIRA was in the nucleus of the rice cell, as shown by bimolecular
fluorescence complementation (BIFC). In addition, this interaction was also
confirmed by a GST pull-down assay. Zinc finger effectors are in nature DNA-
binding proteins, as unveiled by systematic evolution of ligands by exponential
enrichment (SELEX). Moreover, zinc finger effectors inhibited BAX-induced
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programmed cell death (PCD) when transiently expressed in Nicotiana benthamiana.
And rice transgenic lines overexpressing zinc finger effectors exhibited lowered
defense gene expression and increased susceptibility to M. oryzae. A plausible model
has been proposed to elucidate how zinc-finger effectors suppress rice immunity by
recruiting HIRA for chromatin remodeling.
Rice, like Arabidopsis, harbors a family of defense-related genes encoding plant
elicitor peptides (Peps). On one hand, M. oryzae induced the expression of the
OsPep1-encoding precursor gene OsPROPEP1 via the ethylene (ET) and jasmonate
(JA) pathways. On the other hand, synthetic OsPep1 elicited defense response in rice
and thereby enhanced resistance against M. oryzae. Furthermore, rice transgenic lines
overexpressing OsPROPEP1 showed constant defense gene expression and increased
resistance to M. oryzae. All these findings indicate that Pep signaling as an amplifier
of plant defense is an integral part of rice innate immunity against M. oryzae.
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TABLE OF CONTENTS
List of Abbreviations………………………………………………………………...ix
List of figures………………………………………………………………………...xi
List of tables………………………………………………………………………...xiv
Acknowledgements……………………………………………………………….…xv
Chapter 1 Literature review………………………………………………..1
1.1 Plant pathogen effector delivery systems………………………1
1.1.1 Bacterial effecotor secretion/translocation systems………………2
1.1.2 Oomycete and fungal effector delivery venues…………………..5
1.2 The subcellular locations of plant pathogen effectors……...….8
1.3 The host targets of plant pathogen effectors…………………..9
1.3.1 PRRs and Rs…………………………………………………….10
1.3.2 The MAP3K-MAP2K-MAPK cascade…………………………11
1.3.3 The crosstalk between phytohormones…………………………11
1.3.4 The chromatin and transcription machinery……………………13
Chapter 2 A family of M. oryzae zinc-finger effectors are secreted and
translocated inside plant cells………………………………..16
2.1 Introduction…………………………………………………..16
2.2 Results…………………………………………………………21
Indentification and cloning of a family of putative M. oryzae
one-finger zinc finger proteins………………………………...21
Zinc finger effectors accumulate in BIC and translocate into rice
cells during fungal infection…………………………………...23
Motifs L/IPXP and/or L/IXAR may play a role in translocation
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of effectors from fungus to plant cells…………………………27
2.3 Discussion……………………………………………………..34
2.4 Materials and methods……………………………………….35
Genetic constructs……………………………………………..35
Live-cell imaging of zinc finger effectors in rice cells………..36
Protein-lipid overlay assay of zinc finger effector-phospholipid
interaction……………………………………………………...36
Agrobacterium-mediated transformation of N. benthamiana and
biolistic transformation of onion epidermis…………………...37
Chapter 3 Zinc finger effectors are nuclear HIRA recruiters and DNA
binding factors………………………………………………..38
3.1 Introduction…………………………………………………..38
3.2 Results…………………………………………………………45
Zinc finger effectors interact with HIRA and other nuclear
proteins………………………………………………………...46
The interactions between zinc finger effectors and HIRA occur
in the nuclei of rice cells………………………………………50
Zinc finger effectors are DNA binding factors……………….52
3.3 Discussion…………………………………………………….56
3.4 Materials and methods………………………………………60
Yeast two hybrid screen………………………………………60
GST pull-down assay…………………………………………60
Bimolecular fluorescence complementation assay……………60
Systematic evolution of ligands by exponential enrichment
(SELEX) assay………………………………………………..62
Chapter 4 Zinc-finger effectors suppress PTI when expressed
heterologously in plants……………………………………..63
4.1 Introduction………………………………………………….63
4.2 Results………………………………………………………...68
Zinc finger effectors inhibit BAX induced PCD in
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N.benthamiana………………………………………………..68
Zinc finger effectors inhibit BAX induced PCD in rice
Protoplasts…………………………………………………….71
The induction of defense genes is compromised in MGG_07834
or MGG_09035 transgenic lines……………………………....72
MGG_07834 or MGG_09035 transgenic lines are more
susceptible to M. oryzae infection……………………………76
4.3 Discussion……………………………………………………78
4.4 Materials and methods……………………………………...80
Rice protoplast preparation, transfection and FDA staining…80
Rice transformation…………………………………………..81
RNA extraction and cDNA synthesis………………………...81
Quantitative RT-PCR for defense-related genes……………..82
Rice blast inoculation and disease severity assay……………82
Chapter 5 Role of the rice endogenous peptide elicitor OsPep1 in
defense signaling and disease resistance…………………...83
5.1 Introduction…………………………………………………83
5.2 Results………………………………………………………..86
OsPep1 belongs to a 7-member family of potential endogenous
peptide elicitors in rice……………………………………….86
OsPROPEP1 is induced by M. oryzae and JA and ET……….88
OsPep1 induces the expression of its precursor gene and rice
defense genes and enhances disease resistance against
M. oryzae……………………………………………………..91
OsPROPEP1-overexpression lines exhibit growth inhibition
and seed-set reduction………………………………………..95
An array of defense genes are constitutively expressed in
OsPROPEP1-overexpressing lines…………………………...98
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OsPROPEP1-OX lines enhance disease resistance against
M. oryzae…………………………………………………….100
5.3 Discussion…………………………………………………..105
5.4 Materials and methods…………………………………….108
OsPROPEP1 construct and rice transformation……………108
Plant materials and fungal isolates………………………….108
Peptide synthesis and chemical treatments…………………109
Assessment of Agronomic traits and disease severity………109
Quantitative real-time PCR analysis of relative gene expression
Levels……………………………………………………….110
References………………………………………………………………………….112
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LIST OF ABBREVIATIONS
AA Amino Acid
ABA Abscisic Acid
AS1 Asymmetric Leaves 1
AS2 Asymmetric Leaves 2
A. tumefaciens Agrobacterium tumefaciens
AVR Avirulence
BAX BCL-2 Associated X Protein
BIC Biotrophic Interfacial Complex
BiFC Bimolecular Fluorescence Complementation
CEBiP Chitin Elicitor Binding Protein
CERK1 Chitin Elicitor Receptor Kinase
CHIP Chromatin Immunoprecipitation
DAMP Damage Associated Molecular Pattern
ET Ethylene
ETI Effector Triggered Immunity
GST Glutathione S Transferase
HIRA Histone Regulatory Protein A
HDA Histone Deacetylase
HMT Histone Methyltransferase
HR Hypersensitive Response
HTM Host Targeting Motif
IH Invasive Hyphae
JA Jasmonate
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L/IPXP Leucine/Isoleucine-Proline-any amino acid-Proline
L/IXAR Leucine/Isoleucine-any amino acid-Alanine-Arginine
MAMP Microbe Associated Molecular Pattern
M. oryzae Magnaporthe oryzae
N. benthamiana Nicotiana benthamiana
Os Oryza sativa
OX Over-expression
PAMP Pathogen Associated Molecular Pattern
Pep Plant Elicitor Peptide
PEPR Plant Elicitor Peptide Receptor
PPR Pentatricopeptide Repeat
PR Pathogenesis Related Protein
PRR Pattern Recognition Receptor
PTI Pattern Triggered Immunity
qRT-PCR Quantitative Reverse Transcription-Polymerase Chain Reaction
SA Salicylic acid
SELEX Systematic Evolution of Ligands by Exponential Enrichment
STOP1 Sensitive to Proton Rhizotoxicity 1
Ta2A Thosea asigna 2A
T3SS Type III Secretion System
T4SS Type IV Secretion System
T6SS Type VI Secretion System
Xoo Xanthomonas oryzae pv. oryzae
Y2H Yeast Two Hybrid
ZFE Zinc Finger Effector
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LIST OF FIGURES
Figure 1-1 Bacterial T3SS, T4SS and T6SS translocate effectors into host cells……..2
Figure 1-2 Oomycete and fungal effector-exporting specialized structures…………..5
Figure1-3 Subcellular locations and virulence targets in host cells of oomycete and
fungal effectors………………………………………………………………………...9
Figure 2-1 M. oryzae hemibiotrophic life cycle……………………………………...17
Figure 2-2 A family of M. oryzae zinc-finger effectors……………………………...23
Figure 2-3 Zinc finger effectors accumulate in BIC ………………………………...25
Figure 2-4. Zinc-finger effectors translocate inside rice cells……………………….26
Figure 2-5 Protein-lipid overlay assay……………………………………………….30
Figure 2-6 Localizations of MGG_07834 : EGFP and MGG_10020 : EGFP when
expressed in rice protoplasts………………………………………………………….30
Figure 2-7 The role of L/IPXP in BAX fusion protein translocation………………..31
Figure 2-8 The role of L/IPXP in EGFP fusion protein translocation……………….32
Figure 2-9 The role of L/IPXP and L/IXAR in MGG_07834 or its mutant EGFP
fusion protein translocation…………………………………………………………..33
Figure 3.1 Major transcription factors of defense hormone pathways in
Arabidopsis…………………………………………………………………………...39
Figure 3.2 Pathogen effectors and their targets in the nucleus………………………44
Figure 3-3 The repressive chromatin state of the promoter of KNOX genes
established by AS1/AS2/HIRA silencing complex…………………………………..45
Figure 3-4 Y2H screen for MGG_07834 interactors………………………………...48
Figure 3-5 Predicted motifs in HIRA, OsSTOP1 and OsPPR1……………………...49
Figure 3-6 In vitro GST pull-down assay demonstrated zinc-finger effectors interact
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with HIRA in vitro…………………………………………………………………...50
Figure 3-7 Interactions between zinc-finger effectors and HIRA were localized to rice
cell nuclei…………………………………………………………………………….51
Figure 3-8 Coimmunoprecipitation assay for the interaction between effectors and
HIRA in rice protoplasts……………………………………………………………...52
Figure 3-9 3-D structures of zinc finger motifs of MGG_07834, MGG_10020, AVR-
Pii and AS2…………………………………………………………………………...54
Figure 3-10 One-finger zinc finger proteins can specify a sequence of DNA……….55
Figure 3-11 MGG_07834 binds to double stranded DNA probes after five rounds of
SELEX reactions……………………………………………………………………..56
Figure 3-12 A model for establishing a chromatin repressive state at the promoters of
certain defense-related genes by ZFE/HIRA silencing complex…………………….59
Figure 4-1. Zinc-finger effectors suppress BAX-induced PCD when transiently
expressed in N. benthamiana via Agrobacterium infiltration………………………..70
Figure 4-2. Zinc-finger effectors suppress BAX-induced PCD in rice protoplasts….71
Figure 4-3 Suppression of induction of defense related genes in MGG_07834 and
MGG_09035 transgenic lines after M. oryzae inoculation…………………………..76
Figure 4-4 Lesion sizes and Lesion numbers of MGG_07834 and MGG_09035
transgenic lines post M. oryzae inoculation………………………………………….77
Figure 4-5 Disease severity of MGG_07834 and MGG_09035 post spray and punch
inoculation inoculation of M. oryzae strains, respectively…………………………...78
Figure 4-6 Model of M. oryzae ZFEs suppress chitin (GlcNAc)n-CEBiP/OsCERK1
mediated rice immunity………………………………………………………………80
Figure 5-1 OsproPep1 and its mature peptide OsPep1……………………………….88
Figure 5-2 Induction of OsPROPEP1 by various inducers…………………………..90
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Figure 5-3 OsPep1 (1µM) induces expression of its precursor gene and
defense genes…………………………………………………………………………94
Figure 5-4 OsPep (1µM) induces resistance against M. oryzae……………………...95
Figure 5-5 Comparison phenotypes of wild-type and OsPROPEP1-OX plants……..98
Figure 5-6 OsPROPEP-OX lines constitutively express defense genes and enhance
disease resistance against M. oryzae………………………………………………..105
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LIST OF TABLES
Table 2-1 Effectors cloned as AVR proteins in M. oryzae…………………………..18
Table 2-2 Effector accumulation and movement pattern…………………………….20
Table 3-1 Chromatin modulins in plant defense……………………………………..41
Table 3-2 Summary of rice proteins that interact with MGG_07834………………..48
Table 4-1 Rice M/PAMPs and PRRs in PTI…………………………………………66
Table 5-1 OsPROPEPs and the putative mature peptides…………………………...87
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ACKNOWLEDGEMENTS
First and Foremost, I would like to offer my heartfelt gratitude to my advisor Dr.
Yinong Yang. Throughout all these years, without his lasting support, guidance and
patience, I would not have made it so far in my career. I would also like to thank the
members of my dissertation advisory committee: Dr. Teh-hui Kao, Dr. Tim McNellis
and Dr. Dawn Luthe for their helpful suggestions, questions and comments.
Thanks from the bottom of my heart also go to my former and current labmates: Qin
Wang, Dr. Chuansheng Mei, Dr. Xiangjun Zhou, Dr. Jai Rohila, Dr. Gang Ren, Dr.
Kabin Xie, Dr. Zhenyu Liu, Dr. Xiangling Shen, Jianping Chen, Emily Helliwell,
Yueying Chen, Bastian Minkenberg, and Xu Zhao for building a harmonious,
interdependent and even altruistic community from which I obtained valuable advice,
help and encouragement.
I would also like to thank Dr. Barbara Valent of Kansas State University for
collaboration on the rice sheath live-imaging analysis. I also thank Dr. Charles
Anderson for permitting me to use his confocal microscope for BiFC imaging. My
thanks also go to Dr. Chaowen Xiao for discussing techniques on picture and data
handling.
Last but not least, I am indebted to my parents, my wife and my son. Their constant
consideration, support and encouragement are the inexhaustible source of love and
strength that is always with me in my life.
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Chapter 1
Literature review
Plant invading organisms, living in a pathogenic relationship with the host plants,
often produce an array of proteins that can enter or are injected into host cells. These
proteins, predominately living up to the name “effectors”, can turn host biological
processes to the advantage of the invaders. In some cases, upon inside plant cells,
effectors are recognized by the host surveillance system and trigger defense responses.
In this context, effectors are given the name “avirulence” proteins (AVRs) that can
directly or indirectly interact with their cognate resistance proteins (Rs), central
components of the plant surveillance system. The plant defense response, initiated by
AVR-R interaction, is designated as effector-trigger immunity (ETI). Prior to that is
another layer of disease resistance called pathogen associated molecular pattern
(PAMP) triggered immunity (PTI). PTI and ETI are coming in two flavors with the
former being weaker and slower and the latter being much stronger and faster against
pathogen infections (Jones and Dangl, 2006). Successful pathogens deploy a whole
host of effectors that promote infection by targeting signaling components of PTI
and/or ETI pathways from the receptors in the cytosol or at the cell surface all the way
through to the chromatin and gene transcription machineries in the nucleus.
1.1 Plant pathogen effector delivery systems
Plant pathogens have adopted a variety of mechanisms to deliver their effectors
during the process of co-evolution with their hosts. Effectors from viruses are
expressed in plant cells following the whole organism’s cellular entry. Bacteria by
and large use type III and IVsecretion systems to deliver their effectors into plant cells.
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Oomycetes and fungi churn out effectors with N-terminal host targeting motif (HTM),
which may guide effectors into plant cells possibly via HTM-mediated cellular uptake.
Effectors from nematodes are injected into plant cells through the small orifice of the
piercing stylet.
1.1.1 Bacterial effector secretion/translocation systems
Figure 1-1 Bacterial T3SS, T4SS and T6SS translocate effectors into host cells
(adapted from Govers et al., 2008, Citovsky, et al., 2007 and Kapitein et al., 2012).
A. Bacterial T3SS; B. Agrobacterium tumefasciens T4SS; C. Bacterial T6SS.
Type III secretion system (T3SS), also called injectisome, is a nanomachine
specialized in protein secretion and/or translocation in Gram negative bacteria. It
operates not only across bacterial genera but also across host kingdoms, dictating
pathogenicity and virulence in a wide range of animal and plant pathogens such as
Salmonella, Yersinia, Shigella, Pseudomonas, Erwinia, Ralstonia, Xanthomonas and
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Rhizobia. T3SS, having a common evolutionary origin with the flagellum, consists of
up to 25 proteins, of which 9 are conserved across all the seven families found in
plant and animal bacteria (Cornelis, 2006). Plant bacterial pathogen, defective in
T3SS, often forfeits its ability to induce hypersensitive response (HR) in incompatible
plants and pathogenesis in compatible plants. As a result, T3SS is well known as
hypersensitive reaction and pathogenicity (Hrp) pilus in plant pathogens. The Hrp
pilus, projecting from the bacterial envelope all the way up to the plant cell wall, is
home to a hollow channel between the bacterial cytoplasm and the host cell plasma
membrane. A protein, secreted via this channel and dubbed translocon, then forms a
pore through the host plasma membrane that enables finishing up the last step of
assembling the effector delivery machine (Figure 1-1 A).
In comparison with other secretion systems, type IV secretion system (T4SS) is
unique in its ability to deliver nucleic acids in addition to proteins (Figure 1-1 B).
While Type IV functional similarities are present in such a wide range of bacteria as
Helicobacter pylori, Pseudomonas aeruginosa, Bordetella pertussis, Escherichia coli,
Legionella pneumophila and Mesorhizobium loti (Christie et al, 2005; Hubber et al,
2007), T4SS in Agrobacterium tumefaciens has made its way to the stardom in basic
plant research and modern plant biotechnology. Agrobacterium T4SS, also called T-
pilus, is made up of VirB2-VirB11 and VirD4. And VirB1 assists the assembly of T-
pilus by remodeling the peptidoglycan layer. Upon recognition of plant signals by the
bacterial VirA/VirG sensory system, T-pilus is assembled through which T-DNA in
cis with a covalently attached VirD2 and in trans with several other proteins such as
VirE2, VirE3, VirF and VirD5, is exported into the plant cell (Citovsky et al., 2007).
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Once inside the plant cell, the VirD2-T-strand binds to many molecules of VirE2 to
form a mature T-complex. By hijacking many host cell fundamental processes such as
nuclear importing, ubiquitin-26S proteasome system (UPS)-mediated proteolysis and
DNA transcription and repair machineries, VirD2, VirE2, VirF, VirE3 and VirD5
facilitate T-DNA integration and expression in the host genome (Magori et al., 2012).
Type VI secretion system (T6SS), recently discovered in human bacterial pathogens
Pseudomonas aeruginosa and Vibrio cholerae (Mougous et al., 2006; Pukatzki et al.,
2006) is widespread among Gram negative bacteria including plant pathogens and
symbionts such as A. tumafaciens, pseudomonas syringae., Xanthomonas oryzae., and
Rhizobacterium leguminosarum (Records, 2011). T6SS is an inverted phage-like
structure, through which effectors are injected into host cells to promote virulence or
interbacterial outcompetition (Figure 1-1C).
The mechanisms by which bacterial effectors are recognized and delivered by these
systems are poorly understood. However, some insights are emerging from the study
of T3SS effectors. In brief, effector N-terminal peptide, effector RNA and its cognate
chaperone are independently or coordinately involved in effector recognition and
delivery by T3SS. In Pseudomonas syringae, an unusual bias in N-terminal amino
acid composition has been identified and exploited successfully to identify new
effectors, emphasizing the N-terminal signal as a general prerequisite for T3SS
recognition and delivery (Vinatzer et al., 2005; Schechter et al., 2012). In some cases,
the N-terminal signal can be either encoded in the underlying mRNA or the
untranslated leader RNA (Anderson et al., 1999; Niemann et al., 2013). And in some
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other cases, the binding of a cognate chaperone is necessary for an effector to be
delivered by T3SS, but this does not seem to be a universal rule (Guo et al., 2005).
1.1.2 Oomycete and fungal effector delivery venues
Figure 1-2 Oomycete and fungal effector-exporting specialized structures (adapted
from Rafiqi, et al., 2012). (a). Haustorium; (b). Invasive hypha; (c). Arbuscule.
Many biotrophic or hemibiotrophic oomycetes and fungi develop a bulbous feeding
structure, i.e. haustorium or invasive hypha inside the plant cell by invaginating the
host plasma membrane. These specialized structures are bustling venues where
effectors are exported and nutrients are ingested.
Haustorium (Figure 1-2 A) is a globular structure formed from a terminal side branch
of a hypha and surrounded by an extra-haustorial membrane (EHM), which is
continuous with the plasma membrane of the plant cell. In between the pathogen cell
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wall and host plasma membrane rests the biotrophic interface (BI), a matrix of
secreted effectors from the pathogen and nutrients from the host cell. Although
originated from plant plasma membrane, EHM differs in composition from the
remaining plant plasma membrane, suggesting a selection driven by the plant
pathogen (Lu et al., 2012). And this selection for EHM composition may contribute to
effector uptake by the host cell (Bozkurt et al., 2012).
