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Magnaporthe as a Modelfor UnderstandingHost-Pathogen Interactions

Daniel J. Ebbole

Program for the Biology of Filamentous Fungi, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843-2132;email: [email protected]

Annu. Rev. Phytopathol. 2007. 45:437–56

First published online as a Review in Advance on May 9, 2007

The Annual Review of Phytopathology is online atphyto.annualreviews.org

This article’s doi:10.1146/annurev.phyto.45.062806.094346

Copyright c 2007 by Annual Reviews. All rights reserved

0066-4286/07/0908/0437$20.00

Key Words

rice blast, population genetics, signal transduction, fungal

genomics, virulence factors, transposons Abstract

The rice blast pathosystem has been the subject of intense interes

in part because of the importance of the disease to world agriculturebut also because both Magnaporthe oryzae and its host are amenable

to advanced experimental approaches. The goal of this review is toprovide an overview of the system and to point out recent significan

studies that update our understanding of the biology of M. orzyae

The genome sequence of M. oryzae has provided insight into how

genome structure and pathogen population genetic variability hasbeen shaped by transposable elements. The sequence allows system

atic approaches to long-standing areas of investigation, includingpathogen development and the molecular basis of compatible and

incompatible interactions with its host. Rice blast provides an inte-grated system to illustrate most of the important concepts governing

fungal/plant interactions and serves as an excellent starting point for

gaining a broad perspective of issues in plant pathology.

437



INTRODUCTION

The rice blast fungus can be found referenced

in the literature under several names. Pyric-

ularia oryzae was used to refer to the asex-

ual stages of rice blast fungus as it was found

in the field. The rice pathogen was morpho-logically indistinguishable from pathogens of

other hosts, and the entire group was de-fined under the name Pyricularia (or Piricu-

laria) grisea (70). The sexual stage was named

Magnaporthe grisea until it was shown by phy-

logenetic analysis and interstrain fertility teststhat Magnaporthe isolates should be separated

into species that infect Digitaria spp. (crabgrass) ( M. grisea), whereas M. oryzae collec-

tively refers to the other characterized iso-lates, including the rice pathogen (14).

Rice blast has been used as a model to un-

derstand the nature of pathogen populationstructure and the breakdown of plant resis-

tance genes. This has led to efforts to un-derstand the genome of the fungus in or-

der to determine the types of variation thathave allowed it to overcome newly deployed

resistance genes. The lessons learned fromthese studies helped to formulate stategies

for breeding durable resistance in rice. Of-ten in plant pathology, different pathogens are

studied, in part, because they are particularly

suited to examine a specific pathogenic strat-egy. For example, M. oryzae is well known

for its remarkable melanized appressorium, whereas other fungi are best known for

their production of highly selective phyto-toxins (93) or production of plant cell wall–

degrading enzymes (87). With the sequenceof fungal genomes, targeted investigation of

pathogen metabolism, cellular and subcellu-lar differentiation, and signal transduction are

providing a more integrated view of the dif-

ferent strategies employed by an individualfungal pathogen. This can be seen in advances that comparative genomics can bring

to the understanding of developmental pro-

cesses, such as conidiation and appressoriumformation, and in exploring the molecular

events in compatible and incompatible inter-

actions. The tools are now in place to make

rapid advances in understanding the mecha-nisms that are conserved in fungal pathogens

along with the M. oryzae–specific factors thatpermit the fungus to resist and suppress the

innate immunity of its host.

POPULATIONS AND PATHOGENGENOTYPIC VARIATION

The center of origin of rice and rice blastis proposed to be the Uttar Pradesh hills of

the Indian Himalayas (50). Fertile strains ofboth mating types are found from this re-

gion, whereas few studies have found fertilehermaphroditic strains of rice-infecting iso-

lates outside of this region. Furthermore, evi-dence for on-going sexual recombination was

found in this population based on genotyp-ing, despite the fact that the sexual stage has

never been confirmed in the field. The ge-

netic relationship between rice pathogens andpathogens of other hosts suggests a single an-

cient invasion of rice (13). Tracing the centerof origin for M. oryzae prior to its acquisi-

tion of rice as a host remains an interestingquestion. The rice pathogen appears to have

shifted to other grass hosts that grow in asso-ciation with rice cultivation; however, there is

no indication that subsequent shifts have oc-curred from these grasses back to rice. The

amplification of a particular repetitive ele-ment (Pot3) in the rice pathogen population

appears to have occurred after the host shiftfrom rice to grasses since the grass infecting

isolates have few copies of Pot3 (13).