Invasive hypha (Figure 1-2 B) is a bulbous structure and, like haustorium, is also
enveloped by host plasma membrane-derived extra invasive hyphal membrane
(EIHM). Effectors are often observed to accumulate and latter translocate at the
biotrophic interface. In Magnaporthe oryzae, first formed on the tip of the first host-
invading hyphal cell and latter left beside the newly developed bulbous invasive
hypha is a 1-2 µm-diameter dome-shaped biotrophic interfacial complex (BIC), which
is full of effectors waiting for translocation (Kankanala et al., 2007; Mosquera et al.,
2009; Khang, C. H. et al., 2010). Similarly, interfacial bodies, structures of ~ 500 nm
in diameter are also observed at the biotrophic interface as depositories for
Colletotrichum higginsianum effectors (Kleemann et al., 2012).
Arbuscue (Figure 1-2 C) is a highly branched structure in vascular plant root cells and
developed from a hypha of a mutualistic arbuscular mycorrhizal fungus (AMF) during
symbiosis. Like haustorium and invasive hypha, arbuscue is also enclosed by plant
plasma membrane-derived periarbuscular membrane (PAM). In addition to serving as
a nutrient exchange venue, arbuscule is also expected to secret fungal effectors into
plant cells (Kloppholz et al., 2011; Plett et al., 2011).
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Fungal and oomycete effectors are secreted via the classical endoplasmic reticulum
(ER) pathway, i.e. from ER to Golgi apparatus to vesicles to biotrophic interface
where they re-enter plant cell cytosol. The mechanisms by which effectors enter plant
cells are intensively pursued and yet not known. Based on the study of oomycete
RXLR effectors, Kale et al. (2010) proposed a model in which oomycete RXLR motif
or its highly degenerate fungal homologs, binds to phosphatidylinositol-3-phosphate
(PI3P) at the plant cell plasma membrane outer surface and internalizes into plant cell
cytosol via endocytosis. This model, however, is not widely accepted and often
challenged by the oomycete and fungal pathogen communities. In flax rust fungus
Melampsora lini, it is not required for the host targeting motifs of AvrM and AvrL567
to bind phospholipids to guide effectors’ entry into plant cells in the absence of the
pathogen (Gan et al., 2010). In oomycete Phytophthora infestnas, phospholipid
binding of Avr3a is not through its N-terminal RXLR but through its C-terminus, and
this binding is not for effector entry but for its stability and function inside plant cells
(Yaeno et al., 2011). It appears that there exist multiple mechanisms of effector entry
and the search for plant-side receptors continues for this class of effectors. Recently,
in oomycete fish pathogen Saprolegnia parasitica, it was discovered that effector
SpHtp1 enters fish cells via binding of tyrosine-O-sulphate on the host cell surface
(Wawra et al., 2012). This is another twist en route to reveal the mystic and elusive
effector entry mechanisms.
1.2 The subcellular locations of plant pathogen effectors
Once inside the plant cell, effectors can reside in almost all the subcellular
compartments: cytosol, nucleus, chloroplast and mitochondrion (Figure 1-3). In some
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cases the evidence for cytoplasmic localization is directly supported by experiments
showing physical interactions between effectors and R proteins or other proteins in
the plant cytoplasm. Examples include M. oryzae AvrPita. AvrPita was demonstrated
to directly interact with its cognate R protein, Pita in rice cell cytoplasm by transient
gene expression and immunoprecipitation (Jia et al., 2000). In other cases, effectors
have been identified histochemically by antibody staining or via a fluorescent tag.
These include the bacterial effectors AvrPtoB (de Vries et al., 2006) and HopU (Fu et
al., 2007); and the oomycete effectors Avr1b (Dou, 2008) and Avr3a (Armstrong, et
al., 2005). In Xanthomonas spp., a large family of effectors, called transcription
activator-like effectors (TALEs), was identified to function in the host nucleus (Kay
et al., 2007; Yang et al., 2006). This family of effectors contains a central repeat
region for DNA binding, a C-terminal nuclear localization signal and an acidic
activation domain, showing their nuclear localization as plant transcription activators.
Four putative effectors, Nuk6, Nuk7, Nuk10 and Nuk12, from the oomycete P.
infestans were also predicted to be nucleus localized (Kanneganti et al., 2007). SP7
and MiSSP7, two effectors from arbuscular fungi, were found to be translocated into
the plant root cell nucleus (Kloppholz et al., 2011; Plett et al., 2011). The P. syringae
effector HopI1, when expressed in the plant cell, is targeted to the chloroplast where it
remodels the chloroplast and suppresses defenses (Jelenska et al., 2007).
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Figure1-3 Subcellular locations and virulence targets in host cells of oomycete and
fungal effectors. Names of bacterial effectors are in red and names of fungal effectors
in purple.
1.3 The host targets of plant pathogen effectors
The all-over-the-map subcellular locations of plant pathogen effectors suggest that
they perform a variety of roles in promoting infection. In the face of a pathogen, plant
usually mounts two layers of defense responses: i.e. PTI and ETI. Although different
in timing and amplitude, the signaling pathways underlying PTI and ETI to a large
extent are the same, involving recognition of effectors by receptors, activation of
mitogen-activated protein kinases (MAPKs), crosstalk of stress hormones and
Bacteria
JASA
ETABA
Auxin
GA
AvrPtoAvrPtoB
FLS2/EFR/CERK1/
XA21 and BAK1
HopAI1
HopI1
GA, coronatine, auxin, cytokinin
TALEs
XopD/SP7
Fungi
Oomycetes
Nucleus
Chloroplast
Cmu1
TF XH
X
OspF/HopAI1?/NUE?
XHIRA
6b?/ZFs
BIK1 AvrACAvrPphB
Ecp6
Slp1
MAP3KMAP2KMAPK
FEN
HopF2AvrPtoB
10
expression of defense genes. All these fundamental cellular processes are natural
targets of plant pathogen effectors (Figure 1-3).
1.3.1 PRRs and Rs
Pattern recognition receptors (PRRs) are usually receptor-like kinases (RLKs) or
receptor-like proteins (RLPs) in the plant plasma membrane, which recognize PAMPs
and initiate PTI. Rs are largely nucleotide binding-leucine rich repeat proteins (NB-
LRRs) that recognize corresponding Avr proteins and elicit ETI. Therefore, both
PRRs and Rs are the convenient targets for pathogen effectors. Indeed, in Arabidopsis,
tomato and other higher plants, FLS2 (flagellin sensitive 2) is the PRR for flg22 (a
conserved 22 amino-acid N-terminal stretch of flagellin) and the target of P. syringae
effectors AvrPto and AvrPtoB. While AvrPto disrupts PTI by inhibiting the kinase
activity of FLS2 or interfering with the formation of FLS2-BAK1 complexes (Xiang
et al., 2008; Shan et al., 2008), AvrPtoB blocks PTI by eliminating FLS2 via its C-
terminal E3 ligase activity (Gohre et al., 2008). Recently, AvrPtoB has also been
demonstrated to inhibit chitin triggered PTI by marking LysM chitin receptor CERK1
for degradation (Gimenez-Ibanez, et al., 2009). Although its major function is to
target PRRs for degradation, AvrPtoB can overcome ETI by targeting a protein kinase
Fen, an ancient homolog of R protein Pto and a part of a unique ETI pathway
(Rosebrock et al., 2007). Tomato leaf mold fungus Cladosporum fulvum secretes a
lysin motif (LysM) domain-containing effector Ecp6 to compete with PRR receptor
for binding chitin oligosaccharides to avoid eliciting PTI (De Jonge et al., 2010).
Similarly, in M. oryzae, LysM effector Slp1 plays the same role by sequestering chitin
11
oligosaccharides from rice PRR receptors CEBiP and OsCERK1, short-circuiting PTI
(Mentlak et al., 2012).
1.3.2 The MAP3K-MAP2K-MAPK cascade
The perception of PAMPs or Avr proteins by PRRs or Rs is usually followed by the
activation of MAP3K-MAP2K-MAPK signaling transduction cascades. Therefore,
terminating or intercepting MAPK signaling is natural strategy for pathogen effectors
to promote pathogenicity. HopAI1, present in many P. syringae strains, is a
phosphothreonine lyase that irreversibly dephosphorylates MAPK3 and MAPK6 to
terminate PRR signaling (Zhang et al., 2007). HopPtoD2, another effector from P.
syringae, has an in vitro protein tyrosine phosphatase activity and is likely to promote
pathogenicity through inactivation of MAPK signaling (Espinosa et al., 2003). Also in
Peudomonas syringae, HopF2 uses ADP ribosyl-transferase activity to inhibit MKK5
and other MAPKKs, eluding PTI (Wang et al., 2010).
1.3.3 The crosstalk between phytohormones
The crosstalk between plant hormones auxins, salicylic acid (SA), jasmonates (JA),
ethylene (ET), abscisic acid (ABA) and gibberellin (GA) delicately fine-tunes plant
defense reponses in PTI and ETI. In Arabidopsis, the basic crosstalk between SA and
JA/ET is beginning to emerge: SA-dependent resistance is summoned against
biotrophic and hemibiotrophic pathogens, while JA/ET-dependent resistance is
mounted against necrotrophic pathogens and herbivorous insects. The crosstalk
reflects coherent trad-offs between hormone signaling pathways. Pathogen effectors
12
can exploit these trade-offs to their benefits. The P.syringae effector HopI1 resides in
the chloroplast, where it remodels the thylakoid structure and suppresses SA
accumulation. Reduced levels of SA cannot effectively activate SA-dependent
pathway against hemibiotrophic P. syringae (Jelenska et al., 2007). Again, P.
syringae avrPtoB transcriptionally suppresses MIR393, which shuts off auxin
responsive pathway. By going through this delicate, multi-step process, avrPtoB
ultimately activates auxin responsive pathway to inhibit SA-dependent pathway
(Navarro et al, 2006; 2008). In addition to deploying effectors, pathogens also directly
interfere with the crosstalk between hormone signaling pathways by producing
hormones or hormone mimics. A. tumefaciens, the causal agent of crown gall, can
genetically transform its host plant by transferring a segment of the tumor-inducing
(Ti) plasmid into the host’s genome. The transferred T-DNA carries genes that code
for the production of auxins and cytokinins, resulting in uncontrolled host cell
proliferation from which the pathogen can benefit. Necrotrophic fungus Gibberella
fujikuroi, the eponym of GA, was demonstrated to produce significant amount of GA,
possibly to disable JA-dependent necrotroph resistance through GA-mediated
destabilization of DELLA proteins that play a role in controlling the crosstalk
between SA-and JA-dependent defenses (Navarro et al., 2008). P. syringae produces
the phytotoxin coronatine, a JA mimic and suppresses SA-dependent defenses,
thereby promoting susceptibility of the plant to this pathogen (Brooks et al., 2005).
Coronatine also plays a key role in re-opening stomata to facilitate pathogen
penetration after ABA-mediated stomatal closure by antagonizing the SA/ABA
signaling pathways (Melotto et al., 2006). Heterodera glycine, the causal agent of
soybean cyst root-knot, injects into the plant cell an effector HG-SYV46, which
functions as the plant peptide hormone CLAVATA3/ESR (CLE) to alter plant
13
morphology in favor of the pathogen. Effector Cmu1 from maize smut fungus
Ustilago maydis is a functional chorismate mutase, which diverts chorismate
metabolism away from SA production and thereby evade plant defense (Djamei et al.,
2011).
1.3.4 The chromatin and transcription machinery
Both PTI and ETI require extensive transcriptional reprogramming by recruiting
chromatin modulins and transcription machineries. The nucleus, at the heart of the
plant cell, is a major target for pathogen effectors. In Xanthomonas and Ralstonia,
there is a large family of transcription activator-like effectors (TALEs). They bear the
same characteristic structural features: the N-terminal secretion signal for delivery by
the bacterial T3SS; a central repeats of 34 or 35 amino acids for DNA binding, and
the C-terminal region with a nuclear localization signal and an acidic activation
domain. Several members of this TALE family have been demonstrated to function as
transcription factors or by recruiting basal transcription machinery to promote disease.
AvrBs3, the founding member of TALEs from X. campestris pv. vesicatoria (Xcv)
activates the expression of more than 11 genes, causing enlargement of mesophyll
cells in the leaf interior to facilitate pathogen dispersal (Kay, et al., 2007). PthXo1, a
major virulence effector and a TALE from X. oryzae pv. oryzae (Xoo), induces the
expression of a susceptibility gene Os8N3 (also called xa13, OsSWEET11) by binding
to its promoter (Yang et al., 2006). Os8N3, renamed OsSWEET11, encodes a plasma
membrane-bound sugar transporter that exports into the apoplast sucrose which the
pathogen exploits (Chen et al., 2010; 2012). Avrxa5, another TALE from Xoo, acts as
a transcription factor and recruits the γ subunit of the general transcription factor
14
TFIIA for gene transcription to promote pathogenicity (Jiang et al, 2006). XopD, an
effector from Xcv, also has a modular structure: an N-terminal secretion signal for
delivery by the bacterial T3SS, followed by a helix-loop-helix domain for DNA
binding and two ERF (ethylene response factor)-associated amphiphilic repression
(EAR) motifs and a C-terminal small ubiquitin-like modifier (SUMO) protease. XopD
is translocated into the nucleus and regulates gene expression by interacting with
transcription factors. By binding the transcription factor MYB30 and desumoylating
the transcription factor SIERF4, XopD suppresses defense responses in Arabidopsis
and tomato, respectively (Canonne et al., 2011; Kim et al., 2013). SP7, an effector
from arbuscular mycorrhizal fungus Glomus intraradices interacts with the
transcription factor ERF19 in the nucleus to evade plant defense and promote
symbiosis (Kloppholz et al., 2011). Pathogen effectors also regulate host gene
expression to their own benefits by acting as chromatin modulins. Shigella flexneri
OspF has a phosphor-Thr lyase (eliminylase) activity that targets nuclear MAPKs. In
addition, it alters chromatin structure at specific genes by inducing histone
dephosphorylation and deacetylation (Arbibe et al., 2007). It is tempting to propose
that HopAI1, a Pseudomonas syringae effector similar to OspF (Li et al., 2007), also
can affect histone post-translational modifications and limit chromatin access for
specific gene transcription. In Agrobacterium, 6b protein, encoded by T-DNA, is an
effector targeted to the plant cell nucleus where it binds to histone H3 and other
nuclear proteins and probably functions as a histone chaperone to regulate gene
expression (Terakura et al., 2007).
In this study, we try to characterize a family of zinc finger effectors in Magnaporthe
oryzae secretome and answer the following questions. Are zinc finger effectors
translocated into rice cells during fungal infection? What is the mechanism by which
15
zinc finger effectors enter rice cells? What are the targets of zinc finger effectors
inside rice cells? How to understand the role of zinc finger effectors in disease
promotion along the line of suppression of PTI by pathogen effectors?
16
Chapter 2
A family of Magnaporthe oryzae zinc finger effectors are secreted and
translocated inside plant cells
2.1 Introduction
M. oryzae is a hemibiotrophic ascomycete fungus, the causal agent of rice blast
disease. M. oryzae can infect about 50 grasses including some agro-economically
important crops such as rice, wheat, barley and millet (Couch et al., 2005). Each year
10~30% of rice yield worldwide is usually lost to this fungal disease (Zeigler et al.,
1994). In the field, M. oryzae infection cycle is as follows: A conidium from a
conidiophore in the lesion spot falls onto a nearby plant site, where it attaches firmly
and germinates within a few hours. By 12 hours, at the tip of the germination tube,
appressorium forms and within 24 hours appressorium forces a penetration peg into
the underlying cell. From 24 hours to 36 hours, a thin primary filamentous hypha first
appears and subsequently new bulbous invasive hyphae (IH) quickly branch out to fill
the whole cell. From 36 hours on, the IH begin to invade the neighboring cells. Later
at the infection site plant cells die and the fungus enters the necrotrophic phase. About
5~7 days disease lesions appear, from which, conidia are born and the cycle start
anew (Figure 2-1). Great efforts have been focused on the initial infection steps such
as how appressorium forms and how penetration peg forces into plant cells and some
aspects of these initial steps are well understood (Wilson et al., 2009). However, little
is known about how invasive hyphae in the biotrophic phase slyly propagate in living
rice cells under the nose of the plant surveillance systems such as PTI and how
17
transition occurs to a pathogen from a biotrophic life style to a necrotrophic one.
These are fundamental questions and effectors are the keys to the mysteries.
Figure 2-1 M. oryzae hemibiotrophic life cycle
(adapted from Dean et al., 2011)
M. oryzae strain 70-15 (a derivative of the field strain Guy11) has been sequenced and
the genome sequence is available to the public (Dean et al., 2005). Of the predicted
proteome, 739 (7% of 11109), 1360 (12% of 11109), 1546 (12% of 12481), or 2470
(22% of 11069) proteins are independently predicted to comprise the fungus’
secretome based on different signal peptide prediction and false positive filter
softwares (Dean et al., 2005; Yoshida et al., 2009; Soanes et al., 2008; Choi et al.,
2010). M. oryzae secretome is a rich source for effector identification.
0 hr
2~4 hr
12 hr
24 hr
36 hr
5~7 d
18
Table 2-1 Effectors cloned as AVR proteins in M. oryzae
Name Size
(aa)
Molecular
feature
Location Avrulence
function
Virulence
function
ACE1 4035 Secondary
metabolite
enzyme
Fungal
cytoplasm
Pi33 (ETI) None
PWL2 145 None Host
cytoplam
NA (ETI) NA
AVR-CO39 89 None Host
cytoplasm
PiCO39
(ETI)
NA
AVR-Pita1 224 Zinc-
metalloprotease
Host
cytoplasm
Pita (ETI) NA
AVR-Piz-t 108 LXAR Host
cytoplasm
Piz-t (ETI) Destabilize
E3 ubiquitin
ligase and
inhibit PTI
AVR-Pii 70 LXAR;
L/IPXP;
Incomplete
C2H2
Host
cytoplasm
Pii (ETI) NA
AVR-Pia 85 None Host
cytoplasm
Pia (ETI) NA
AVR-
Pik/km/kp
113 None Host
cytoplasm
Pikm (ETI) NA
Due to the large pool of effectors and gene redundancy in M. oryzae, direct gene
knock-out mutations usually give few clues to effectors’ functions. To date, only a
few effectors were cloned by exploiting their functions as AVR proteins in the rice- M.
oryzae pathosystem (Table 2-1). ACE1 triggers hypersensitive response (i.e. ETI) in
rice cultivars containing the resistance gene Pi33. It encodes a hybrid polyketide
synthase-nonribosomal peptide synthase, a putative secondary metabolite biosynthetic
enzyme (BÖhnert et al., 2004; Collemare J., et al., 2008). PWL2, a cysteine rich 145aa
protein, belongs to a small family with different number of members in different M.
oryzae strains (Kang et al., 1995). During fungal biotrophic growth, it is expressed
and secreted into biotrophic interfacial complex (BIC) and later enters cytoplasm and
moves from the first infected cell to the neighboring cells in advance of invasive
hyphae (Khang et al., 2010). AVR-CO39 gene is cloned from weeping lovegrass-
infecting M. oryzae strain and encodes an 89 aa protein that can be recognized in rice
19
cultivars containing PiCO39 resistance gene (Farman et al., 1998). AVR-CO39 can be
translocated into rice cell cytosol in the absence of M. oryzae (Ribot et al., 2012).
AVR-Pita1 encodes a 224 aa protein with similarity to zinc metalloprotease (Orbach
et al., 2000). Besides its signal peptide, it contains a propeptide that must be cleaved
for its activity to produce a mature 176 aa protein. AVR-Pita176 was demonstrated to
interact directly with its cognate resistance protein Pita in vivo (Jia et al., 2000).
AVR-Pita is accumulated in BIC and translocated into plant cytoplasm during fungal
invasive growth (Khang et al., 2010).
AVR-Piz-t is a 108 aa protein with no similarity to any known proteins and
recognized by the cognate R protein Piz-t to fulfill its avirulence function (Li et al.,
2009). AVR-Piz-t was recently also assigned a virulence function of suppressing
chitin elicited PTI by interacting with and destabilizing the E3 ubiquitin ligase APIP6
(Park et al., 2012). AVR-Pii, AVR-Pia and AVR-Pik/km/kp were cloned by re-
sequencing of a Japanese rice blast isolate Ina 168 and the ensuing association genetic
analysis (Yoshida et al., 2009). Of note, AVR-Pia was also independently isolated
using map-based cloning (Miki et al., 2009). AVR-Pii is 70 aa protein with a LXAR
motif and an incomplete monodactyl C2H2 zinc-finger. AVR-Pia and AVR-Pik/km/kp
are typical fungal effectors (a.k.a novel proteins) with no similarity to any known
proteins. All three proteins are tanslocated into rice cells during infection.