Genotyping of strains is facilitated by fin-gerprinting of the MGR586 repetitive el-

ement (24, 34). Populations of the ricepathogen in different regions of the world

consist of groups of distinct clonal lineageseach exhibiting particular fingerprint pat-

terns.The Himalayanpopulation contains thegreatest number of lineages at 59. In con-

trast, there are eight clonal lineages in theUnited States (30, 50, 59). Even in popula-

tions that appear to display the potential for

438 Ebbole



sexual recombination, the preponderance of

reproduction of the pathogen is through asex-ual sporulation. The lack of recombination

in the pathogen population in Texas allowedfor combining of rice resistance genes (pyra-

miding) to yield the “Jefferson” cultivar thathas retained resistance over the past decade

(25). This may represent an example of thelineage exclusion concept (3, 100). The lin-

eage exclusion concept is based on severalhypotheses generalized from empirical obser-

vation. First, rice blast populations are com-

posed of sets of discrete clonal lineages thateach have a limited virulence spectrum. Sec-

ond, the virulence characteristics of each lin-eage are such that “high-quality” resistance

gene(s) may be found in the host. This meansthat for an isolate of that fungal lineage to

evolve to overcome that host resistance geneitmustpayafitnesscost(53).Thus,theappro-

priatecombinationofresistancegenesthatex-cludes all local pathogen lineages and requires

each lineage to pay a fitness cost to overcomethe resistance will be durable. Finding high-

qualityresistancegenesinthehostisalimiting

factor.Clonality appears to be the rule for the

structure of populations of M. oryzae associ-ated with rice; however, the pathogen is well

known to display genetic variation, particu-larly in laboratory studies. Rapid genetic re-

arrangement in the field might jeopardize suc-cessful application of the lineage exclusion

concept. Of course, rare sexual recombina-tion is possible, although other sources of

pathogen genetic variability have been noted.In the laboratory, heterokaryosis and parasex-

uality have been demonstrated (16, 29, 99).

As in many fungal species, mycoviruses of riceisolates are known (51), although the contri-

bution of mycoviruses to the biology of M.

oryzae is largely unexplored. In other fun-

gal pathogens, supernumerary chromosomeshave been shown to play a role in host-

microbe interactions (15, 82) and such chro-mosomes have been observed in M. oryzae (11,

63). A chimeric 1.6-Mb chromosome con-taining a portion of chromosome 1 that car-

ries a copy of the avrPik avirulence gene is

fused to sequences unique to supernumerary chromosomes (63).

The genome sequence revealed an abun-dance of repetitive elements representing

9.7% of the genome (18). Sequencing of anordered clone library spanning the small-

est chromosome revealed a larger amountof repetitive sequence (14%) than was de-

termined by the whole genome shotgun ap-proach used for the full genome sequence

(85). Repetitive sequences are difficult to as-semble, and so repetitive sequences are dif-

ficult to place into an overall genome se-

quence assembly; thus, genome sequencesslightly underestimate the level of repetitive

sequences. Most of the repetitive sequence in M. oryzae is derived from transposable ele-

ments. Seven types of retrotransposons andthree types of DNA transposons are preva-

lent in M. oryzae. An analysis of these el-ements reveals that gene duplication, re-

arrangement, and sequence diversification arecorrelated with the presence of repetitive se-

quences. Genome rearrangement promotedby repetitive elements might lead to aberrant

chromosome pairing that would reduce pro-

duction of viable progeny resulting from sex-ual recombination (86). Furthermore, meiotic

recombination between repetitive elementsleads to deletions of certain regions at high

frequency and may play a role in the in-stability of genes for melanin synthesis in