20
Table 2-2 Effector accumulation and movement pattern
Name BIC Location movement
AVR-Pita1 Yes cytoplasm NA
PWL2 Yes cytoplasm Move ahead of IH
BAS1 Yes cytoplasm Move ahead of IH
BAS2 Yes plasmodesmata NA
BAS3 Yes Plasmodesmata NA
BAS4 No Lining IH NA
An alternative way to isolate putative M. oryzae effectors was created in Dr. Barbara
Valent’s lab at Kansas State University (Mosquera et al., 2009). By using hand-
trimmed sheath tissue containing first invaded cells as RNA materials, Mosquera and
collegues obtained RNA with ~20% RNA from invasive hyphae. M. oryzae whole
genome microarray hybridization showed that 59 genes encoding putative secreted
proteins are upregulated by at least 10-fold, and of the 59 genes one is a known
effector gene PWL2. They named the induced and secreted proteins during fungal
biotrophic growth BAS proteins (biotrophy-associated, secreted proteins). They
further characterized four BAS proteins, BAS1-4, and some known efectors such as
AVR-Pita1 and PWL2 by carefully observing their accumulation at the biotrophic
interface and cell-to-cell movement from first invaded cell to neighboring cells. The
accumulation and movement patterns of these effectors fall into four classes (Table 2-
2). First, effectors like AVR-Pita1 accumulate in BIC and translocate into plant
cytoplasm. Second, effectors like BAS2 and BAS3 accumulate in BIC and later in
invasive hyphal host cell wall crossing points (likely plasmodestama). Third, effectors
like PWL2 and BAS1 accumulate in BIC and later move to neighboring cells ahead of
21
invasive hyphae. And Forth, effectors like BAS4 do not accumulate in BIC but
outline the growing invasive hyphae. It is tempting to ascribe some virulence
functions to these different classes of effectors. Effectors that outline invasive hyphae
are fungal cell wall protectors, say, like C. fulvum Avr4 which bind fungal cell wall
component chitin to prevent degradation from host chitinases. Effectors accumulate at
plasmodesmata are fungal movement proteins that like virus movement proteins
modify plamodesmata to allow hyphal crossing. Effectors that move ahead of invasive
hyphae are heralds that prime neighboring cells for imminent fungal invasion. All this
is hypothetic and the fact will be revealed with the finding of the virulence targets of
these effectors.
In this study, a family of one finger zinc finger proteins was identified as putative
effectors. Zinc finger effectors accumulate first in BIC and then translocate into rice
cells. Once inside rice cells, zinc finger effectors can move to neighboring cells. And
the translocation of zinc finger effectors may depend on two putative host targeting
motifs L/IPXP and L/IXAR.
2.2 Results
Identification and cloning of a family of putative M. oryzae one-finger zinc finger
proteins
In the predicted proteome of M. oryzae strain 70-15, there is a family of putative
effector proteins with seven paralogs: MGG_07834, MGG_09035, MGG_08230,
MGG_10556, MGG_12038, MGG_13665 and MGG_10020 (Figure 2-2 A). They are
modular proteins with an N-terminal signal peptide (SP), an immediate region of two
22
conserved motifs L/IPXP and L/IXAR and a C-terminal one-finger C2H2 motif. It
should be noted that MGG_15926 with similar motifs except for two C2H2 fingers is
not included in this family. AVR-Pii, an ortholog from a Japanese strain Ina168, was
demonstrated to perform avirulence function inside rice cells, suggesting that this
protein is translocated into rice cells during fungal infection (Yoshida et al., 2009).
This host targeting property might be extended to its orthologs in M.oryzae isolate 70-
15. Another indication that this family of zinc finger proteins might be functional
effectors is that MGG_07834 and MGG_09035 were upregulated 74 and 35 folds,
respectively, in invasive hyphae of the first invaded rice cells in comparison to axenic
mycelium grown in vitro (Mosquera et al., 2009). We cloned this family of putative
zinc finger effector genes for further study. Sequence alignment and phylogenetic
analysis (Figure 2-2 B) revealed that MGG_10238, the closet member to AVR-Pii,
also has an incomplete zinc finger motif, suggesting zinc-finger motif may play a role
in deciding effector’s virulence and avirulence functions.
A
AVR-Pii MQLSKITFAIALYAIGIAALPTPASLNGNTEVATISDVKLEAR
MGG_07834 MQLHNVCSILALLAAGVFALPAPVNPSEIQAR
MGG_09035 MQIFNIVQVLGLLAVGASALPTPANVGAVQPVEGSQLQAR
MGG_08230 MQIFKIVQVLGLLAVGTSALPAPANPAAVQPAQGGQLAVHGQPASCPPECRDVQGAKPGHLQAR
MGG_10556 MQFSKITLAIVLYALGTAALPTASRCAGAPGRDVAATRGAKLQAR
MGG_12038 MHFFKTVQIVTMFAVGIMALPTPGYTPTNQNTGDIVAR
MGG_13665 MQIFKIVQVLGLLAVGTSALPTPAGVDVDGGQLQAR
MGG_10020 MHAFKFVQIWALLAIGAAAIPTPINLPAALTSQLEPR
AVR-Pii SDTTY
MGG_07834 SAADKAKPKPQTERVWMDEPEAAQYLSDPRWKLVKKK
MGG_09035 SSNFYSAGWTQYPSANSGYPSNSASTYY
MGG_08230 SRFYATSDTGRPLANSGYPTHGYRDA
MGG_10556 EEDKPTPQ
MGG_12038 SDTVKGKDVATSTDANKKKRTKDYY
MGG_13665 AEGDKVRPPS
MGG_10020 AEGGASAGDANNIQPQSQEKSP
AVR-Pii HKCSKCGYGSDDSDAYFNHKCN
MGG_07834 YSCGYCDSSSSKVDKINKHRDEVHGTRQAQVDHTIYPGETRLTFERVSGYP
MGG_09035 YKCNHCGKHTKEESQQKYHQENSHKGKESNYSEVQA
MGG_08230 YRCLYCGAVRDDVSAVQDHITYRHSNRGGDTSNYDTTTVRDDR
MGG_10556 YRCDKCEKEFVKGNDFFNHGGRGHCKMSGY
MGG_12038 WRCANCQAKFSDYESFNKHGNECKSV
MGG_13665 IKCLTCEQGGFPNVDEYEQHYKQKHPEIVQQVSSPNPHLNKGKKAN
MGG_10020 FICVWCGKDKGGSSALIGHEIHNHQDDEMVAWTGRDRRLNKYETDKEGYRYFKKYPGVRLPNRTWKGKGKRPE
23
B
Figure 2-2 A family of M. oryzae zinc-finger effectors. A. Alignment of protein
sequences of 8 members of zinc-finger effectors. B. phylogenetic tree of zinc-finger
effectors.
Zinc finger effectors accumulate in BIC and translocate into rice cells during
fungal infection
The biotrophic interfacial complex (BIC) is initially developed at the tip of the first
host-invading hyphal cell and later left on the side of the newly developed bulbous
invasive hypha. BIC is the depository for most, if not all, of fungal effectors that
24
function inside plant cells. Under the constitutive promoter P27 of M. oryzae
ribosomal protein 27, MGG_07834 : EGFP and MGG_10020 : EGFP were observed
to accumulate in BIC 27 hours and 30 hours post inoculation, respectively (Figure 2-
3).
To further study the translocation and movement of effectors inside rice cells during
fungal infection, we made new constructs PPWL2 : MGG_07834 : mCherry : NLS: Ter :
PBAS4 : BAS4 : EGFP and PPWL2 : MGG_10020 : mCherry : NLS: Ter : PBAS4 : BAS4 :
EGFP. We exploited the promoter of a known effector gene PWL2 instead of their
native promoters to drive the expression of fusion proteins MGG_07834 : mCherry:
NLS and MGG_10020 : mCherry : NLS. Three tandem repeats of the nuclear
localization signal (NLS) of the simian virus large T-antigen were added to enrich the
fusion proteins in the nucleus in order to observe more clearly their translocation and
movement inside rice cells. BAS4 : EGFP, an effector fusion protein outlining
invasive hyphae, was used to distinguish fungal growth inside rice cells.
MGG_07834 : mCherry : NLS and MGG_10020 : mCherry : NLS were translocated
into the nuclei of rice cells when observed 28 hours and 33 hours post inoculation,
respectively (Figure 2-4). Furthermore, traces of MGG_07834 : mCherry : NLS and
NGG_10020 : mCherry : NLS began to appear in the nuclei of neighboring cells of
the first invaded cell in advance of the invasive hyphae (Figure 2-4), suggesting this
family of zinc finger effectors may play a role in priming the rice cells for imminent
fungal invasion.
25
Figure 2-3 Zinc finger effectors accumulate in BIC. A. MGG_07834 : EGFP
accumulates in BIC in rice sheath epidermal cell unplasmolyzed (first row) or
plasmolyzed (second row). B. MGG_10020 : EGFP accumulates in BIC in rice sheath
epidermal cell unplasmolyzed (first row) or plasmolyzed (second row). Arrows point
to BIC.
A
B
26
Figure 2-4. Zinc-finger effectors translocate inside rice cells. A. MGG_07834 :
mCherry : NLS translocates into the nuclei of rice sheath epidermal cells
unplasmolyzed (first row) or plasmolyzed (second row). B. MGG_10020 : mCherry :
NLS translocates into the nuclei of rice sheath epidermal cells unplasmolyzed (first
row) or plasmolyzed (second row). Arrows point to the nuclei of the uninvaded
neighboring cell.
A
B
27
Motifs L/IPXP and/or L/IXAR may play a role in translocation of effectors from
fungus to plant cells
Finding host-targeting motifs is one of the major goals in pathogen effector biology
study. In the proteomes of oomycete pathogens including Phytophthora,
Hyaloperonospora, Pythium and Albugo, hard on the heel of the signal peptide, comes
a certain conserved motif such as RXLR, LXLFLAK, or CHXC. These motifs are
involved in translocation across the host cell plasma membrane and proteins
containing one of these motifs are effectors functioning inside plant cells (Rehmany et
al., 2005; Haas et al., 2009; Kemen et al., 2011). In fungal pathogens, to date, host-
targeting motifs were only identified in four effectors: ToxA from Pyrenophora
tritici-repentis, MiSSP7 from Laccaria bicolor and AvrL567 and AvrM from
Melampsora lini (Manning et al., 2008; Plett et al., 2011, Rafiqi et al., 2010). Unlike
oomycete RXLR, LXLFLAK and CHXC motifs, the motifs identified in these four
fungal effectors are not widespread in the pathogen’s proteome. Recently, in barley
powdery mildew Blumeria graminis f. sp. hordei, the tripeptide Y/F/WXC four-amino
acid away from the signal peptide cleavage site were found in many secreted proteins,
but experiments demonstrating its involvement in host translocation is still lacking
(Godfrey et al., 2010).
We set out to find some possible host targeting motifs in this family of zinc finger
effectors. There are two conserved motifs following the signal peptide but preceding
the zinc finger domain: L/IPXP and L/IXAR. We searched the Broad Institute fungal
database and found L/IPXP exist in another 52 proteins in M. oryzae’s secretome and
at the same position as in zinc finger effectors, i.e. immediately after the signal
peptide cleavage site (Figure 2-1 A). In addition, L/IPXP is found in the secretomes of
28
a wide range of fungal pathogens such as Melampsora larici, Botryotinia fuckeliana
and Puccinia graminis f. sp. tritici. The second motif L/IXAR is also widely present
in dozens of M. oryzae secreted proteins including a known effector AVR-Piz-t. The
relatively ubiquitous presence of L/IPXP and/or L/IXAR suggests a conserved
function possibly in secretion and/or translocation.
We demonstrated that this family of proteins can bind to various phospholipids with
different affinity by using protein-lipid overlay assay (Figure 2-5). GST-MGG_07834
can bind strongly with Phosphatidylinositol-4-phosphate (PI4P), PI3P, PI5P and
phosphatidylinositol-3,5-bisphosphate (PI(3,5)P), while GST-MGG_09035 can bind
to all –phosphates, -bisphosphates, -triphospate and phosphatidylserine (PS).
Phospholipids are major components of cell membranes and involved in protein
cross-membrane trafficking. From this result, we can infer that zinc finger effectors
may interact with plant cell membrane components to enter plant cells.
When expressed in rice protoplasts, MGG_07834 : EGFP and MGG_10020 : EGFP
accumulated largely in the nucleus, although we cannot rule out the effect of EGFP
because EGFP is all over the map including the nucleus and cytoplasm (Figure 2-6).
But these results clearly revealed two interconnected events: first, zinc finger effectors
are secreted in rice cells via the ER secretary pathway, and second, zinc finger
effectors can re-enter rice cells via host targeting motifs. In other words, zinc finger
effectors, like most oomycete and fungal effectors including one known M. oryzae
effector AVR-CO39 (Ribot et al., 2012), may enter plant cells in the absence of the
pathogen.
29
Knowing zinc finger effectors possibly translocate into plant cells in the absence of
the pathogen, we started to test the two most conserved motifs L/IPXP and LXAR for
potential host-targeting activity. We chose BAX as a macroscopic indicator for fusion
proteins being in or out of pant cells based on the assumption that BAX induces cell
death inside but not outside plant cells. Using pMDC32 as a backbone vector, five
constructs, MGG_07834SP : BAX, MGG_07834SP-LPAPVN : BAX, MGG_07834SP-
AAAAVN : BAX , MGG_07834SP-LPAP-IQARSA : BAX and BAX were made. They
were introduced by agroinfiltration and expressed in Nicotiana benthamiana. In
comparison with BAX, MGG_07834SP : BAX and MGG_07834SP- AAAAVN :
BAX no longer induced cell death, whereas MGG_07834SP- LPAPVN : BAX and
MGG_07834SP-LPAP-IQARSA : BAX did (Figure 2-7). The plausible explanation is
that due to a lack of or a mutation of the LPAP motif, MGG_07834SP : BAX and
MGG_07834SP- AAAAVN : BAX were not capable of re-entering into plant cells
when secreted via the plant ER secretary pathway. By observing the localizations of
EGFP fusion proteins, we tested L/IPXP for host-targeting function again. Constructs
EGFP, MGG_10020SP : EGFP, MGG_10020SP : IPTP : EGFP, and
MGG_10020SP : AAAA : EGFP were recombined into vector pMDC32 and
introduced biolistically into onion epidermal cells. Again, different localization
patterns were observed between IPTP and its alanine substitution mutant AAAA
EGFP fusion protein (Figure 2-8). Finally, we tested both L/IPXP and L/IXAR motifs
for host-targeting activity using wild-type full-length effector or mutant EGFP fusion
proteins. Six constructs EGFP (control), MGG_07834 SP : LPAP : IQAR : ZF :
EGFP (Wild type), MGG_07834 SP : AAAA : IQAR : ZF : EGFP (LPAP single
mutant), MGG_07834 SP : LPAP : AAAA : ZF : EGFP (IQAR single mutant),
MGG_07834 : AAAA : AAAA : ZF : EGFP (LPAP and IQAR double mutant) and ZF :
30
EGFP (deletion of SP, LPAP and IQAR) were recombined into pMDC32 and
transiently expressed in N. benthamiana. In comparison to wild type that is localized
largely in the nucleus and deletion mutant that is in the cytoplasm, single and double
mutants appeared at a different location, probably outside of the cells (Figure 2-9).
This indicates that both L/IPXP and L/IXAR motifs may play a role in effector
translocation into plant cells.
Figure 2-5 Protein-lipid overlay assay. From left, Spot positions of phospholipids on
PIP strips; GST control; GST-MGG_07834 and GST-MGG_09035.
Figure 2-6 Localizations of MGG_07834 : EGFP and MGG_10020 : EGFP when
expressed in rice protoplasts
PIP strips GST GST-MGG_07834 GST-MGG_09035
EGFP MGG_07834-EGFP MGG_10020-EGFP
31
Figure 2-7 The role of L/IPXP in BAX fusion protein translocation
1. MGG_07834SP : BAX
2. MGG_07834SP-LPAPVN : BAX
3. MGG_07834SP-AAAAVN : BAX
4. MGG_07834SP-LPAP-IQARSA : BAX
5. BAX
1
2
3
4
5
1
2
3
4
5
32
Figure 2-8 The role of L/IPXP in EGFP fusion protein translocation.
A. EGFP
B. MGG_10020 SP : EGFP
C. MGG_10020 SP : IPTP : EGFP
D. MGG_10020 SP : AAAA : EGFP
33
Figure 2-9 The role of L/IPXP and L/IXAR in MGG_07834 or its mutant EGFP
fusion protein translocation
A. GFP.
B. MGG_07834 SP : LPAP : IQAR : ZF : EGFP (WT).
C. MGG_07834 SP : AAAA : IQAR : ZF : EGFP.
D. MGG_07834 SP : LPAP : AAAA : ZF : EGFP.
E. MGG_07834 : AAAA : AAAA : ZF : EGFP.
F. ZF : EGFP.
A
B
C
D
E
F
34
2.3 Discussion
We have identified a family of zinc finger effectors from M. oryzae strain 70-15.
Live-cell imaging showed that they first accumulate in BIC, then are translocated into
rice cells, and probably move from the first invaded cell to neighboring cells prior to
invasive hypal invasion. To figure out if there are conserved motifs that can be
recognized for secretion and/or translocation, we have tested two relatively
widespread conserved motifs in this family of zinc finger effectors for host-targeting
activity. Our results showed that L/IPXP and L/IXAR may play a role in host
translocation. Based on these results, we propose a model for the secretion,
translocation and movement of this family of zinc finger effectors during M. oryzae
infection: First, zinc finger effectors are expressed and secreted via the ER secretary
pathway out of the hyphal cell into BIC. Second, certain receptors in the plant plasma
membrane-derived BIC membrane recognized host targeting motifs L/IPXP and
L/IXAR and mediate tanslocation of effectors into the plant cell. And finally, inside
the plant cell, zinc finger effectors localize to a subcellular compartment or continue
the journey to neighboring cells where they reprogram the host cells to the benefits of
fungal biotrophic growth.
Although we demonstrated that this family of zinc finger effectors can bind
phospholipids, we cannot conclude that phospholipids are the receptors mediating the
translocation of this family of zinc finger effectors. Phospholipid-mediated
endocytosis was proposed as a mechanism by which pathogen effectors enter host
cells (Kale et al., 2010), but has since been challenged. Yaeno et al. (2011)
demonstrated that phospholipid binding is required not for effector entry but for
35
effector’s function inside host cells. Another study showed that effector SpHtp1 enters
host cells by binding tyrosine-O-sulphate but not phospholipids on the host cell
surface (Wawra et al., 2012). An early study reported that L/V/IPXP motif in
peroxidases is a binding site for aromatic amino acids (Veitch et al., 1994). It will be
interesting to investigate if L/IPXP in fungal effectors can also bind some aromatic
amino acids or their derivatives like tyrosine-O-sulphate.
2.4 Materials and methods
Genetic constructs
Gene fusions P27 : MGG_07834/10020, PPWL2 : MGG_07834/10020 and gene mutants
L/IPXP/AAAA, L/IXAR/AAAA were obtained by overlap extension PCR.
EcoRI- P27 : MGG_07834/10020-BamHI were ligated with vector fragment EcoRI-
pBV126-BamHI to obtain MGG_07834/10020 : EGFP expression constructs. XbaI-
PPWL2 : MGG_07834/10020-BamHI, XbaI-mCherry : Ter : PBAS4 : BAS4-BamHI
(PCR from pBV591), and EcoRI-pBV126-BamHI were ligated together to obtain
MGG_07834/10020 : mCherry expressing constructs. All these 4 constructs were
used to transform blast isolate O-137. MGG_07834/10020 : EGFP fragments were
recombined into vector pUGW11 by the gateway cloning strategy. These two
constructs were used to transfect rice protoplasts. L/IPXP : BAX fusion gene
fragments: G_07834SP : BAX , MGG_07834SP-LPAPVN : BAX, MGG_07834SP-
AAAAVN : BAX, and MGG_07834SP-LPAP-IQARSA : BAX were obtained by overlap
extension PCR. These L/IPXP : BAX fusion genes were recombinant into gateway
vector pMDC32 and used for transient expression in N. benthamiana. L/IPXP : EGFP
36
frusion genes MGG_10020 SP : EGFP, MGG_10020 SP : IPTP : EGFP and
MGG_10020 SP : AAAA : EGFP were also constructed in pMDC32 vector and used
to tansform onion epidermal cells by particle bombardment. Full-length effector :
EGFP gene constructs MGG_07834 SP : LPAP : IQAR : ZF : EGFP, MGG_07834
SP : AAAA : IQAR : ZF : EGFP, MGG_07834 SP : LPAP : AAAA : ZF : EGFP,
MGG_07834 : AAAA : AAAA : ZF : EGFP and ZF : EGFP were constructed in
pMDC32and used to transform N. benthamiana by agroinfiltration.
Live-cell imaging of zinc finger effectors in rice cells
Fungal spores from O-137 strains containing MGG_07834/10020 : mCherry : NLS :
BAS4 : EGFP were inoculated into the hollow interior of detached leaf sheath of rice
cultivar YT16 at a concentration of 3X104 spores/ml in 0.25% of gelatin. 24~36 hours
post inoculation, inner epidermal layers were trimmed off and observed with Zeiss
Axiovert 200M confocal microscope. Excitation/emission wave lengths were
488nm/505~550 for EGFP and 543nm/560nm for mCherry.