certain genetic backgrounds (22). Conceiv-ably, this mechanism could lead to loss of

avirulence determinants as well. In addition,ends of chromosomes are subject to deletion,

and this has been shown to result in lossof avirulence genes located at chromosome

ends (23, 67). Finally, active transposable el-ements also contribute to genotypic variabil-

ity. Ectopic recombination of transposons at

telomeric regions increases genetic variabil-ity (69). Insertional mutation events caused

by retrotransposons have been noted, for ex-ample, leading to conidiation defects (64) and

inactivation of avirulence genes (27, 45). Iden-tification of strains lacking active transposable

www.annualreviews.org • Magnaporthe in Host-Pathogen Interactions 439



elements would facilitate laboratory studies of

Magnaporthe.

THE DISEASE CYCLE

Aerial conidiophores produce a sympodial ar-

rangement of conidia and these spores form

the inoculum for secondary infection cycles(Figure 1). Conidia attach to the host sur-face, germinate, and respond to host cues to

form an appressorium that firmly adheres tothe host surface and functions in direct pene-

trationof thehost cell. Followingpenetration,the fungus ramifies intra- and intercellularly

in susceptible host tissue before lesion forma-tion initiates with host cell death starting 4 to

5 days after infection. The next generation of

conidia is then produced. The remainder ofthis review focuses primarily on this disease

cycle. A number of genes are discussed andthey are listed in Table 1 in order of their

appearance in the text.

Conida Formation and Dispersal

Dissemination of the fungus during an epi-

demic occurs by aerial dispersal of conidia.Overseasoning of hyphae in plant material

is possible, although the fungus does notsurvive freezing temperatures well. It was

recently shown that Magnaporthe is able toinfect roots of rice and colonize the plant

Conidium

Spore tip mucilage

Pitfields

Membrane capon invasive hypha

Penetrationpeg

Plant cell

Sympodialconidia

Germling with extracellular matrix

Melanizedappressorium

Autophagy

a

b

c

d

e

Figure 1

Disease cycle of Magnaporthe oryzae. (a) Conidia are arranged in a sympodial fashion on the aerial

conidiophore. (b) Conidia adhere to the host surface using mucilage stored in a compartment at the sporetip. (c ) Conidia germinate and produce a hyphal filament that is sheathed in an extracellular matrix thatadheres to the host surface. (d ) The melanized appressorium is formed and contains theautophagocytized contents of the conidium and germ tube. (e) A penetration peg employs turgor pressureto penetrate the plant cuticle and cell wall. The penetration peg gives rise to infection hyphae that have abulbous appearance and ramify within the cell. Movement into the next cell occurs specifically at clustersof plasmadesmata, called pit fields. The hyphae constricts to a narrow diameter as it passes through theplant cell wall. Invasive hyphae have a membrane cap composed of membrane lamellae that may functionin protein secretion into the cytoplasm of the living host cell.

440 Ebbole



two-hour exposure to darkness and decreases

with continued darkness. The mgwc-1 mutantdoes not release the spores it produces since

it cannot detect the light-to-dark transitionthat triggers release (56). Thus, the release of

spores is an active process, and it has beensuggested that the small stalk cell formed at

the base of the conidium (Figure 1 a

) buildsturgor pressure until it ruptures, thereby

launching the conidium (41). However, themoment of spore release has not been photo-

documented, and this mechanism remains

hypothetical.Spore traps confirm the relevance of the

laboratory findings since the concentration of conidiaintheairoveraninfectedricefieldfol-

lows the diurnal pattern of spore release (68). The peak of spore release occurs at midnight,

allowing the spores to disseminate while hu-midity is highand dew is forming. This timing

for release ensures germination in free water.Of course, rain splash and strong wind also

lead to dispersal at other times during the day.Conidia are coated with a rodlet layer of hy-

drophobin protein making them hydropho-

bic (48, 80, 81), such that spores are not eas-ily wetted and thus do not readily fall to the

ground with the water droplets. Rather, they are launched into the air by the force of rain

dropping on the leaf surface.Geneticanalysis of conidiation reveals sev-

eral conidiation “con” loci that each have dis-tinct effects on control of conidiation and

conidial morphology (73, 74). The epistaticrelationships between several of these loci

have been examined, but sequence is only available for the CON7 gene. The con7 mutant

produces a mixture of normal and aberrantly

shaped conidia. con7 mutants are unable toform appressoria and are nonpathogenic. Al-

though alternative splicing occurs that wouldproduce different polypeptides, both forms of