Protein-lipid overlay assay of zinc finger effector-phospholipid interaction
GST-MGG_07834 and GST-MGG_09035 were purified by Glutathione sepharose 4B
(GE Healthcare Life Sciences). PIP strips were purchased from Echelon Biosciences
Inc (Salt Lake City, UT). Protein-overlay assay was conducted according to Echelon’s
procedures. PIP strips were blocked in 3% fatty-acid free BSA in TBS for one hour at
room temperature. The blocking solution was discarded and PIP strips were incubated
37
with 1µg/ml GST-MGG07834/09035 in TBS for 1 hr at RT. The strips were washed
with TBST three times for 10 minutes each and then incubated with an anti-GST
antibody 1: 8,000 in blocking solution for 1 hr at RT. The strips were washed three
times for 10 minutes each and incubated with an anti-mouse HRP antibody diluted 1:
10,000 in blocking solution for 1hr at RT. After the strips were washed 3 times for 10
minutes each, chemiluminescence was detected using SuperSignal®West Pico
Chemiluminescent substrate (Thermo Scientific) and the ChemiDoc XRS system
(Bio-Rad).
Agrobacterium-mediated transformation of N. benthamiana and biolistic
transformation of onion epidermis
A. tumefasciens GV3101 carrying gene constructs were grown on YEP media plates
(5 g of Yeast extract, 10 g of NaCl, 10 g of bacteriological peptone, 15 g of agar in 1
liter) overnight. Cells were harvested and resuspended in MMA media (5g of MS salts,
1.95g of MES, 5g of glucose in l liter, pH 5.6, filter sterilized) containing 200 µM of
acetosyringone at an OD600 of 0.3. Cell suspensions were placed at RT for at least 3
hours before infiltration. Cells were infiltrated into leaves with a 1ml disposable
syringe without a needle. For biolistic transformation of onion epidermis, gold
particles (1 micron) were coated with gene constructs (~5µg) and bombarded into
onion epidermal cells via Model PDS-1000/He biolistic particle delivery system (Bio-
Rad, Hercules CA).
38
Chapter 3
Zinc finger effectors are nuclear HIRA recruiters and DNA binding factors
3.1 Introduction
Receptors of pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) are
usually at the cell surface and are a class of receptor-like kinases (RLKs) or receptor-
like proteins (RLPs), whereas receptors for avirulence (AVR) proteins are usually
nucleotide binding leucine-rich repeat (NB-LRR) proteins in the cytoplasm.
RLKs/RLPs are called pattern recognition receptors (PRRs) and NB-LRR proteins
resistance (R) proteins. Upon recognition of their cognate ligand, PRR initiates
pattern triggered immunity (PTI), and R initiates effector triggered immunity (ETI)
usually with a hypersensitive response (Jones and Dangl, 2006). Although different in
timing and amplitude at the infection site, both types of defenses can trigger systemic
acquired resistance (SAR) in the neighboring and distal tissues to prime the cell for
secondary infection (Mishina and Zeier, 2007). Signaling for PTI, ETI or SAR leads
to extensive transcriptome reprogramming in the nucleus. For example, SAR in
Arabidopsis involves upregulation or downregualtion of ~2000 genes, i.e., ~10% of
the transcriptome (Fu and Dong, 2013). This feat is accomplished to a large extent by
plant transcription (co)factors and chromatin modulins.
Transcription (co)factors regulating plant defenses are major components of plant
defense hormone signaling pathways or interlocutors between these pathways. In
Arabidopsis, plant defenses are mainly determined by three plant defense hormone
39
pathways and their crosstalks (Figure 3.1). SA largely regulates defense against (hemi)
biotrophic pathogens and functions in SAR. Recently, NPR3 and NPR4, paralogs of
NPR1 (nonexpressor of PR1 genes), were shown to be SA receptors and act as BTB
domain-containing adaptors for cullin3 ubiquitin E3 ligase to regulate the level of
transcription cofactor NPR1 in the nucleus. In infected cells, SA enhances NPR3-
NPR1 interaction by binding to NPR3 and promotes NPR1 degradation. In
neighboring or distal cells, SA disrupts NPR4-NPR1 interaction and release NPR1 for
gene activation. Transcription cofactor NPR1, along with transcription factors TGAs
activates defense gene expression such as PR1 (Fu et al., 2012). JA alone activates
responses to wounding and herbivory, but in the presence of ET, it activates defense
against necrotrophs. Transcription factors ERF1 and ORA59 consolidate the JA/ET
signaling to one pathway and control disease resistance to necrotrophs (Pre et al.,
2008). Components in SA signaling such as transcription (co)factors NPR1, TGAs
and WRKY70 can suppress components in the JA/ET signaling pathway, resulting in
a negative crosstalk between SA and JA/ET pathways.
Figure 3.1 Major transcription factors of defense hormone pathways in Arabidopsis
SA signaling
SA
NPR3,4
NPR1
TGAsWRKY70
SA-responsive genese.g., PR1
Defense against(hemi)biotrophs
JA signaling
JAs
SCF COI1
JAZ
MYC2ERF1ORA59
JA-responsive genese.g.,VSP2 e.g.,PDF1.2
ET signaling
ET
EIN2
SCFEBF1/2
EIN3
ERF1
ET-responsive genese.g., PDF1.2
Defense againstnecrotrophs
40
Chromatin modulins are the other essential part of the chromatin/transcriptional
regulatory machinery and play important roles in plant immunity. Chromatin
modulins are a plethora of regulators of chromatin structure such as histone
chaperones, histone modifying enzymes and ATP-dependent remodelers. Histone
modifications include acetylation, methylation, phosphorylation, ubiquitination,
sumoylation, carbonylation and glycosylation (Kouzarides, 2007). In general, histone
acetylation is associated with gene activation, whereas deacetylation is linked to gene
suppression. Histone methylation or ubiquitination can either activate or suppress
transcription (Smolle and Workman, 2013). Epigenomic mapping of histone
modifications in Arabidopsis showed that tri-methylations of H3K4 (H3K4me3) and
H3K36 (H3K36me3) are correlated with active genes, while H3K27me3 and
H3K9me2 are connected to repressed genes (Zhang et al., 2009; Roudier et al., 2011).
Net histone acetylations result from antagonistic histone acetyltransferases and
histone deacetylases (HDAs), while histone methylations are catalyzed by histone
methyltransferase (HMTs). Chromatin modulins play important roles in plant disease
resistance (Table 3.1). In Arabidopsis, HDA6 and HDA19 increase basal level
expression of JA/ET responsive genes and enhance resistance to fungal pathogen
Alternaria brassicicola (Zhou et al., 2005; Wu et al., 2008). Interaction of HDA19
with WRKY38 and WRKY62 leads to an increase of PR gene expression and
resistance to P. syringae DC3000 (Pst DC3000) (Kim et al., 2008). In rice, HDT701,
a H4 deacetylase, negatively regulates histone acetyaltion levels at the promoters of
two PRRs, CEBiP and FLS2, and one R chaperone complex unit SGT1, thereby
conferring resistance to M. oryzae and X. oryzae pv. oryzae (Xoo) (Ding et al., 2012).
In Arabidopsis, SDG8, a major HMT for H3K36me3, increase the level of
41
H3K36me3 at the loci of R genes, such as RPM1 and LAZ5, and is thus required for
R initiated ETI (Palma et al., 2010). SDG8 also mediates H3K36me3 enrichment in
the loci of MKK3, MKK5 and several PR genes, thereby establishing a permissive
state for defense-related gene expression (Berr et al., 2010). H3K4me3 established by
an unknown HMT at WRKY6, WRKY29 and WRKY53 serves as a stress memory in
SAR (Jaskiewicz et al., 2011). And H3K9 acetylation as a stress memory in SAR
even can be passed on to the next generation (Luna et al., 2012).
Table 3-1 Chromatin modulins in plant defense
Name Plant Consequence Association Effect on plant defense
HDA6 Arabidopsis Histone
deacetylation
transcription
repression
Increase basal expression
of JA/ET responsive genes;
enhance resistance to A.
brassicicola
HDA19 Arabidopsis Histone
deacetylation
Transcription
repression
Same as HDA6; Increase
PR gene expression and
enhance resistance to Pst
DC3000
HDT701 Rice H4
deacetylation
Transcription
repression
Negative regulator of PTI
and ETI
SDG8 Arabidopsis H3K36me3 Transcription
activation
Negative regulator of ETI;
Increase JA/ET responsive
genes and resistance to A.
brassicicola
HMT? Arabidopsis H3K4m3 Transcription
activation
Positive regulator of SAR
HAT? Arabidopsis H3K9ac Transcription
activation
Positive regulator of
transgenerational SAR
As mentioned above, the nucleus is a major venue for the host cell to interpret cues
and rapidly respond to an ever changing environment by transcriptional
reprogramming. Many pathogens deliver effectors into the nucleus to manipulate the
chromatin/transcriptional machinery in order to suppress host defense and/or siphon
off nutrients (Figure 3.2). In addition to transferring T-DNA, A. tumefasciens also
delivers several effectors along the way. In the nucleus, the T-complex, a compound
of T-DNA, VirD2 and VirE2, is pulled to the chromatin by the interaction of the
42
VirE2 interacting protein 1 (VIP1) with core histones. Then, T-DNA seems to be
integrated into the host genome by the non-homologous end-joining (NHEJ)
mechanism (Magori and Citovsky, 2011). T-DNA transformed plant cells undergo
neoplastic growth and synthesize opines as a carbon and nitrogen source for the
bacteria. In some strains of Xanthomonas spp. and Ralstonia solanacearum, several or
dozens of transcription activator-like effectors (TALEs) are delivered into the nucleus,
where they act as eukaryotic transcription factors. TALEs specifically bind to a
stretch of DNA sequence in the promoters of their target genes in a simple and
decodable manner (Boch et al., 2009; Moscou and Bogdanove, 2009). AvrBs3 from
Xcv, acting as a transcription factor, regulates dozens of genes, of which some are
expansin genes. The activation of AvrBs3 target genes changes host cell morphology
to facilitate bacterial dispersal for the next cycle of infection (Marois et al., 2002; Kay
et al., 2007). PthXo1 and AvrXa7/PthXo3/Talc from Xoo, functioning as transcription
factors, target OsSWEET11 and OsSWEET14, respectively, two genes encoding sugar
transporters for normal sugar flow between host cells. The bacteria co-opt these sugar
transporters to pump more sugars into the apoplast where the bacteria thrive (Yang et
al., 2006; Chen et al., 2010; 2012). XopD from Xcv interacts with two transcription
factors Myb30 and SIERF4, suppressing their roles in plant disease resistance
(Canonne et al., 2011; Kim et al., 2013). SP7, an effector from arbuscular mycorrhizal
fungus Glomus intraradices interacts with the transcription factor ERF19 to evade
plant defense and promote symbiosis (Kloppholz et al., 2011). RKN16D10, a 13 aa
secretory effector from a root knot nematode Meloidogyne incognita, acts as a ligand
for two plant scarecrow-like transcription factors, but the biological role of this
binding is yet to be known (Huang et al., 2006). OspF, an effector from human
pathogen Shigella flexneri and a homolog of pseudomonas syringae HopAI1,
43
functions as a phospothreonine lyase, preventing histone H3 phosphorylation and
acetylation (Arbibe et al., 2007). In addition, it also interacts with a chromatin
remodeling factor Rb to suppress NF-КB regulated immune responsive genes. NUE,
an effector from another human pathogen Chlamydia trachomatis, acts as a histone
modifier, using its SET domain-containing methyltransferase activity to induce
histone methylation (Pennini et al., 2010). LntA, an effector from human pathogen
Listeria monocytogenes, interacts with a heterochromatin-forming BAHD1silencing
complex to regulate interferon-stimulated gene expression (Lebreton et al., 2011).
Characterization of LntA unveiled the role of this novel BAHD1 silencing complex in
interferon signaling pathway. Agrobacterium T-DNA encoded 6b protein is localized
to the nucleus and interacts with several nuclear proteins such as H3. It was first
proposed that 6b might affect gene expression by acting as a chromatin chaperone or a
transcription regulator (Terakura et al., 2007). Recently, 6b was demonstrated to have
ADP-ribosylating activity and to function as a RNA silencing suppressor by targeting
two components in the miRNA pathway for ADP-ribosylation (Wang et al., 2011).
44
Figure 3.2 Pathogen effectors and their targets in the nucleus. Names of bacterial,
fungal/oomycete and nematode effectors are in red, purple and green, respectively.
The transcriptional repression of KNOX genes mediated by the AS1/AS2/HIRA
silencing complex is a model that smart pathogen effectors are likely to emulate in
suppressing plant defense genes (Figure 3-3). KNOX genes encode homeodomain
proteins that promote stem cell activity, and their expression must be repressed during
lateral organogenesis such as in the development of leaves. In Arabidopsis, this
process is initiated by the formation of a DNA binding complex of ASYMMETRIC
LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2). AS1, the ortholog of maize
ROUGH SHEATH2 (RS2), is an unusual myb domain-containing protein and AS2 is
a zinc finger and leucine zipper-like domain-containing protein (Timmermans et al.,
1999; Byrne etal., 2000; Iwakawa et al., 2002). AS1/AS2 binds to two sites in the
BacteriaFungi
Oomycetes
TF X
H X
OspF/HopAI1?/NUE
XHIRA
6b?/ZFs
TALEsXopD/SP7/RKN16D10
MiSSP7CRNs
BAHD1
LntA
H
VirD2-T-DNA/VirE2/VIP1
Nucleus
Nematodes
45
promoter of KNOX gene and recruits HIRA to form an AS1/AS2/HIRA silencing
complex (Phelps-Durr et al., 2005; Guo et al., 2008). This silencing complex interacts
with histone methyltransferase PRC2 and/or histone deacetylase HDA6 to establish a
repressive chromatin state at the promoter loci of KNOX genes (Lodha et al., 2013;
Luo et al., 2012). This repressive chromatin state is stably maintained all the way
through the many rounds of cell divisions during leaf development.
Figure 3-3 The repressive chromatin state of the promoter of KNOX genes
established by AS1/AS2/HIRA silencing complex
In this study, the interaction between zinc finger effectors and rice HIRA is
reminiscent of the interaction between AS1/AS2 and HIRA. Furthermore, like
AS1/AS2, zinc finger effectors bind to DNA with specificity. Therefore we
hypothesize that zinc finger effectors promote disease by recruiting HIRA for defense
gene expression.
3.2 Results
HIRAAS1 AS1
AS2AS2
PRC2
HDA6
H3k27
me3
H3K27
me3
H3
ac
AS1/AS2/HIRA
Silencing complex
X
46
Zinc finger effectors interact with HIRA and other nuclear proteins
To find the host virulence targets of this family of zinc finger effectors, we screened
the rice yeast two hybrid (Y2H) cDNA libray made from transcriptome induced by M.
oryzae. In total, 2X107 independent clones were screened for their interactions with
MGG_07834. Yeast transformants harboring the bait pDB-Trp/MGG_07834 and the
prey pAD-GAL4-2.1/cDNA were tested for all three Y2H markers (His+, Ura
+, Laz
+).
In total, four independent clones were obtained as MGG_07834 interactors (Figure 3-
4; Table 3-2).
HIRA (histone regulatory protein A) is one of the three interactors of MGG_07834.
HIRA is encoded by a single-copy gene in most eukaryotes except yeast. In yeast, two
proteins HIR1 and HIR2 function together complementarily as homologues of HIRA.
HIRA is known not only to establish and maintain heterochromatin loci but also to
control the spatial and temporal expression of some euchromatic genes (Spector et al.,
1997; Magaghi et al., 1998; Phelps-Durr et al., 2005). The interaction between
MGG_07834 and HIRA suggests a new role that HIRA may play in plant immunity.
Rice HIRA (OsHIRA) is a 975 aa nuclear proteins. Like its orthologs in Arabidopsis
(AtHIRA) and maize (ZmHIRA), OsHIRA contains 7 canonical or degenerate WD40
repeats and one C-terminal HIRA motif conserved in all eukaryotes. One nuclear
localization signal (NLS) is predicted in the near middle of the protein. OsHIRA is
closer to Zm HIRA than is AtHIRA in the primary structure (Figure 3-5 A).
The second interactor of MGG_07834 is an ortholog of Arabidopsis STOP1 and
named OsSTOP1 (Figure 3-5 B). OsSTOP1 contains four C2H2 zinc finger motifs in
47
its C-terminus, suggesting it may function as a transcription factor in rice. In
Arabidopsis, STOP1 regulates multiple genes associated with detoxifying photon and
aluminum toxicities (Sawaki et al., 2009). In rice, ART1, a paralog of OsSTOP1, also
plays a role in regulating genes linked to detoxifying aluminum toxicity (Yamaji et al.,
2009). Due to its interaction with MGG_07834, it is tempting to investigate whether
OsSTOP1 also plays a role in plant immunity.
Another interactor of MGG_07834 is OsPPR. Pentatricopeptide repeat (PPR)
containing proteins comprise a large family of proteins in plants. There are 450 and
470 PPR protein-encoding genes in Arabidopsis and rice, respectively. A few studied
PRR proteins are implicated in various plant developmental processes via their
functions in RNA editing in plant organelles. But some might also have novel
functions such as in disease resistance and abiotic stress tolerance (Laluk et al., 2011).
Two reports prompted us to focus our attention on HIRA being an important regulator
of plant immunity. Yang et al. (2008) found that βC1, a pathogenicity factor from
tomato yellow leaf curl China virus, associated with AS1 to suppress JA responsive
genes by competing with AS2 for AS1 binding. And AS1 was demonstrated to be a
negative regulator of plant immunity to necrotrophic fungus Botrytis cinerea by
suppressing JA responsive genes (Nurmberg et al., 2007). These studies suggested
that a similar silencing complex to AS1/AS2/HIRA may play a role in plant immunity
by selectively suppressing a set of defense genes. We conducted in vitro GST pull-
down assay with GST-MGG_07834 and His-HIRA. His-HIRA can be co-precipitated
with GST-MGG_07834 by Glutathione sepharose 4B beads (Figure 3-6). HIRA in the
fusion protein has no N-terminal WD40 domain and it is encoded by the orignal
48
partial ORF we identified in Y2H screening. The C-terminal region of HIRA is
sufficient for it to interact with MGG_07834.
Figure 3-4 Y2H screen for MGG_07834 interactors. Clones 1. STOP1. 2. PPR. 3.
STOP1. 4. HIRA. A. SC- Trp-Leu. B. SC-Trp-Leu-His+3AT (25mM). C. SC-Trp-
Leu-Ura. D. SC-Trp-Leu+5-FOA (0.2%). E. β-Gal filter lift assay.
Table 3-2 Summary of rice proteins that interact with MGG_07834
Interacting Proteins
MGG_07834-DB(25mM3AT)
MGG_07834-DB (0.2% 5-FOA)
Number of Colonies
Accession Number
HIRA + + - - 1 AK065913
STOP1 + + - - 2 AK072417
TPR + + - - 1 AK065769
++ demonstrates strong growth. - - indicates no or weak growth.
A
B
C
D
E
B
C
D
E1
2
34
49
Figure 3-5 Predicted motifs in HIRA, OsSTOP1 and OsPPR1. Marks in brown
indicate nuclear localization signal (NLS). Marks in pink indicate low-complexity
sequence. Mark in green shows a coiled-coil motif. BLAST, degenerate WD40.
AtHIRA 1040aa
ZmHIRA 964aa
OsHIRA 975aa
OsSTOP1
OsPPR
A
B
C
IB: α-His
Protein loading
His-HIRA (75kD)
GST-MGG07834 (36kD)
GST (25kD)
50
Figure 3-6 In vitro GST pull-down assay demonstrated zinc-finger effectors interact
with HIRA in vitro.
The interactions between zinc finger effectors and HIRA occur in the nuclei of
rice cells
HIRA and the other two MGG_07834 interactors OsSTOP1 and OsPPR are all
predicted to have at least one nuclear localization signal, suggesting that this family of
effectors interact with their virulence targets in the nucleus. And the zinc finger
effector MGG_10020 was observed to be localized into the nucleus during fungal
infection with live-cell imaging microscopy (Barbara Valent, personal
communication). We set out to test the possible nuclear localization of the interaction
between this family of zinc finger effectors and HIRA using bimolecular fluorescence
complementation (BIFC). Three zinc finger effectors MGG_07834, MGG_10020 and
AVR-Pii were N-terminally fused to cEYFP, and HIRA was both N-terminally and C-
terminally fused to nEYFP. BiFC constructs for effector and HIRA were
appropriately combined and transfected into rice protoplasts. As expected, the
interaction between MGG_07834 and HIRA is almost exclusively in the nucleus
(Figure 3-7 A). The interaction between MGG_10020 is to a large extent in the
nucleus (Figure 3-7 B). The interaction between AVR-Pii and HIRA is in the
cytoplasm of rice cells (Figure 3-7 C). Compared with MGG_07834 and MGG_10020
that are expected to be virulent effectors, AVR-Pii is avirulence protein (AVR) that
can be recognized by its cognate resistance (R) protein Pii in rice cells. The difference
in their functions is likely to be used to explain the different patterns of their
51
interaction with HIRA, vice versa. The molecular mechanism underlying this
difference will be proposed in the following section. Coimmunoprecipitation (CoIP)
was attempted unsuccessfully to demonstrate the interaction between MGG_07834
and HIRA in rice protoplasts (Figure 3-8). It seems that the fusion protein HIRA-HA
was not appropriately expressed in rice protoplasts.
Figure 3-7 Interactions between zinc-finger effectors and HIRA were localized to rice
cell nuclei. A, MGG07834-cEYFP+HIRA-nEYFP. B, MGG10020-cEYFP+nEYFP-
HIRA. C, AvrPii-cEYFP+HIRA-nEYFP. Each row from left to right: GFP, bright and
composite. Scale bar, 20 µM.
A
B
C
52
Figure 3-8 Coimmunoprecipitation assay for the interaction between effectors and
HIRA in rice protoplasts. 10% input was left as control for western blot. Left panel,
blotted membrane was detected with α-HA. Right panel, blotted membrane was
detected with α-FLAG.
Zinc finger effectors are DNA binding factors
One-finger zinc finger proteins are usually known as protein interactors, and not as
DNA binding factors. But our study showed that in addition to interacting with
proteins, one-finger zinc finger proteins such as this family of zinc finger effectors
can be DNA binding factors.
AS2 in the AS1/AS2/HIRA silencing complex is a one-finger zinc finger protein and
its zinc finger motif shares similarities with zinc finger effectors MGG_07834,
MGG_10020 (Figure 3-9 A and B). AS2 contributes a major part to the DNA binding
Input
HIRA-HA
MGG_07834-FLAG
+
-
Elute Input Elute
+
+
+ + + + + +
- - -+ + +
α-HA α-FLAG
MGG_07834-FLAG
53
activity of the AS1/AS2 complex, because AS1 has changed the conserved amino
acids in the third helix of the R3 MYB motif that are essential for MYB-DNA
interaction. Furthermore, mutant as2 is genetically epistatic to as1, suggesting AS1
function depends on AS2 (Phelps-Durr et al., 2005; Guo et al., 2008). From the above
analysis, it is plausible to propose that fungal zinc finger effectors are mimics of AS2
or AS1/AS2 complex in DNA-binding.
There is a precedent that a one-finger zinc finger protein specifies a stretch of DNA
sequence. GAGA transcription factors function either as transcription activators or
repressors by inducing changes of chromatin composition and conformation at
promoters (Adkins et al., 2006). A typical GAGA protein has an 82 aa segment
containing a C2H2 motif and two stretches of basic amino acids and this segment is
sufficient for it to bind to consensus sequence GAGAG (Pedone et al., 1996).
Specifically, amino acids in the zinc finger motif make contacts with GAG in the
DNA major groove, while amino acids in basic region 1 (BR1) interact with A in the
minor groove and those in BR2 make contact with G in the minor groove (Figure 3-10
B). Through these interactions, the C2H2 zinc finger and the basic stretches of amino
acids make high-affinity specific DNA-binding. Zinc finger effectors MGG_07834,
MGG_10020, MGG_08230, MGG_13665 and MGG_10238 have flanking stretches
of basic amino acids (Figure 3-10 A), suggesting that these zinc finger effectors can
specifically bind to certain DNA sequences with their C2H2 motif and nearby basic
stretches of amino acids.
We conducted systematic evolution of ligands by exponential enrichment (SELEX) to
try to find some consensus DNA sequences for this family of zinc finger effectors.
54
Random DNA oligomers of 16 nucleotides were synthesized and used as SELEX
probes. GST- MGG_07834 was used to bind the probes and pull down specific probes.
GST-MGG_07834-SELEX probe complexes were washed and SELEX probes were
eluted. Eluted SELEX probes were PCR amplified and added to the next round of
binding reaction. After 5 rounds of SELEX reactions, GST-MGG_07834, like
transcription factor EIL1 (positive control), pulled down DNA, while GST alone
(negative control) could not precipitate DNA (Figure 3-11). This result showed that
zinc finger effectors are DNA binding factors.
Figure 3-9 3-D structures of zinc finger motifs of MGG_07834, MGG_10020, AVR-
Pii and AS2. A. Sequence arrangement of zinc finger motifs of effectors and AS2.
Shaded letters indicate conserved amino acids. B. 3-D structures of zinc finger motifs
of effectors and AS2 modeled on homologous zinc finger proteins with known
structures.
MGG_07834
MGG_10020
AS2
MGG_07834 MGG_10020 AVR-Pii AS2
AVR-Pii
A
B
55
Figure 3-10 One-finger zinc finger proteins can specify a sequence of DNA. A.
Sequence alignment of zinc finger effectors MGG_07834, MGG_10020,
MGG_08230, MGG_13665, MGG_10238 and GAGA-DB. Basic stretches of amino
acids are indicated in red, zinc finger motif in blue. B. 3-D structure of complex of
GAGA-DB and DNA sequence GAGAG. BR, basic region. (Adapted from
Omichinski et al., 1997).
GNTSGVLSTPKAKRAKHPPGTEKPRSRSQSEQPATCPICYAVIRQSRNLRRHLELRHFAKPGVKKEKKSKSGNDTTLDSSME
LPAPVNPSEIQARSAADKAKPKPQTERVWMDEPEAAQYLSDPRWKLVKKKYSCGYCDSSSSKVDKINKHRDEVHGTRQAQVDHTIYPGETRLTFERVSGYP
INLPAALTSQLEPRAEGGASAGDANNIQPQSQEKSPFICVWCGKDKGGSSALIGHEIHNHQDDEMVAWTGRDRRLNKYETDKEGYRYFKKYPGVRLPNRTWKGKGKRPE
LPAPANPAAVQPAQGGQLAVHGQPASCPPECRDVQGAKPGHLQARSRFYATSDTGRPLANSGYPTHGYRDAYRCLYCGAVRDDVSAVQDHITYRHSNRGGDTSNYDTTTVRDDR
LPTPAGVDVDGGQLQARAEGDKVRPPSIKCLTCEQGGFPNVDEYEQHYKQKHPEIVQQVSSPNPHLNKGKKAN
LPTPGYTPTNQNTGDIVARSDTVKGKDVATSTDANKKKRTKDYYWRCANCQAKFSDYESFNKHGNECKSV
GAGA-DB
MGG_07834
MGG_10020
MGG_08230
MGG_13665
MGG_10238
A
BR1: A
BR2: G
Zinc-finger: GAG
5’
3’
N
C
B
56
Figure 3-11 MGG_07834 binds to double stranded DNA probes after five rounds of
SELEX reactions.
3.3 Discussion
From yeast two hybrid screening, we found that HIRA is an interactor of zinc finger
effectors. HIRA is well known to induce a suppressive chromatin state in promoters
of genes such as the KNOX genes. This prompted us to ask these questions: Whether
does HIRA play a role in suppression of plant defense genes? If so, how is it recruited
to the promoters of certain defense genes? The latter question led us to the discovery
of the molecular/biochemical function of zinc finger effectors, i.e., DNA binding
factors with specificities. Most fungal effectors are usually small novel proteins with
no similarities to any known proteins. And fungal gene knock-out mutants usually
have no changes in disease symptoms due to redundancy of genes or functions. As a
result, it is not easy to characterize their molecular/biochemical functions, much less
the mechanisms by which they interact with their virulence targets in host cells. To
date, only a few virulence targets and/or functions have been ascribed to fungal
M
100bp
72bp
57
effectors. Cmu1, an effector from maize smut fungus U. maydis, acts as a chorismate
mutase to divert the shikimate pathway towards production of aromatic amino acids
and away from production of SA (Djamei et al., 2011). SA is a key plant defense
hormone in maize. In rice-M. oryzae pathosystem, an E3 ubiquitin ligase APIP6 was
found to be a virulence target of AVR-Piz-t, but the mechanism by which AVR-Piz-t
destabilizes APIP6 to suppress PTI is not known (Park et al., 2012). Our finding of
HIRA as a virulence target of zinc finger effectors will provide new insights into how
fungal effectors defeat plant immunity.
The function of AS1/AS2/HIRA silencing complex is well established in suppression
of KNOX genes. AS1 and AS2 interact to form an AS1/AS2 complex and this
heterodimer complex specifically binds to two sites in the promoters of KNOX genes
(Guo et al., 2008). Although neither AS1 nor AS2 alone can bind DNA in vitro, the
zinc finger protein AS2 likely contributes more to the activity and specificity of the
DNA binding complex AS1/AS2. In this study, zinc finger effector MGG_07834 has
been demonstrated to bind DNA in vitro, suggesting that zinc finger effectors fulfil
the function of the AS1/AS2 complex in DNA binding. It needs to be pointed out that
differences in DNA binding likely exist in this family of zinc finger effectors based on
the amino acid sequence of zinc-finger motif and the flanking basic regions.
MGG_07834, MGG_10020, MGG_08230, and MGG_13665 contain an intact C2H2
zinc finger motif and basic regions that precede, follow, or flank this motif (Figure 3-
10 A). These four zinc finger effectors are presumably active DNA-binding factors. In
contrast, AVR-Pii contains an incomplete zinc finger motif and cannot form a typical
α-helix in the modeled structure that is critical for zinc finger motif to make contacts
with bases in the DNA double helix (Figure 3-9). In addition, AVR-Pii lacks basic
58
stretches of amino acids that also take part in determining DNA binding specificity as
in GAGA transcription factors and TALEs (Pedone et al., 1996; Nga-Sze Mak et al.,
2013). In this study, the BiFC assay demonstrated that the localization of the
interaction between AVR-Pii and HIRA is in the cytoplasm, while MGG_07834 and
MGG_10020 interplay with HIRA in the nucleus. The prediction that AVR-Pii has no
DNA binding activity is a possible explanation for this observation.
Well known as two conserved factors functioning in leaf development in plants, AS1
and AS2 were also demonstrated to be involved in suppression of JA responsive
defense genes (Nurmberg et al., 2007; Yang et al., 2008). However, the picture of
AS1 or AS2 in suppression of plant defense genes is incomplete. Our findings that
fungal zinc finger effectors were AS2 or AS1/AS2 mimics, and that HIRA was
recruited by these DNA binding factors make this picture clearer, richer and more
colorful. To explain the mechanism by which zinc finger effectors suppress plant
defense genes, we propose the following model. Zinc finger effectors bind
specifically to the promoters of certain defense-related genes and recruit HIRA to the
promoters to form a zinc finger effector (ZFE)/HIRA silencing complex. This
ZFE/HIRA silencing complex then interacts with histone modifiers such as histone
methyltransferase PRC2 and histone deacetylase HDA6 to establish a repressive
chromatin state at the promoters. As a result, the promoters are closed to the
transcriptional machinery and defense genes are silenced (Figure 3-12).
59
Figure 3-12 A model for establishing a chromatin repressive state at the promoters of
certain defense-related genes by ZFE/HIRA silencing complex
It is pressing to map the binding sites of zinc finger effectors across the rice genome.
Chromatin immunoprecipitation-squencing (CHIP-seq) will help us obtain this goal.
Recently, CHIP-exo, a new technique that combines standard CHIP and DNase
footprinting, was used to map the binding sites of transcription factors at one base
resolution (Rhee and Pugh, 2011). One-finger zinc finger proteins are likely to bind
shorter sequences, say, 3~8 bp as demonstrated by the GAGA transcription factors.
CHIP-exo will help us narrow and pin down the binding sites of this family of fungal
zinc finger effectors in the rice genome. Other must-do things are to find the
interactors of the ZFE/HIRA silencing complex by Y2H screening and map the
histone methylation and acetylation status at promoters of some key players in plant
immunity. All these efforts will lead to a deeper understanding of how fungal
effectors act to avoid, suppress and defeat plant defenses.
HIRAZFE
PRC2
HDA6
H3k27
me3
H3K27
me3
H3
ac
ZFE/HIRA
Silencing complex
X
60
3.4 Materials and methods
Yeast two hybrid screen
MGG_07834 was amplified using forward and reverse primers with SalI and NotI
sites, respectively. PCR fragment of MGG_07834 was cut with SaI and NotI and
inserted into the bait vector pDB-Trp such that the GAL4 DNA binding domain and
MGG_07834 were fused in frame. The cDNA library contains cDNAs that were
synthesized from M. oryzae induced mRNAs and cloned into HybriZAP-2.1vector.
The cDNA library was converted to pAD-GAL4-2.1 phagemid library by mass
excision using ExAssist helper phage. Approximately 2×107 pAD-GAL4-2.1-cDNA
phagemids were used to transform yeast strain MaV203 carrying pDB-
Trp/MGG_07834 according to the protocol described by Gietz and Schiestl (2007).
Transformants were grown on SC-Trp-Leu-His+25mM 3-AT plates for 3-8 days. His
prototropic clones were selected for further tests. His+ colonies were transferred to
SC-Trp-Leu-Ura plates to screen for Uracil prototrophy. His+ Ura
+ colonies were
spotted on SC-TrP-Leu+5-FOA (0.2%W/V) media for counter-selection of Ura+. β-
galactosidase filter lift assay was performed to test for activity of lacZ gene. In total,
all three marker genes were tested for MGG_07834 interactors. Plasmids were
extracted from His+ Ura
+ lacZ
+ clones by using the glass bead method.
GST pull-down assay
MGG_07834 was cloned into pGEX-4T-2 (GE healthcare), making an N-terminal
fusion with GST. HIRA was cloned into pDEST17 (Invitrogen), creating an N-
terminal fusion with 6×His. 10 µg of GST or GST-07834 was incubated with 50 µl of
61
glutathione sepharose 4B beads (GE healthcare) for 1 h at 4ºC in 500 µl of 20 mM
Tris, pH7.5, 500 mM NaCl and 0.5% NP40. After washing once, the beads were
incubated with 20 µl of bacterial extract His-HIRA at 4ºC for 2 h in 500 µl of the
same buffer. Beads were washed three times in 200 µl of 10 mM Tris, pH7.5, 150
mM NaCl and 0.1% NP40. Precipitated proteins were separated by SDS-PAGE and
blotted onto PVDF membrane. Anti-His antibody (H1029, Sigma, St. Louis, MO) was
used to detect the pull-downed 6×His-HIRA.
Bimolecular fluorescence complementation assay
MGG_07834, MGG_10020 and AVR-Pii were cloned into Gateway BiFC vector
pUGW2/cEYFP such that MGG_07834, MGG_10020 and AVR-Pii were N-terminally
fused to cEYFP. HIRA was cloned into Gateway BiFC vectors pUGW0/nEYFP and
pUGW2/nEYFP for N-terminal and C-terminal fusion to nEYFP, respectively. Rice
protoplasts were prepared from the stems of 10-day-old seedlings. Approximately
1X105 protoplasts were transfected with 5µg of MGG_07834-cEYFP and 5 µg of
HIRA-nEYFP. Two combinations: 5 µg of MGG_10020-cEYFP and 5µg of nEYFP-
HIRA, 5 µg of AVR-Pii-cEYFP and 5µg of HIRA-nEYFP, were also transfected into
the same amount of protoplasts. Protoplast transformation was conducted as described
in standard protocol for PEG/Ca2+
method. Fluorescence was detected by using a
Zeiss Cell Observer SD Spinning Disk Confocal microscope equipped with a
YoKogawa CSU-X1 spinning disk head and a Photoetrics QuantEM 512SC EMCCD
camera. Excitation/emission wave lengths for EGFP were 488nm/525-550nm,
respectively. The objective was Apochromat 100X 1.4 NA oil immersion objective.
62
Systematic evolution of ligands by exponential enrichement (SELEX) assay
Random sequence Oligo DNAs of 72 nucleotides 5´-GGA TTT GCT GGT GCA
GTA CAG TGG ATC C-(N)16-GGA TCC TTA GGA GCT TGA AAT CGA GCA
G-3´ were synthesized and purified by HPLC (Integrated DNA Technologies).
Random sequence Oligo DNAs were made double stranded by Taq DNA polymerase
and reverse primer. Double-stranded DNAs were separated by native PAGE and
recovered from the gel. 10 ng of dsDNA and 5 ng of GST-MGG_07834 (GST and
GST-EIL1 as negative and positive controls, respectively) were incubated on ice for
30 m in 50 µl of protein-DNA binding buffer: 10 mM of Tris, pH7.5, 50 mM of NaCl,
1 mM of EDTA, 5% of glycerol, 10 mM of DTT, 10mg/ml of BSA, 1µg/µl of poly
(dI/dC). Equilibrated glutathione sepharose 4B beads (GE healthcare) were used to
pull down GST-MGG_07834 and its binding probes. After washing, beads were
collected and suspended in 20 µl water. 5µl were used for PCR at the following
program: 5 m at 94ºC; 20 cycles (round 1 of SELEX) or 15 cycles (other rounds) of 1
m denaturation at 94ºC; 1 m at 50ºC; 30 s at 72ºC; and a final extension of 20 m at
72ºC, followed by 4ºC incubation. After 5 rounds of SELEX reactions, GST-
MGG_07834 (GST and GST-EIL1) binding DNAs were separated by PAGE and
recovered from the gel for sequencing.
63
Chapter 4
Zinc-finger effectors suppress PTI when expressed heterologously in plants
4.1 Introduction
Pattern triggered immunity (PTI) is the first line of plant active defense response.
Potential pathogens fail to cause disease on a certain plant are usually due to PTI and
these pathogens are called nonadapted pathogens to the plant. Adapted pathogen
usually delivers a plethora of effectors into its host to defeat PTI. PTI is activated
when conserved microbe/pathogen associated molecular patterns (M/PAMPs) are
recognized by pattern recognition receptors (PRRs) at the host cell surface. The past
decade has witnessed the identification of many M/PAMPs and PRRs. In rice, several
pairs of M/PAMP and PRR provide innate immunity to rice bacterial and fungal
pathogens (Table 4-1).
Xanthomonas oryzae pv. oryzae (Xoo) Ax21 is type I secreted sulfated 194aa quorum
sensing protein that is conserved in all Xanthomoads and related genera. A sulphated
17 aa peptide synthesized based on Ax21 N-terminal sequence is an active epitope for
rice XA21 recognition (Lee et al., 2009). To function properly, XA21 interacts with
several proteins such as XB3, XB15, XB24 and XB10 (Seo et al., 2011). XB3 is an
E3 ubiquitin ligase and phosphorylated by XA21. The biological relevance of this
interaction is not known (Wang et al., 2006). XB15 is a PP2C phosphatase and
dephosphorylates XA21, and is thus a negative regulator of the XA21-mediated
immunity (Park et al., 2008). XB24, a protein with intrinsic ATPase activity, is likely
64
to keep XA21 in an inactive state by promoting autophosphorylation of XA21 (Chen
et al., 2010b). XB10 is the transcription factor WRKY62. Upon activation, the
intracellular domain of XA21 is cleaved and translocated into the nucleus where it
directly interacts with WRKY62 for gene regulation (Park and Ronald, 2012).
Chitin, a conserved component of the fungal cell wall, is a homopolymer of N-
acetylglucosamine (GlcNAc)n. Although it was well documented that chitin is a
potent elicitor of plant defense response, it was only recently that its receptor, the
chitin elicitor binding protein (CEBiP) was first identified in rice (Kaku et al., 2006).
CEBiP is a lysin motif (LysM) containing receptor-like protein (LysM-RLK),
consisting of a signal peptide, three consecutive LysMs, and a C-terminal region
without any noticeable signature, followed by a glycosylphosphatidylinositol (GPI)
anchor motif. Due to its lack of cytoplasmic signaling domain, CEBiP forms a
heterodimer with another LysM containing receptor-like kinase (LysM-RLK) to
transduce the signal (Shimizu et al., 2010). This LysM-RLK is called rice chitin
elicitor receptor-like kinase (OsCERK1), an ortholog of Arabidopsis CERK1. In
Arabidopsis, CERK1 binds chitin and forms homodimer to transduce signal, as
opposed to OsCERK1 which has low affinity to chitin and must form heterodimer
with CEBiP for signaling (Liu et al., 2012). Adding some complexity to the rice chitin
signaling is the finding that two other LysM-RLPs, LYP4 and LYP6, also play a role
in chitin signaling (Liu et al., 2012). In addition to binding chitin, LYP4 and LYP6
also bind peptidoglycan (PGN). PGN, a cell wall component of Gram positive and
negative bacteria, consists of heteroglycan chains with alternating GlcNAc and N-
acetymuramic acid (MurNAc) units. Heteroglycan chains are crosslinked via a stem
peptide connected to two MurNAc moieties. PGN and its degraded soluble derivatives
65
muropeptides are potent plant defense elicitors. In Arabidopsis, two LysM proteins
LYM1 and LYM3 are identified as PGN receptors and need CERK1 for PGN
signaling (Willmann et al., 2011). In rice, LYP4 and LYP6 can be PGN receptors, but
whether they need OsCERK1 for signaling is not known.