CON7 contain a putative Cys2-His2 zinc fin-ger transcription factor. Recently, CON7 was

reisolatedasapathogenicitygene(65)thatwasfound to regulate multiple members of a gene

family thought to encode G-protein coupledreceptors (49). In addition, regulation of sev-

eral genes involved in cell wall remodeling was

altered in con7 and the mutant cell walls hadreduced chitin content (65).

Another spore morphology locus, smo, isdefective in conidium and appressorium mor-

phology, andresulted from a screen to identifymutants that fail to produce the spore tip mu-

cilage needed for spore attachment to the hostsurface (36). Despite these cell shape defects,

smo conidia are able to germinate to form ap-pressoria that are capable of penetrating the

host and causing disease. Acropetal (acr ) is an

interesting conidiophore morphogenesis mu-tant that is orthologous to medA of Aspergillus

nidulans , a gene involved in cell type pattern-ing in the conidiophore (52, 64). In the acr

mutant, the pattern of conidia formation is al-teredsuchthatinsteadofasympodialarrange-

ment of conidia on the conidiophore, there isan acropetal pattern (one conidia produced by

budding directly from the top of the previousone). This observation suggests that medA or-

thologs play a conserved role in conidiophorepatterning across ascomycete lineages. This

view is supported by recent work in Fusar-

ium oxysporum, in which theequivalentmutantstrain displays novel spore shape and produc-

tion patterns (66). RGS1 is a regulator of G-protein signal-

ing that affects conidiation and pathogenesis(60). RGS1 is the ortholog of the flbA gene of

A. nidulans . FlbA controls the switch between vegetative growth and development by reg-

ulating the FadA G-alpha subunit, and flbA

mutants do not produce conidia (37, 98). Mu-

tants of the G-protein gene fadA are able tosporulate normally, thus the role of FlbA pro-

teinistodownregulatetheactivityoftheFadA

G-protein. MAGB encodes a G-alpha subunitof M. oryzae that is orthologous to fadA. In

contrast to the fadA mutant of A. nidulans

magB mutants conidiate poorly or not at all

(60). Also in direct contrast to the pheno-type of the flbA mutants of A. nidulans , rgs1

mutants have increased levels of conidiation Thus, the G-protein signaling pathways in

A. nidulans and M. grisea have opposite rolesin regulating conidiation. However, in both

442 Ebbole



systems, Rgs1/FlbA downregulate the activ-

ity of MagB/FadA with respect to sporulation. The rgs1 mutant strain also displays consti-

tutive appressorium formation on both in-ductive and noninductive surfaces. This point

is discussed further below. Overall, there areboth similarities and differences in how con-

served regulators function to control develop-ment. Understanding the mechanisms in the

evolution of developmental processes in fungiis an area that is now open to investigation due

to the availability of fungal genome sequences

for comparative functional analysis.

Prepenetration

The first level of attachment is the reversibleinteraction of the hydrophobic spore to the

leaf surface. However, as mentioned above,conidia contain a spore tip mucilage that is

released upon hydration and forms a strongadhesive to irreversibly attach the conidium

to the surface prior to germination. Spore tip

mucilage is an example of passive adhesion be-causeitisproducedduringsporedevelopment

and active metabolism is not needed for thespore to adhere to the surface. The spore tip

mucilage is thought to be composed of man-nose containing carbohydrates and glycopro-

teins. The importance of spore tip mucilagefor adhesion was tested by exposing spores to

concanavalin A, a lectin that binds to man-nose (35). This treatment blocked adhesion

of conidia to surfaces until after germinationand the synthesis of new adhesives.