Flagellin is the major component of the bacterial flagellum and a potent elicitor of
plant defense. Flagellin or its active epitope flag22 at the conserved N-terminus serves
as a ligand of Arabidopsis flagellin sensitive 2 (FLS2). Arabidopsis FLS2 is a leucine-
rich repeat receptor-like kinase (LRR-RLK) and has orthologs in tomato, N.
benthamiana and rice. Flagellin from two rice bacterial incompatible pathogens P.
avenae and Acidovorax avenae can induce immune response in rice similar to that
mediated by flg22-FLS2 in Arabidopsis, suggesting OsFLS2 in rice is also a
functional sensor for bacterial flagellin (Che et al., 2000; Tanaka et al., 2003).
In rice, two R proteins mediate wide-spectrum resistance against pathogens and may
also serve as PRRs, although their cognate M/PAMPs are not known. Xa26 from Xoo
is an LRR-RLK and required for broad-spectrum resistance against Xoo. Pi-d2 is a
bulb-type mannose specific binding lectin (B-lectin) receptor-like kinase and mediates
wide-spectrum resistance to M. oryzae (Chen et al., 2006).
66
Table 4-1 Rice M/PAMPs and PRRs in PTI
Pathogen M/PAMP/epitope Virulence
function
PRR Overall
structure
Ligand
binding
domain
Xoo Ax21/AxYs22 Quorum
sensing
XA21 LRR-
RLK
LRR
Fungi Chitin/(GlcNAc)n Cell wall
integrity
CEBiP LysM-
RLP
LysM
Bacteria;
Fungi
PGN/(GlcNAc-
MurNAc)n;
Chitin/(GlcNAc)n
Cell wall
integrity
LYP4/LYP6 LysM-
RLP
LysM
Fungi;
Bacteria
PGN/(GlcNAc-
MurNAc)n;
Chitin/(GlcNAc)n
Cell wall
integrity
OsCERK1 LysM-
RLK
LysM
Bacteria Flagellin/flg22 mobility OsFLS2 LRR-
RLK
LRR
Fungi Unknown
unknown Pid2 B-
Lectin-
RLK
Lectin
Xoo unknown unknown XA26 LRR-
RLK
LRR
Upon binding to M/PAMP, PRR is activated and PTI signaling is initiated. These are
followed by the production of reactive oxygen species (ROS), inflow of Ca2+
, and
activation of MAPK cascades. In rice, there are 17 MAPKs (Reyna and Yang, 2006),
of which OsMAPK5, OsMAPK6 and OsMAPK12 were demonstrated to be involved
in plant immunity. OsMAPK5 is induced by several biotic and abiotic stresses, and
acts as a positive regulator of drought tolerance and a negative regulator of plant
defense (Xiong and Yang, 2003). OsMAPK6 is chitin-induced and involved in the de
novo synthesis of diterpenoid phytoalexin against M. oryzae (Kishi-Kaboshi et al.,
2010). OsMAPK12 (a.k.a., BWMK1) is induced by both M. oryzae and Xoo and
implicated in plant innate immunity to these two pathogens (He et al., 1999; Seo et al.,
2011).
Two major types of transcription factors WRKYs and TGAs are implicated in rice
immunity. More than 100 WRKYs are among the rice proteome, several of them such
67
as WRKYs 62, 28, 71, 76 and WRKYs 53, 89 were investigated for their involvement
in plant defense. WRKY62 is a negative regulator of XA21-mediated basal immunity
and physically interacts with the cleaved intracellular domain of XA21 in the nucleus
(Park and Ronald, 2012). In contrast, overexpression of all four WRKYs 62, 28, 71,
76 leads to enhanced resistance against Xoo, suggesting their roles in regulating plant
immunity negatively or positively (Peng et al., 2010). WRKY53 is induced by chitin
and overexpression of WRKY53 increases resistance against M. oryzae (Chujo et al.,
2007). Overexpression of WRKY89 also results in enhanced resistance to M. oryzae
(Wang et al., 2007). In Arabidopsis, it is well established that NPR1 and TGAs
regulated gene expression that is associated with SAR. In rice, TGAs also play a role
in rice immunity. OsTGAP1 is chitin inducible and regulates the expression of genes
related to diterpenoid phytoalexin biosynthesis (Okada et al., 2009). And rTGA2.1
plays a negative role in regulating disease resistance against Xoo (Fitzgerald et al.,
2005).
Phytohormone signaling often crosstalks with and fine-tunes plant immunity against
pathogens. Brassinosteroids (BRs) regulate plant growth and development and also
have a role in plant disease resistance. Rice plants treated with brassinolide are more
resistant to M. oryzae and Xoo (Nakashita et al., 2003). Several PRRs such as FLS2,
EFR, EIX1/2, VE1, likely as well as LYM1/ LYM3/CERK1 all require BAK1 (BRI1-
associated kinase; a. k. a., SERK3) for PTI signaling. BAK1 was originally identified
in the BR pathway as an essential component for BR signaling. The involvement of
BAK1 in BR signaling and PTI suggests a likely inherent crosstalk between BR and
plant defenses. In rice, ET and ABA often act antagonistically to regulate disease
resistance and abiotic stress. Usually, ET enhances disease resistance, whereas ABA
68
weakens it. JA and SA generally have positive roles in regulating resistance against
pathogens in rice (Mei et al., 2006; Jiang et al., 2010).
To exert PTI effect, plants mount an extensive reprogramming of their physiology,
including production of ROS, deposition of callose, synthesis of phytoalexin and
expression of PR genes. There are 17 family of PR genes (PR1-PR17), encoding
hydrolytic enzymes (such as glucanase and chitinase) and defensins that hydrolyze
pathogen cell wall and disrupt pathogen membrane, respectively. Phytoalexins are
secondary metabolites that are de novo synthesized in response to pathogen infection.
Callose deposition often occurs in the cell walls at the infection site to restrict
pathogen further growth. ROS is produced rapidly also at the infection site to directly
kill pathogen and/or functions as a signal for defense response.
In this study, we demonstrated that rice plants over-expressing zinc finger effectors
showed more severe disease symptoms and that the induction of defense-related genes
was moderately inhibited. Because the induction of defense-related genes is
characteristic of PTI, we hypothesized that zinc finger effectors suppress PTI to
promote disease.
4.2 Results
Zinc finger effectors inhibit BAX induced PCD in N. benthamiana
Due to the functional redundancy between zinc finger effectors, M. oryzae mutants
with a single knock-out effector gene are likely to show no detectable changes in their
virulence. We thus investigated the roles of zinc finger effectors in virulence by
69
heterologously expressing them in plants. BAX is a proapoptotic factor of the Bcl-2
family and promotes programmed cell death (PCD) in animals. Although the key
regulators of animal apoptosis (a typical PCD), such as Bcl-2 family proteins and
caspases, have not been found in plant proteomes, some aspects of the PCD molecular
machinery are shared by plants and animals. BAX can cause cell death in plants very
similar to the hypersensitive response, a mechanism by which plants sacrifice cells at
the infection site to restrict pathogen growth and spread. To test whether zinc finger
effectors can inhibit BAX- induced PCD in N. benthamiana, we adopted two
strategies. First, we cloned effector gene and murine BAX gene in the viral vector
pGR106, respectively. Agrobacterium GV3101 carrying the effector gene was
infiltrated into a spot of N. benthamiana and 24 hours later Agrobacterium GV3101
harboring the BAX gene was infiltrated into the same spot. A period of 24 hours is a
hiatus for zinc finger effectors to be expressed first in the primarily transformed cells
and and then in the neighboring cells. All zinc finger effectors MGG_07834,
MGG_09035, MGG_08230, MGG_10238, MGG_13665, MGG_10020 and
MGG_10556 inhibited BAX-induced PCD, while the EGFP control did not (Figure 4-
1 A). Second, in order to express the effector and BAX genes in the same cell and at
the same time, we inserted a ribosomal stuttering signal from Thosea asigna virus
between the effector gene and BAX gene. The effector gene : Ta2A : BAX fusions were
cloned into pMDC32 vector and introduced into N. benthamiana by agroinfiltration.
MGG_07834, MGG_10020 completely inhibited BAX-induced PCD, while
MGG_09035, MGG_08230, MGG_10238, MGG_13665, MGG_10556 and AVR-Pii
did not (Figure 4-1 B). This time, we added AVR-Pii and it did not inhibit BAX-
induced PCD. Compared with the results from the first strategy, MGG_09035,
MGG_08230, MGG_10238, MGG_13665 and MGG_10556 did not inhibit BAX-
70
induced cell death. And this may be due to the second strategy being more sensitive
and strict.
Figure 4-1. Zinc-finger effectors suppress BAX-induced PCD when transiently
expressed in N. benthamiana via Agrobacterium infiltration.
A. 1. pGR106/MGG_07834+pGR106/BAX. 2. pGR106/MGG_09035+pGR106/BAX. 3.
pGR106/MGG_08230+pGR106/BAX. 4. pGR106/MGG_10238+pGR106/BAX. 5.
pGR106/MGG_13665+pGR106/BAX. 6. pGR106/MGG_10020+pGR106/BAX. 7.
pGR106/MGG_10556+pGR106/BAX. 8. pGR106/EGFP+pGR106/BAX.
A1
2
3
45
6
7
8
8
B 1
2
3
4
5
9
6
7
8
9
71
B. 1. pMDC32/MGG07834:Ta2A:BAX. 2. pMDC32/MGG09035:Ta2A:BAX. 3.
pMDC32/MGG08230:Ta2A:BAX. 4. pMDC32/ MGG10238:Ta2A:BAX. 5.
pMDC32/MGG13665:Ta2A:BAX. 6. pMDC32/ MGG10020:Ta2A:BAX. 7.
pMDC32/MGG10556:Ta2A:BAX. 8. pMDC32/ AvrPii:Ta2A:BAX. 9.
pMDC32/EGFP:Ta2A:BAX.
Zinc finger effectors inhibit BAX induced PCD in rice protoplasts
To further demonstrate that zinc finger effectors are genuine BAX-induced PCD
inhibitors, we cloned the effector gene : Ta2A : BAX fusions into pUGW11 vector. We
chose MGG_07834 and MGG_10020 for this experiment, because they consistently
showed BAX-induced PCD inhibition in N. benthamiana in previous experiments.
Rice protoplasts transfected with pUGW11/MGG_07834 : Ta2A : BAX,
pUGW11/MGG_10020 : Ta2A : BAX and pUGW11/GST : Ta2A : BAX (control) were
observed for fluorescence after fluorescein diacetate (FDA) staining. FDA
accumulates inside the plasmalemma of viable protoplasts can be detected by
fluorescence microscopy. Compared with the control, BAX-induced cell death was
largely inhibited by MGG_07834 and MGG_10020 (Figure 4-2). These results
demonstrated that zinc finger effectors have virulent functions inside N. benthamiana
and rice cells.
A B C
72
Figure 4-2. Zinc-finger effectors suppress BAX-induced PCD in rice protoplasts. A.
pUGW11/GST:Ta2A:BAX. B. pUGW11/MGG_07834:Ta2A:BAX. C.
pUGW11/MGG_10020:Ta2A:BAX
The induction of defense genes is compromised in MGG_07834 or MGG_09035
transgenic lines
We cloned MGG_07834 and MGG_09035 into pMDC32 (Curtis and Grossniklaus,
2003) and made transgenic rice lines stably expressing MGG_07834 and MGG_09035.
We obtained 24 and 20 lines for MGG_07834 and MGG_09035, respectively.
For each gene, three lines were chosen for the following experiments. MGG_07834
and MGG_09035 transgenic lines and their genetic background rice cultivar Kitaake
(Wide type, WT) were inoculated with M. oryzae virulent isolate 70-15 and leaf
tissues were harvested 0, 24 and 48 hours post inoculation for RT-PCR. Five rice
defense-related genes, OsPAL, OsCPS4, OsBBI2-3, OsPR-5 and OsJIPR10, were
tested for their relative expression levels in wild type and transgenic lines. In plants,
phenylalanine ammonia lyase (PAL) is the first and committed step in the
phenylpropanoid pathway and involved in the biosynthesis of polyphenol compounds
such as flavonoids, phenylpropanoids and ligin, which are antimicrobial and effective
against various pathogens. Pathogens and PAMPs such as chitin, PGN and flagellin
can induce PAL gene expression. When induced by M. oryzae, OsPAL levels in
MGG_07834 and MGG_09035 transgenic lines were lower by about 2 folds than in
WT rice plants (Figure 4-3 A). Upon pathogen infection, diterpene phytoalexins such
as momilactones, oryzalexins and phytocassanes are de novo synthesized in cells at
the infection site. OsCPS4 is the first enzyme in the phytoalexin biosynthesis pathway
73
leading to production of momilactone A, B and oryzalexin S from acommon
precursor geranylgeranyl diphosphate. Induction of OsCPS4 gene in MGG_07834 and
MGG_09035 transgenic lines were compromised and the expression levels were
lower by ~3-6 folds in these transgenic lines than in WT (Figure 4-3 B). OsBBI2-3 is
a Bowman–Birk serine protease inhibitor and induced by wounding, pathogens and
herbivores in rice. Upon M. oryze infection, induction of OsBBI2-3 was decreased by
about 2 folds in MGG_07834 and MGG_09035 transgenic lines than in WT plants
(Figure 4-3 C). PR5 and PR10 are two families of pathogenesis-related genes and
encode thaumatin and ribonuclease-like structure proteins, respectively. In rice,
OsPR5 and OsJIPR10 are induced by pathogens, herbivores and phytohormones such
as ET and JA. After inoculation of M. oryzae, the induction of OsPR5 and OsJIPR10
were slightly compromised. The relative expression of OsPR5 and OsJIPR10 are
lower by ~0.5 fold in MGG_07834 and MGG_09035 transgenic lines than in WT
plants (Figure 4-3 D, E). In summary, the trend is evident that the induction of rice
defense related genes by M. orzyae was moderately compromised in zinc finger
effector transgenic lines.
74
0
1
2
3
4
5
6
Rela
tive q
uan
tity
OsPAL
A
0
1
2
3
4
5
6
7
8
Rela
tive q
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tity
OsCPS4
B
75
OsBBI2-3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Rela
tive q
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tity
C
OsPR5
0
0.5
1
1.5
2
2.5
Rela
tive q
uan
tity
D
76
Figure 4-3 Suppression of induction of defense related genes in MGG_07834 and
MGG_09035 transgenic lines after M. oryzae inoculation. A. OsPAL. B. OsCPS4. C.
OsBBI2-3. D.OsPR5. E. OsJIPR10. Values are the means of three replications and
error bars represent the SE (n=3).
MGG_07834 or MGG_09035 transgenic lines are more susceptible to M. oryzae
infection
We examined disease severity of MGG_07834 and MGG_09035 transgenic lines
caused by M. oryzae isolates 70-15 and Guy11-GFP. Six days post inoculation of M.
oryzae 70-15, disease severity indices such as lesion size and lesion number were
measured. In comparison with those of WT plants, lesion size and lesion number of
MGG_07834 and MGG_09035 transgenic lines were significantly increased (n=6,
P<0.01) (Figure 4-4 A, B). The macroscopic disease severity of WT, MGG_07834
and MGG_09035 transgenic lines is shown in Figure 4-5 A. We also inoculated WT
and transgenic lines by the punch method that is more suitable to measure the subtle
difference caused by basal level disease resistance (Ono et al., 2001). Compared with
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Rela
tive q
uan
tity
OsJIPR10E
77
WT, MGG_07834 and MGG_09035 transgenic lines are more susceptible to Guy11-
GFP, as shown by the inoculation spot size and GFP intensity (Figure 4-5 B).
Figure 4-4 Lesion sizes and Lesion numbers of MGG_07834 and MGG_09035 transgenic
lines post M. oryzae inoculation. A. Lesion sizes. B. Lesion numbers. Values are the means
0
1
2
3
4
5
6
7
WT MGG_07834 MGG_09035
Lesio
n S
ize (
mm
)
* ** *
A
0
5
10
15
20
25
30
35
40
WT MGG_07834 MGG_09035
Lesio
n n
um
ber
**
**
B
78
of three replications and error bars represent the SE (n=6, **P<0.01).
Figure 4-5 Disease severity of MGG_07834 and MGG_09035 post spray and punch
inoculation of M. oryzae strains, respectively. A. Spray inoculation. B. Punch inoculation.
4.3 Discussion
This study demonstrated that zinc finger effectors suppress rice innate immunity by
silencing a wide variety of defense-related genes. The products of these defense genes vary
from enzymes of phenylpropanoid synthesis to enzymes of phytoalexin production, from
serine protease inhibitors to thaumatins to ribonucleases. All these low molecular weight
products, either proteins or secondary metabolites, contribute to the basal defense against
pathogens. Is the expression of these defense genes controlled by several master
transcription factors or by disparate regulators? Mapping the binding sites across the rice
genome for this family of zinc finger effectors is the key to solving this question. Primary
SELEX results for MGG_07834 are promising and CHIP-exo mapping of MGG_07834
and MGG_10020 binding sites throughout the rice genome is underway.
WT
MGG07834
MGG09035
AB
79
Pathogen effector biology study often serendipitously reveals novel components or
mechanisms of plant signaling pathways that otherwise defy exploring. In this study,
HIRA is identified as an interactor of M. oryzae zinc finger effectors, not only expanding
the functions of HIRA in gene silencing, but also revealing chromatin status changing as
an important mechanism of defense gene regulation in plant innate immunity. The next
challenge is to find out all essential players in the ZFE/HIRA silencing complex such as
the predicted histone modifiers and transcription repressors. Yeast two hybrid screens and
in planta coimmunoprecipitation followed by mass spectrometry will be the chosen
practices to find these ZEF/HIRA interactors.
Based on the data obtained in this study, we propose a simple model for how M. orzyae
zinc finger effectors suppress rice defense, in particular, the chitin (GlcNAc)n-CEBiP
mediated PTI (Figure 4-6). Upon M. oryzae infection, rice cells are alerted to the
recognition of the fungal chitin fragments by CEBiP and deploy the CEBiP/OsCERK1
initiated PTI against the pathogen. To defeat PTI, zinc finger effectors from M. oryzae
accumulate in the BIC and later translocate into rice cells. Inside rice cells, zinc finger
effectors are localized in the nuclei where they bind specifically to promoters of their target
genes such as defense-related genes in the PTI pathway and recruit HIRA to form
ZEF/HIRA silencing complex. The ZFE/HIRA silencing complex creates a repressive
chromatin state at the promoters of these genes possibly by directing the activities of
histone modifiers such as HAD6 and PRC2. Eventually defense-related genes are silenced
and PTI suppressed. Rice sheath live-imaging revealed that zinc finger effectors, like M.
oryzae BAS1, PWL2 and U. maydis Cmu1, can move from infected cells to neighboring
cells ahead of invasive hyphae. This phenomenon can be explained by this model: zinc
finger effectors enter neighboring cells to create a chromatin repressive state at the
80
promoters of defense-related genes, thereby priming the neighboring cells for an ensuing
fungal invasion.
Figure 4-6 Model of M. oryzae ZFEs suppress chitin (GlcNAc)n-CEBiP/OsCERK1
mediated rice immunity.
4.4 Materials and methods
Rice protoplast preparation, transfection and FDA staining
Fusion fragments of GFP:Ta2A:BAX, MGG_07834:Ta2A:BAX and
MGG_10020:Ta2A:BAX were made by overlap extension PCR and cloned into vector
pUGW11. Rice protoplasts were prepared and transfected as described in the Materials
and methods of Chapter 3. Twenty four hours after transfection, protoplasts were stained
HIRAZFE
PRC2
HDA6
H3k27
me3
H3K27
me3
H3
ac
X
CEBiP
OsCERK1
(GlcNAc)n
MAPK
cascade
TF
Permissive chromatin Repressive chromatin
ZFEs
ZFE/HIRAsilencing complex
BIC
Nucleus
81
with a solution of 0.01% (w/v) fluorescein diacetate (FDA) in acetone. Fluorescence was
observed on an epifluorescence stereomicroscope (Leica) with UV filter. Excitation and
emission wave lengths were 360/40 nm and 420 nm, respectively.
Rice transformation
MGG_07834 and MGG_09035 were recombined into the gateway vector pMDC32 under
the control of the CaMV 35S promoter. Constructs were introduced into A. tumafesciens
strain EHA 105 by electroporation. Vigorously growing rice calli induced from embryos of
the Japanese cultivar Kitaake were used for Agrobacterium-mediated gene transformation
as described by Xiong and Yang (2003). T0 lines were self-pollinated and the resulting T1
seeds were germinated in 50 µg/ml of hygromycin solution. Hygromycin resistant seeds
were planted in Metro Mix 360 (Sun Gro) soil for T2 seeds. A fraction of T2 seeds were
soaked in 50 µg/ml of hygromycin solution for germination. The rest of T2 seeds with
100% germination rate in hygromycin solution were kept as lines with homozygous
genotype for MGG_07834 or MGG_09035.
RNA extraction and cDNA synthesis
Approximately 100 mg of rice leaf tissues were harvested at 0, 24 and 48 hours post
inoculation of M. oryzae strain 70-15 and ground in liquid nitrogen to powders. RNA was
extracted using 1 ml of TRIzol reagent (Invitrogen) according to the manufacturer’s
instructions. Traces of DNA were removed by treating RNA samples with DNaseI (New
England Biolabs). cDNA was synthesized using High Capacity Reverse Transcription Kit
(Applied Biosystems) according to the manufacturer’s protocol.
82
Quantitative RT-PCR for defense-related genes
Quantitative RT-PCR was conducted using Step One Plus Real-Time PCR system
(Applied Biosystems). PCRs were conducted following the protocol of DyNAmo SYBR
green PCR Kit (New England Biolabs). Programs for qRT-PCR were as follows: 15 min at
94ºC for denaturation; 40 cycles of 10 sec at 94ºC for denaturation and 1 min at 60ºC for
amplification and extension. Gene expression levels were calculated using the ∆∆Ct
method and normalized relative to rice ubiquitin gene.
Rice blast inoculation and disease severity assay
M. oryzae isolates 70-15 and Guy11 were used in this study. Fungi were grown on oatmeal
agar under fluorescent light at room temperature. After 7~8 days, conidia were harvested
and adjusted to a concentration of 5×105 spores/ml in 0.1% (v/v) of Tween 20 and 0.25%
of (w/v) gelatin. 2-week-old plants were used for spray inoculation or punch inoculation.
The spray or punch inoculated plants were placed at a container with water to keep
moisture for 24 hours at room temperature and then moved to a Conviron growth chamber
set at 12:12 photoperiod with temperature of 28ºC/24ºC (day/night). Six to seven days
later, disease indices of the spray inoculated plants were scored by measuring and counting
the sizes and numbers of lesions on 6 leaves, respectively. Leaves from punch inoculated
plants were photographed using fluorescence stereomicroscope (Leica) with a GFP filter.
The excitation and emission wave lengths of GFP were 425/60nm and 480nm,
respectively.
83
Chapter 5
Role of the rice endogenous peptide elicitor OsPep1 in defense signaling and disease
resistance
5. 1 Introduction
Plant endogenous elicitors, also known as damage-associated molecular patterns
(DAMPs), are usually molecules released from broken components of plant cell walls or
intracellular precursor proteins processed upon pathogen attack and/or wounding. Like
pathogen-associated molecular patterns (PAMPs), DAMPs also elicit pattern triggered
immunity (PTI) against pathogens or insect herbivores.
Oligogalacturonides (OGs) are released from broken pectin of plant cell walls by bacterial
or fungal secreted polygalacturonases and can induce defense response in various plants
(D’Ovidio et al., 2004). Exogenous application of OGs protects grapevine leaves against
the necrotrophic fungal pathogen Botrytis cinerea (Aziz et al., 2004). In Arabidopsis, the
pattern recognition receptor (PRR) for OGs is identified as WAK1 (Brutus et al., 2010).
WAK1 is a wall-associated epidermal growth factor (EGF)-domain containing receptor-
like kinase and its ectodomain specifically binds to the elicitor active form of “egg-box”
type of OGs to transduce the signal (Cabrera et al., 2008). Arabidopsis plants
overexpressing WAK1 exhibited increased resistance against necrotrophic pathogens B.
cinerea and Pectobacterium carotovorum (De Lorenzo et al., 2011).
In addition to carbohydrate DAMPs, proteinaceous DAMPs are also discovered as strong
84
plant endogenous elicitors. Systemin, an 18-amino acid (aa) peptide, is processed from the
C-terminal region of its precursor prosystemin and can induce defense response against
herbivorous insects in many solanaceous plants (Pearce et al., 1991). Systemin is the first
peptide signal reported in plants. The receptor for systemin in tomato was isolated as
SR160, the tomato ortholog of Arabidopsis brassinosteroid receptor BRI1 (Scheer et al.,
2002). However, BRI1/SR160 being the common receptor for both brassinosteroids and
systemin is debatable, as demonstrated by the finding that tomato BRI1/SR160 cu-3/curl-3
mutants, while being insensitive to brassinosteroid, were still fully responsive to systemin
(Holton et al., 2007).
AtPeps, a 6-member family of 23-aa plant elicitor peptides (Peps) in Arabidopsis, were
found to induce expression of pathogenesis-related (PR) gene PR1, defensin gene PDF1.2
and their precursor genes AtPROPEPs (Huffaker et al., 2006; Huffaker and Ryan, 2007).
Arabidopsis plants overexpressing PROPEP1 showed enhanced resistance to the
necrotrophic oomycete root pathogen Pythium irregular (Huffaker et al., 2006) and wild-
type plants pretreated with AtPep1 became more resistant to the hemibiotrophic bacterial
pathogen Pseudomonas syringae pv. tomato DC3000 (Yamaguchi et al., 2010).
Orthologous genes of AtPROPEP were found in many plant species including canola,
soybean, potato, maize and rice (Huffaker et al., 2006), suggesting that Pep signaling may
be widely involved in plant defense against various biotic stresses. Indeed, ZmPROPEP1,
the Arabidopsis AtPROPEP1 gene ortholog in maize, was induced by JA, ZmPep1 and a
fungal pathogen Cochliobolis heterostrophus and pretreatment with ZmPep1 of maize
leaves and stems enhanced resistance to necrotrophic anthracnose stalk rot pathogen C.
heterostrophus and hemibiotrophic southern leaf blight pathogen Colletotrichum
graminicola, respectively (Huffaker et al., 2011). Furthermore, ZmPep3, another Pep from
85
the 5-member family of maize Peps, was shown to induce herbivore-specific defense
responses against insect Spodoptera exigua and its precursor gene ZmPROPEP3 was
induced by the oral secretions of S. exigua (Huffaker et al., 2013). These new findings
expand the spectrum of resistance elicited by Peps from against pathogens to insects. Like
systemin, lacking a typical N-terminal signal peptide, Peps are likely to be released into the
apoplast from injured cells upon pathogen/insect attack and/or wounding. The apoplastic
Peps then function as DAMPs, binding receptors at the surface of neighboring cells to
elicit defense against pathogens or insects. Two pattern recognition receptors (PRRs) for
AtPeps were identified as PEPR1 and PEPR2 with overlapping functionalities (Yamaguchi
et al., 2006; Yamaguchi et al., 2010; Krol et al., 2010).
Like the perception of PAMPs by PRRs, upon interaction of AtPeps and PEPR1/2, plant
cells rapidly activate defense responses by undergoing a cascade of cellular and molecular
events such as Ca2+
influx, production of reactive oxygen species (ROS), MAPK
activation, ethylene (ET) production and defense gene expression. Aside from the
induction of defense genes, AtPeps as well as many PAMPs such as flg22 also activate the
expression of the precursor genes AtPROPEPs, generating more AtPeps to act as DAMPs.
There is a positive feedback loop inherent in plant innate immunity. A current view is that
the AtPep/PEPR system plays the role of an amplifier of the PAMP/PRR initiated plant
immunity by facilitating the rapid buildup and maintenance of plant defense responses in
the face of invading pathogens (Huffaker and Ryan, 2007).
In rice, it is well known that several PAMP perception systems function as surveillance
systems over bacterial and fungal pathogens. PAMPs such as bacterial flagellin, quorum
sensing protein Ax21 and fungal chitin are all potent elicitors of rice defense responses. In
86
rice, PRR for flagellin was identified as OsFLS2, an ortholog of the Arabidopsis FLS2 and
heterologous expression of OsFLS2 in Arabidopsis fls2 mutants restores flg22
responsiveness (Takai et al., 2008). Interestingly, the rice Ax21/XA21 system also seems
to exist in Arabidopsis where Ax21 is recognized by the same flagellin receptor FLS2
(Danna et al., 2011). Arabidopsis and rice also share chitin elicitor receptor-like kinase
(CERK1) in chitin signaling. In Arabidopsis, upon perception of chitin, CERK1 forms
homodimers to transduce chitin signaling, whereas in rice chitin elicitor binding protein
CEBiP and OsCERK1 interact to signal intracellularly (Liu et al., 2012; Shimizu et al.,
2010). In addition, OsWAK1, a distant ortholog of AtWAK1, plays an important role in
mediating disease resistance against rice blast pathogen Magnaporthe oryzae (Li et al.,
2009), although evidence for OGs as DAMPs of OsWAK1 is yet to be obtained. Therefore
many P/DAMP/PRR systems are well conserved across divergent plant taxa and it is
plausible to propose Peps may also function in rice and other plants. In this study, we make
the first step to demonstrate that rice endogenous elicitor OsPep1 is an active DAMP and
plays a role in defense signaling and disease resistance.
5.2 Results
OsPep1 belongs to a 7-member family of potential endogenous peptide elicitors in rice
Based on the 23-aa AtPep1 sequence, we blasted the Rice Genome Annotation Project
Database and identified 7 predicted proteins with a C-terminal amino acid sequence close
to that of AtPep1(Table 5-1). The genes encoding these 7 proteins are designated
OsPROPEP1-7, and the putative mature peptides from the C-termini of these proteins
OsPep1-7. OsPROPEP1 resides alone on chromosome 4, while the other 6 OsPROPEPs
87
are located in tandem within a 33 kb region on chromosome 8. Furthermore, we checked
Genevestigator and RED microarray databases for expression of these genes. Except for
OsPROPEP4, which we cannot find any information on its expression, the others are
activated by 2~3 by different inducers such as pathogen, insect and phytohormones (Table
5-1). Of the 7 OsPROPEPs, OsPROPEP1 is induced by M. oryzae and ethylene (Table 5-
1), and more importantly, OsPROPEP1 has all the conserved motifs typical of precursors
of established bioactive peptide elicitors AtPep1 and ZmPep1 (Figure 5-1 A; Huffaker et
al., 2006; Huffaker et al., 2011). Like AtPep1 and ZmPep1, OsPep1 is 23-aa peptide
processed from the C-terminus of its precursor OsproPep1 likely through a biogenesis
pathway similar to systemin. OsPep1 is closely related to ZmPep1 (Figure 5-1 B),
suggesting that it probably also functions in rice. We chose OsPep1 and its precursor
encoding gene OsPROPEP1 for further study.
Table 5-1 OsPROPEPs and the putative mature peptides
Gene name Gene locus Annotated
length of
precursor
protein (AA)
Predicted processed C-terminal
23- aa elicitor peptide
Inducers of
expression*
OsPropep1 Os04g54590 159 ARLRPKPPGNPREGSGGNGGHHH M. oryzae; ET
OsPropep2 Os08g07600 93 DDSKPTRPGAPAEGSGGNGGAIH M. oryzae; Chitin; JA
OsPropep3 Os08g07630 168 ADSAPQRPGAPAEGAGGNGGAVH M. oryzae
OsPropep4 Os08g07640 137 ADSAPQRPGAPAEGAGGNGGDVH N/A
OsPropep5 Os08g07660 182 ADSAPQRPGSPAEGAGGNGGAVH Chitin
OsPropep6 Os08g07670 172 SKPKPEPPGYPREGGGGNGGVVD N. lugens
OsPropep7 Os08g07690 111 RAMPRSERPVLREGNGGKGGAHH M. oryzae
*Gene induction information is from Genevestigator and RED microarray databases. N/A, not available.
88
Figure 5-1 OsproPep1 and its mature peptide OsPep1. A. The amino acid sequence of
OsproPep1 and OsPep1. Conserved motifs are KEKE motif (in blue), amphipathic helix
motif (in purple) and elicitor active OsPep1 (in red). B. The relationship between OsPep1
and other Peps. The dendrogram was generated based on a multiple sequence alignment of
7 OsPeps, 5 ZmPeps and 7 AtPeps.
OsPROPEP1 is induced by M. oryzae and JA and ET
OsPROPEP1 have two transcripts as a result of alternative splicing. The splicing occurs
OsproPep1
MDRVEEKEGN RFQEPASDRC EDNEDKEQDN SEESSSVDQR KEEEEEEKEG CEEATPAAAA 60
AAAAPSFFAH PCSLLQYIAR VCACCLGLSD SFCDPKASSV LVPEPEPAAA DPSQEGEEDM 120
KSSYFYMQEA TTRVRAARLR PKPPGNPREG SGGNGGHHH* 159
A
B
89
immediately upstream of the C-terminal region encoding OsPep1, producing a precursor
with 5 fewer amino acids. The seemingly frequent occurrence of alternative splicing in
PROPER gene transcription may have a role in tightly regulating the biogenesis of Peps
from their precursors (Huffaker et al., 2011). Time-course analysis of the expression of
OsPROPEP1 after M. oryzae 70-15 inoculation showed that the level of OsPROPEP1 in
rice leaves increased 1 day post inoculation (DPI) and peaked 2 DPI and then gradually
fell. In comparison with mock (water) inoculation, M. oryzae induced just over 2.5-fold
change in OsPROPEP1 expression but with statistical significance (P<0.05; Figure 5-2 A).
The expression of AtPROPEP1 changed little upon treatment with various pathogens or
PAMPs, whereas ZmPROPEP1 and ZmPROPEP3 were highly induced by pathogen and
insect oral secretions, respectively (Huffaker et al., 2006; 2011; 2013). The observation of
the difference in induction of PROPEPs indicates that Peps as DAMPs play different roles
in eliciting defenses against pathogens or insects.
We further tested the responsiveness of OsPROPEP1 to JA and ET. 100 µM of methyl
jasmonate (MeJA) or 100 µM of ET precursor 1-aminocyclopropane-1-carboxylic acid
(ACC) was sprayed onto 2-week-old rice seedlings. Rice leaves were harvested at the
indicated time points and used to prepare RNA for detection of OsPROPEP1 transcripts by
qRT-PCR. In comparison with water, both JA and ACC induced the expression of
OsPROPEP1 to a moderately high level at 1 hour post treatment. At its peak, OsPROPEP1
transcripts increased 2.5- and 3.5-fold with statistical significance by JA and ACC,
respectively (Figure 5-2 B). In rice, defense hormones SA, JA and ET play different roles
in disease resistance. With a high basal level in rice, SA changes little under various
abiotic and biotic stresses and is regarded as a minor regulator of rice defense (Silverman
et al., 1995; Yang et al., 2004). In contrast, ET and JA play prominent roles in rice defense
90
against pathogens (Singh et al., 2004; Helliwell et al., 2013; Mei et al., 2006).
OsPROPEP1 is induced by JA and ET (Figure 5-2 B) but not by SA (data not shown),
suggesting that ET and JA play major roles in OsPep1 signaling.
Figure 5-2 Induction of OsPROPEP1 by various inducers. A. Induction of OsPROPEP1
by M. oryzae. B. Induction of OsPROPEP1 by JA (100 µM) and ACC (100 µM). Values
represent means ± SE from three replications. Asterisks indicate statistically significant
0
0.5
1
1.5
2
2.5
3
3.5
4
0 1 2 3 4
Rela
tive q
uan
tity
Days post M. oryzae inoculation
Water
Blast
OsPROPEP1
*
*
A
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 2 4
Rela
tive q
uan
tity
Hours post hormone treatment
Water
JA
ACC
OsPROPEP1
*
**B
91
differences compared with water treatment (* and ** indicate P<0.05 and 0.01,
respectively; Student’s t test).
OsPep1 induces the expression of its precursor gene and rice defense genes and
enhances disease resistance against M. oryzae
In addition to OsPROPEPs, two putative OsPep receptors OsPEPR1 and OsPEPR2 are
also encoded in the rice genome at loci Os08g34640 and Os10g06760, respectively,
lending another support to the hypothesis that the Pep/PEPR system also functions in rice.
To test whether OsPep1 is perceived in rice and whether OsPep1 is biologically active in
eliciting rice defense response, the 23-aa OsPep1 was synthesized and used to feed rice
seedlings at a concentration of 1 µM. Rice seedlings at the indicated time points were
harvested and RNA were extracted and used to detect the expression of OsPROPEP1,
OsPR5 and OsPAL. At 6 h, the expression of OsPROPEP1 was 4.5 fold higher in the
OsPep1-supplied plants compared with water-supplied control. OsPROPEP1 transcripts
continued to accumulate and reached a peak at 12 h where they were 6.5-fold richer in
OsPep1-supplied plants in comparison with water-supplied plants. And at 24 h,
OsPROPEP1 expression was still 5-fold higher in OsPep1-treated plants relative to control
plants (Figure 5-3 A). As a result, consistent with the early findings with AtPep1 and
ZmPep1 (Huffaker et al., 2006; 2011), OsPep1 signals via a positive feedback loop. OsPR5
and OsPAL are two well-established pathogenesis-related (PR) genes that are induced by
ET, JA and M. oryzae. We tested the effect of OsPep1 on the expression of these two
genes. The transcripts of OsPR5 accumulated to a peak level 8-fold higher in the OsPep1-
supplied plants compared with water-supplied plants at 6 h. And at 12 h, the expression of
OsPR5 was still higher by 5-fold (Figure 5-3 B). The expression of OsPAL is 4-fold higher
92
at 12 h in OsPep1-supplied plants compared with water-supplied plants (Figrue 5-3 C). In
rice, upon pathogen attack, ET and JA often cooperate to regulate the expression of a
plethora of defense genes including OsPR5 and OsPAL. As such, it is unclear whether
OsPep1 induces the expression of its precursor gene and specific defense genes through a
distinct JA or ET pathway or both pathways.
Because M. oryzae induced the expression of OsPROPEP1 and because OsPep1-supplied
plants had elevated PR gene expression, we inferred that OsPep1 would enhance disease
resistance against M. oryzae. To test this hypothesis, 2-week-old rice seedlings were
sprayed with 1µM OsPep1 and 12 h after OsPep1 treatment, rice seedlings were inoculated
with M. oryzae 70-15. Six days after M. oryzae inoculation, disease symptoms and severity
were compared between OsPep1- and water-treated plants. For water-sprayed plants, 70%
of leaf tips were wilted, whereas only 10% of leaf tips of OsPep1-sprayed plants were
withered (Figure 5-4 A). More quantitatively, lesion size and lesion number were evaluated
on water- and OsPep1-sprayed plants. Both disease severity indices were lower with
statistical significance for OsPep1-sprayed plants compared with water-sprayed plants
(P<0.01; Figure 5-4 B and C).
93
**
*
A
*
0
2
4
6
8
10
12
0 0.5 1 6 12 24
Rela
tive q
uan
tity
Hours post treatment
Water
OsPep1
OsPROPEP1
B
**
**
*
0
5
10
15
20
25
0 0.5 1 6 12 24
Rela
tive q
uan
tity
Hours post treatment
Water
OsPep1
OsPR5
C
**
*
0
2
4
6
8
10
12
14
0 0.5 1 6 12 24
Rela
tive q
uan
tity
Hours post treatment
Water
OsPep1
OsPAL
94
Figure 5-3 OsPep1 (1µM) induces expression of its precursor gene and defense genes. A.
Time-course induction of OsPROPEP1. B. Time-course induction of OsPR5. C. Time-
course induction of OsPAL. Values represent means ± SE from three replications. Asterisks
indicate statistically significant differences compared with water treatment (* and **
indicate P<0.05 and 0.01, respectively; Student’s t test).
OsPep1Water
A
0
0.5
1
1.5
2
2.5
3
3.5
4
Water OsPep1
Lesio
n s
ize (
mm
)
**
B
95
Figure 5-4 OsPep (1µM) induces resistance against M. oryzae. A. Comparison of effects of
water and OsPep1 on M. oryzae infection. B. Lesion size 6 days post inoculation of M.
oryzae. C. Lesion number 6 days post inoculation of M. oryzae. Values are the means of
three replications and error bars represent the SE (n=6, **P<0.01).
OsPROPEP1-overexpression lines exhibit growth inhibition and seed-set reduction
One way to ascertain a functioning DAMP/PRR system in plants is to characterize
transgenic plants constitutively producing DAMP. Plants overexpressing prosystemin
established the role of systemin in plant defense against herbivores (McGurl et al., 1994).
Similarly, overexpression of a fungal polygalacturonase in Arabidopsis and tobacco helped
to elucidate the role of the OG/WAK1 system in plant defense against pathogens (Ferrari et
al., 2008; Capodicasa et al., 2004). Likewise, Arabidopsis plants overexpressing
PROPEP1 and PROPEP2 facilitated the revelation of the role of the Pep/PEPR system in
plant defense against pathogens (Huffaker et al., 2006). To further establish that an active
Pep/PEPR system also exists in rice, we sought to make transgenic plants overexpressing
0
5
10
15
20
25
Water OsPep1
Lesio
n n
um
ber
**
C
96
OsPROPEP1. The construct CaMV 35S:PROPEP1 was introduced into a fast-growing
Japanese rice variety Kitaake by Agrobacterium-mediated transformation. Three
independent OsPROPEP1-overexpression (OX) lines, designated OsPROPEP1-OX1, 2
and 3, were chosen for further analysis.
Compared with wild-type (WT) Kitaake, OsPROPEP1-OX lines were dwarf and yellowish
(Figure 5-5A). Tracking the growth week by week, we found that the height of
OsPROPEP1-OX lines was about half that of the wild-type plants at weeks 7 and 8 when
the plants completed the vegetative growth and entered the reproductive stage (Figure 5-5
B). In addition, seed-set rate of OsPROPEP1-OX lines was lowered approximately by half
in comparison with that of wild-type Kitaake (Figure 5-5 C). Little difference in plant
height or seed-set rate was found between the three OsPROPEP1-OX lines. The dwarf
phenotypes were also observed with transgenic tobacco plants overexpressing an OG-
generating polygalacturonase and tomato plants overexpressing prosystemin (Capodiscasa
et al., 2004; McGurl et al., 1994). Although Arabidopsis plants overexpressing PROPEP1
and PROPEP2 exhibited increased root and aerial growth (Huffaker et al., 2006), seedlings
growing in MS medium supplemented with 1µM AtPep1 showed pronounced growth
retardation (Krol et al., 2010). Consistent with these early findings, our results revealed a
tradeoff in rice between plant development and OsPep/OsPEPR-mediated plant defense.
97
WT OsPROPEP1-OX
A
B
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Pla
nt h
eig
ht
(cm
)
Week
WT
OX-1
OX-2
OX-3
98
Figure 5-5 Comparison phenotypes of wild-type and OsPROPEP1-OX plants. A. Statures
of wild-type and OsPROPEP1-OX plants at 10-week-old stage. B. Growth curves of wild-
type and OsPROPEP1-OX plants. C. Seed-set of wild-type and OsPROPEP1-OX plants.
An array of defense genes are constitutively expressed in OsPROPEP1-overexpressing
lines
Constant presence of DAMPs in plants usually leads to constitutive expression of an array
of plant defense genes. Tomato plants overexpressing prosystemin accumulated high levels
of serine proteases, peroxidases and many other defense-related proteins (Bergey et al.,
1996). Similarly, tobacco plants overexpressing polygalacturonase, which generates OGs
by degrading pectin, constitutively expressed POX (peroxidase). And Arabidopsis
overexpressing the same polygalacturonase had high basal levels of transcripts for PR-
1(unknown), PDF1.2 (defensin) and AtPGIP1 (Polygalacturonase inhibiting protein)
(Ferrari et al., 2008). Likwise, Arabidopsis plants overexpressing PROPEP1 and
PROPEP2 expressed PR-1 and PDF1.2 at higher levels than those found in wild-type
plants (Huffaker et al., 2007).
0
10
20
30
40
50
60
WT OX-1 OX-2 OX-3
Seed
s p
er
pan
icle
C
99
Eight rice defense-related genes, OsPAL, OsCPS4, OsBBI2-3, OsBBI3-3, OsOsPR-5,
OsVSP, OsJIPR10 and PXO22.3, were tested for their relative expression levels in wild
type and OsPROPEP1 transgenic lines (Figure 5-6 A). In plants, phenylalanine ammonia
lyase (PAL) is the first and committed step in the phenylpropanoid pathway and is
involved in the biosynthesis of polyphenol compounds such as flavonoids,
phenylpropanoids and lignin, which are antimicrobial and effective against various
pathogens. Pathogens and PAMPs such as chitin, peptidoglycan and flagellin can induce
PAL gene expression. The level of OsPAL expression was 2.5~4.5-fold higher in
OsPROPEP1-OX lines as compared with wild-type plants. Upon pathogen infection,
diterpene phytoalexins such as momilactones, oryzalexins and phytocassanes are de novo
synthesized in cells at the infection site. OsCPS4 is the first enzyme in the phytoalexin
biosynthesis pathway leading to production of momilactone A, B and oryzalexin S from
acommon precursor geranylgeranyl diphosphate. OsCPS4 transcripts were 2.5~3.8-fold
more abundant in OsPROPEP1-OX lines than in wild-type plants. OsBBI2-3 and OsBBI3-
3 are two Bowman–Birk serine protease inhibitors and induced by wounding, pathogens
and herbivores in rice. The transcripts of OsBBI2-3 were 3.2~7.8-fold higher and those of
OsBBI3-3 were 1.8~7.5-fold more in OsPROPEP1-OX lines compared with wild-type
plants. PR5 and PR10 are two families of pathogenesis-related genes and encode thaumatin
and ribonuclease-like proteins, respectively. VSP2, i.e., vegetative storage protein 2, is an
acid phosphatase. In rice, OsPR5, OsJIPR10 and OsVSP2 are induced by pathogens,
herbivores and phytohormones such as ET and JA. OsPROPEP1-OX lines exhibited a
2.8~15-fold greater expression of OsPR5 than did wild-type plants. Compared with wild-
type plants, OsPROPEP1-OX lines displayed a 4.2~9.2-fold increase in OsJIPR10
transcript. Similarly, OsVSP2 transcripts were 5.5~8.8-fold more abundant in comparison
100
with wild-type plants. PXO22.3, encoding a peroxidase, is induced by Xanthomonas
oryzae pv. oryzae (Xoo) and M. oryzae. OsPROPEP1-OX lines showed a 3.2~6.3-fold
increase in PXO22.3 transcript compared with wild-type plants.
OsPROPEP1-OX lines enhance disease resistance against M. oryzae
The positive correlation between constitutive expression of defense-related genes and
disease resistance prompted us to test OsPROPEP1-OX lines for basal resistance to M.
oryzae. One M. oryzae highly virulent isolate IE1K was used to inoculate wild-type plants
and OsPROPEP1-OX lines. Although OsPROPEP1-OX lines showed no R-mediated
qualitative resistance, disease symptoms were less severe on OsPROPEP1-OX lines as
compared with wild-type plants when plant leaves were either spayed or spot-inoculated
with IE1K (Figure 5-6 B). Measured by lesion size and lesion number, both disease indices
were lower by nearly half with statistical significance on OsPROPEP1-OX lines in
comparison with wild-type plants (P<0.01; Figure 5-6 C).
0
1
2
3
4
5
6
7
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
OsPALA
101
0
1
2
3
4
5
6
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
OsCPS4
0
2
4
6
8
10
12
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
OsBBI2-3
0
1
2
3
4
5
6
7
8
9
10
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
OsBBI3-3
102
0
2
4
6
8
10
12
14
16
18
20
WT OX-1 OX-2 OX-3
Rela
tive e
xp
ressio
n l
evel
OsPR5
0
2
4
6
8
10
12
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
OsJIPR10
103
0
2
4
6
8
10
12
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
OsVSP2
0
1
2
3
4
5
6
7
8
9
WT OX-1 OX-2 OX-3
Rela
tive q
uan
tity
PXO22.3
104
WT OsPROPEP1-OX WT OsPROPEP1-OXB
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
WT OX-1 OX-2 OX-3
Le
sio
n s
ize
(m
m)
**
** *
*
C
105
Figure 5-6 OsPROPEP-OX lines constitutively express defense genes and enhance disease
resistance against M. oryzae. A. Fold changes of defense gene expression in 2-week-old
OsPROPEP1-OX lines compared with wild-type plants. B. Comparison of disease severity
of wild-type and OsPROPEP1-OX lines 6 days post spray (left) and spot (right)
inoculation of M. oryzae. C. Lesion sizes 6 days post inoculation of M. oryzae. D. Lesion
numbers 6 days post inoculation of M. oryzae. Values are the means of three replications
and error bars represent the SE (n=6, **P<0.01).
5.3 Discussion
Pep signaling is activated upon pathogen or insect attack, suggesting that it plays an
essential role in plant defense against various biotic agents (Huffaker et al., 2006; 2011;
2013). This seemingly widespread component of plant defense mechanism is demonstrated
again by our finding that OsPep1 signaling is also operational in rice basal resistance
against M. oryzae. A current view holds that Pep signaling works as an amplifier of PTI to
help mount and sustain a rapid and prolonged defense at the local infected or infested sites
0
5
10
15
20
25
30
WT OX-1 OX-2 OX-3
Lesio
n n
um
ber
** *
* **
D
106
(Huffaker and Ryan, 2007; Yamaguchi et al., 2010). In line with this model and based on
the experimental data in this study, our explanation for the role of OsPep1 in rice basal
resistance against M. oryzae could be as follows. Upon M. oryzae infection, chitin
oligomers or other yet to be discovered fungal PAMPs promote a series of stereotypical
PTI events, chief among them being induction of an array of rice defense-related genes
encoding PR proteins and signaling molecules. Among the induced signaling molecules
are OsPROPEPs and their receptors OsPROPEP1/2. Released into the apoplast from
OsPROPEP1, OsPep1, together with its receptors, elicits new waves of PTI in neighboring
cells at the infection site. In this way OsPep1 signaling contributes to the basal resistance
against M. oryzae. Most likely, PTI initiated by chitin and strengthened by OsPep may also
play a role in rice basal resistance against another fungal pathogen Rhizoctonia solani.
Furthermore, because rice also senses PAMPs from bacteria such as flagellin, Ax21 and
peptidoglycan, it is plausible to expect a role for OsPep signaling in disease resitance
against bacterial pathogens such as Xoo. In addition, Huffaker and colleagues found that
OsPep2 induced emission of the same spectrum of volatile organic compounds as did
ZmPep3 in maize, indicating a role of OsPep2 signaling in herbivore defense (Huffaker et
al., 2013). Besides OsPep2, OsPep6 also most likely shares a role of eliciting herbivore
defense, as suggested by the observation that OsPROPEP6 was induced by Nilaparvata
lugens (table 5-1).
Pep signaling as an amplifier of PTI is mediated by plant defense hormones, i.e., SA, JA
and ET. PAMPs such as flg22 and elf18 often induce the production of SA and ET (Tsuda
et al., 2008; Felix et al., 1999), which in turn induce the expression of PROPEPs (Huffaker
et al., 2006; Huffaker and Ryan, 2007). Recently, the detailed interconnections between ET
and Pep pathways were revealed. On one hand, Arabidopsis ein2 mutants were elf18
107
insensitive due to the loss of PROPEP2 induction by ET (Tintor et al., 2013). On the other
hand, pepr1/2 mutants were compromised in ET-mediated defense response and growth
inhibition as a result of the defective Pep signaling (Liu et al., 2013). In rice, SA, JA and
ET play differential roles in disease resistance. Due to a relatively high basal level of SA
and lack of measurable induction upon pathogen infection, SA is usually regarded as a
minor contributor to rice disease resistance by serving as an antioxidant (Silverman et al.,
1995; Yang et al., 2004). Compared with SA, ET and JA play major roles in regulating rice
defenses. ET is inducible by M.oryzae and overexpression of OsACS2, a key enzyme of
ET biosynthesis, leads to enhanced basal disease resistance against M. oryzae and R. solani
(Iwai et al., 2006; Helliwell et al., 2013). Moreover, ET antagonizes ABA to promote
biotic defense over abiotic tolerance (Xiong and Yang, 2003; Bailey et al., 2009).
Similarly, rice plants overexpressing OsAOS2, a key enzyme in JA biosynthesis, exhibited
increased PR gene expression and enhanced disease resistance against M. oryzae (Mei et
al., 2006). In rice, JA also antagonizes gibberellin (GA) to prioritize defense before growth
during biotic stress (Yang et al., 2012). Realizing that ET and JA are important players in
rice defense, we tested their roles in OsPep1 signaling. The results that OsPROPEP1 was
induced by ET and JA and that OsPROPEP1-OX lines constitutively expressed ET-and
JA-induced PR genes point to a looming conserved PTI amplification loop of PAMP-
ET/JA-Pep in rice defense. Future dissection of OsPep1 signaling by using ET/JA mutants
such as OsEIN2-RNAi and OsCOI1-RNAi will provide more insights into the Pep pathway.
In this study, we demonstrated that Pep signaling is conserved and functional in rice
defense, promising a prospect of exploiting this integral part of PTI for basal resistance
against devastating rice pathogens such as M. oryzae, R. solani and Xoo. We obtained
OsPROPEP1-OX lines with constitutive PR gene expression and enhanced M. oryzae
108
resistance. Expectedly, OsPROPEP1-OX lines also showed stunted stature and lowered
seed-set rate due to the constant activation of defense. This tradeoff between defense and
growth will be overcome by directing OsPROPEP expression with pathogen inducible
promoters such as PBZ1 promoter (Mei et al., 2006; Helliwell et al., 2013). To make the
most of the Pep signaling in disease resistance, it is also tempting to construct chimeric
receptors by swapping ectodomains or intracellular domains between OsPEPR1/2 and
other rice PRRs such as OsCERK1 and XA21. Rice transgenic lines harboring these
chimeric receptors are likely to provide a new, broadened and strengthened spectrum of
basal resistance against pathogens.
5.4 Materials and methods
OsPROPEP1 construct and rice transformation
OsPROPEP1 full-length cDNA clone (AK062886) was obtained from the Rice Genome
Resource Center, Tsukuba, Japan and used as template to amplify OsPROPEP1 fragment
with primers: 5´-CAC CAT GGA TCG GGT CGA GGA AAA G-3´ and 5´-CTA GTG
ATG GTG TCC TCC ATT G-3´. OsPROPEP1 fragment was first incorporated into vector
pENTRTM
/D-TOPO and then combined into the binary Gateway vector pMDC32. Plasmid
pMDC32/OsPROPEP1 was introduced into Agrobacterium tumefasciens strain EHA 105
by electroporation. Agrobacterium-mediated rice transformation was conducted using calli
derived from rice cultivar Kitaake according to a protocol as described by Xiong and Yang
(2003).
Plant materials and fungal isolates
109
Rice cultivar Kitaake and its OsPROPEP1-OX lines were grown in MetroMix 360 soil
(Sun Gro, Bellevue, WA) in greenhouse with alternating 12 h of light (day) at 28 ºC and 12
h of dark (night) at 24 ºC. M. oryzae isolates 70-15 and IE1K were grown on oatmeal agar
under constant fluorescent light at room temperature. After 7~8 days, conidia were
harvested and adjusted to 5 × 105 spores/ml in a solution containing 0.1% (v/v) of Tween
20 and 0.25% of (w/v) gelatin. For analysis of the effect of OsPep1 treatment on M.
oryzae infection, two-week-old Kitaake seedlings were sprayed with 10 µM OsPep1 and
inoculated with M. oryzae 70-15 24 h post elicitor treatment. For characterization of
OsPROPEP1-OX lines, highly virulent M. oryzae IE1K was used to inoculate two-week-
old seedlings.
Peptide synthesis and chemical treatments
OsPep1 (RAARLRPKPPGNPREGSGGNGGHHH) was synthesized by GenScript
(Piscataway, NJ) and dissolved in water at 1 mM as stock solution. Two-week-old rice
seedlings grown in soil were sprayed with 100 µM of JA or ACC and rice leaves were
harvested at the time points 0, 0.5, 1, 2 and 4 h post hormone treatment. Ten-day-old rice
seedlings grown in liquid MS were transferred to fresh liquid MS medium supplied with 1
µM of OsPep1 and leaves were harvested at the time points 0, 0.5, 1, 6, 12 and 24 h post
elicitor treatment.
Assessment of Agronomic traits and disease severity
The heights of Kitaake and OsPROPEP1-OX lines were measured every week until
110
maturity. Height was scored as the length of the plant aerial part and three plants from each
line were measured. The seed-set rate was indicated by seeds per panicle and three plants
from each line were scored. Two independent biological replicates were conducted in two
growing seasons. Disease severity was assayed on the third leave of each plant 6 days after
M. oryzae inoculation. Each leaf was scored for the number of necrotic lesions and the size
of each of the three largest lesions. Three independent experiments were carried out to
evaluate disease severity.
Quantitative real-time PCR analysis of relative gene expression levels
RNA was isolated from 100~200 mg rice leaves using 1 ml of TRIzol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions. Traces of DNA were removed
by treating RNA samples with DNase I (New England Biolabs, Ipswich, MA). cDNA was
synthesized using High Capacity Reverse Transcription Kit (Applied Biosystems, Foster
City, CA) according to the manufacturer’s instructions. Quantitative real-time PCR was
performed on Step One Plus Real-Time PCR system (Applied Biosystems) with DyNAmo
SYBR green PCR kit (New England Biolabs). Programs for quantitative real-time PCR
were as follows: 15 min at 94ºC for denaturation; 40 cycles of 10 sec at 94ºC for
denaturation and 1 min at 60ºC for amplification and extension. Gene specific primers
were used to amplify the fragments of OsPROPEP1 (AK062886), OsPR5 (AK241419),
OsPAL (AK068993), OsCPS4 (AK100631), OsBBI2-3 (AK064050), OsBBI3-3
(AK243607), OsJIPR10 (AK121376), OsVSP2 (AK100363), PXO22.3 (AK073202) and
OsUBQ1 (AK059011). These primers are as follows: OsPROPEP1 forward primer, 5´-
GAC ATG AAG AGC AGT TAT TTC TAC-3´, and reverse primer, 5´-CTA GTG ATG
GTG TCC TCC ATT G-3´; OsPR5 forward primer, 5´-GCA ACA GCA ACT ACC AAG
111
TC-3´, and reverse primer, 5´-TGT CCC ATG ATA CAT ATA CTA C-3´; OsPAL
forward primer, 5´-TGA ATA ACA GTG GAG TGT GGA G-3´, and reverse primer, 5´-
AAC CTG CCA CTC GTA CCA AG-3´; OsCPS4 forward primer, 5´- CCC CAC CTC
CAC TAC CAA TTC CC-3´, and reverse primer, 5´-AAA ACA CGA GGT ACA CCA
CAA CAA AC-3´; OsBBI2-3 forward primer, 5´- TGT GTT GTC TCG TGT GAA CGA
TGG-3´, and reverse primer, 5´-ACG ATG ACA GCG CAA CAT GGC-3´; OsBBI3-3
forward primer, 5´-CTG CAA GGA CCG CTT CAC CG-3´, and reverse primer, 5´-AGC
TGC AAG CTA GGG CGA GT-3´; OsJIPR10 forward primer, 5´- GAC GCT TAC AAC
TAA ATC GTC-3´, and reverse primer, 5´-CAA TCA CTG CTT GGA AGC AG-3´;
OsVSP2 forward primer, 5´-CCA ACC CTG CCT ACT ACA TCG-3´, and reverse primer,
5´-TAG CTA GGC ATC GTT TGC TTC A-3´; PXO22.3 forward primer, 5´-ATG GCT
TCT GCA ACT AAT TCT TC-3´, and reverse primer, 5´- TTA GGA GTT CAC CTT
GGA GCA GCT G-3´; and OsUBQ1 forward primer, 5´-AAC CAG CTG AGG CCC
AAG AAG-3´, and reverse primer, 5´-ACG ATT GAT TTA ACC AGT CCA TG-3´. Gene
expression levels were calculated using the ∆∆Ct method and normalized relative to the
level of rice ubiquitin gene transcripts.
112
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VITA
Wenhua Liu
Education
2006-present Ph.D. Student in Plant Biology Program, The Pennsylvania State
University.
2005-2006 Ph.D. Candidate, Department of Plant Pathology, University of Arkansas
1990-1993 M.Sc., Department of Plant Pathology, Nanjing Agricultural University
1986-1990 B.Sc., Department of Biology, Southwest Agricultural University
Research Experience
1993-2005 Research Assistant and Lecturer, Biotechnology Research Center,
Zhongshan University. Projects: Map-based Cloning and Characterization of Rice
Wide Compatibility Gene; Study on Rice Disease Resistance Gene Analogs and Their
Applications in Marker-assisted Rice Breeding for Pathogen Resistance; Research on
Molecular Markers specific for Prostate Cancer and Benign Prostate Hyperplasia.
1990-1993 Graduate Student, Department of Plant Pathology, Nanjing Agricultural
University. Thesis: Study on the Virulence Genes of Rice Bacterial Blight Pathogen
Xanthomonas oryzae pv. oryzae.
1986-1990 Undergraduate Student, Department of Biology, Southwest Agricultural
University. Graduation Thesis: Application of Biostatistical Techniques to Assist
Barley Breeding.
Teaching experience
Plant Molecular Biology. Section-The Molecular Biology of Plant-Pathogen
Interactions, yearly, 2003-2005.
Pharmaceutical Biotechnology. Sections-Vaccine, Gene Test and Gene Therapy, 2004.
Publications
Liu, W., Shen, X., Chen, Y., Wang, Q. and Yang, Y. A family of Magnaporthe
oryzae zinc-finger effectors suppresses rice immunity by recruiting in host cells HIRA
for chromatin remodeling. In preparation.
Liu, W., Shen, X., Zhao, X., Wang, Q. and Yang, Y. Role of the rice endogenous
peptide elicitor OsPep1 in defense signaling and disease resistance. In preparation.
Liu, W., Xie, K. and Yang, Y. 2012. Genomic and bioinformatic resources for rice
research. In “Methods in Molecular Biology: Rice Protocols” Ed. Y. Yang, Humana
Press.
Liu Wenhua. 2003. The molecular biology of plant-pathogen interactions. Pp530-564.
in: Plant Molecular Genetics (2nd
edition). Liu Liangshi (ed.) Science Press, Beijing,
China.
Luo Wenyong, Li Xiaofang, Hu Jun, Liu Wenhua, Xiao Xin and Liu Liangshi. 2004.
Application of ordered differential display to isolate rice cDNAs induced by
Magnaporthe grisea. Prog. Biochem. Biophysi. 31(7): 635-641.
Zhong Weide, He Huichan, Liu Liangshi, Liu Wenhua, Jiang Funeng and Wei
Honge. 2004. Primary study on the application of cDNA microarray to diagnose
prostate cancer. Natl. Med. J. China. 84(7): 564-565.