As mentioned above, the hydrophobin

rodlet protein Mpg1 was shown to coat thesurface of conidia and imparts its strong hy-

drophobicity. The MPG1 gene is expressedduring germination and appressorium forma-

tion and plays a role in adhesion to the hostsurface (80, 81). The mpg1 mutant is greatly

reduced in appressorium formation on some,but not all, substrates (6). Hydrophobins self-

assemble to form monolayers of rodlet pro-teins, with one side being hydrophobic and

the opposite side being hydrophilic. On anextremely hydrophobic surface, such as a rice

leaf, the Mpg1 hydrophobin may contribute

to strong attachment to the leaf surface by providing an adaptor surface between the hy-

drophilic surface of the germ tube and the hy-drophobic rice leaf.

Adhesion of germ tubes to the substrateinvolves synthesis of an adhesive extracellular

matrix. The extracellular matrix is producedduring germ tube growth and this has been

visualized using fluorescently labeled lectinsand by scanning electron microscopy (94).

Our understanding of the genes contribut-ing to the extracellular matrix is limited. An

extracellular matrix protein encoding gene,

FEM1, was identified in F. oxysporum (71). FEM1 is anchored in the cell wall of F. oxys-

porum. The ortholog in M. oryzae of FEM1

( EMP1) has been characterized (1) and the

geneis induced during germinationconsistent with its proposed role as an extracellular ma-

trix protein. The emp1 mutant displayed re-duced appressorium formation (∼50%), sug-

gesting that the protein contributes to germtube adhesion that is an important signal to

induce appressorium development. The ap-pressoria formed by the mutant were func-

tional, suggesting that Emp1 is not a major

contributor to the adhesives involved in an-choring the appressorium to the leaf surface.

The emp1 mutants were able to colonize rice,although lesion sizes were reduced in the mu-

tants, suggesting a role for the protein duringcolonization.

The Appressorium

The basic structure of the melanized appres-sorium of M. oryzae has been examined in

detail (38). The appressorium is formed asa dome-shaped cell with a complex cell wall

structure. At the base of the appressorium, incontact with the plant cuticle, is the appres-

sorium pore, where the cell wall is extremely thin. The pore at the base of the appressorium

has a diameter of approximately 5–10 micronsand is wider in diameter than the penetration

peg, which penetrates directly through thehost cuticle. The penetration peg diameter




isintherangeof0.5microns,focusingthetur-

gor pressure produced in the appressorium ona very small area of the host cell surface. The

appressorium is melanized, and the melaninis derived from a polyketide precursor (38).

The cell wall is approximately 100 nm thick, with the inner melanin layer being perhaps

20 nm thick. The melanin layer does not ex-tend into the appressorial pore region in con-

tact with the host cuticle. The melanin layer is remarkable in that

it forms an effective barrier to solute move-

ment. Water can diffuse across the melaninlayer, but the melanin serves as a semiperme-

able barrier that prevents ions and other smallmolecules from moving into or out of the cell.

Carbohydrates and lipids stored in the conid-ium are mobilized to the appressorium. Sub-

sequent breakdown of the lipid and carbohy-drate stores results in a large increase in solute

concentration inside the appressorium. Glyc-erol is the major solute, reaching a concentra-

tion of approximately 3 M (38, 40). Maintain-ing such a high concentration of polyols in

solution requires significant amounts of wa-

ter, which can travel into the appressoriumthrough the melanin layer. Since the glyc-

erol cannot escape the melanin layer, tremen-dous turgor pressure is generated when wa-

ter is available. This turgor pressure has beenmeasured (39) and is generally accepted to

be approximately 80 atmospheres of pressure, which is approximately equivalent to 1200 psi

or the pressure felt at 800 meters under water. Thus, the cell wall must be very rigid to with-

stand this considerable pressure. If the extra-cellular solute concentration is raised so that

the solute concentration is higher outside the

cell than inside, water is withdrawn from thecell and the resultant higher external pressure

causes the appressorium to collapse (38).Clearly, the coordination of morphogene-

sis with metabolism is important for properappressorium function. Trehalose is a glu-

cose disaccharide that accumulates in coni-dia. A trehalose synthase mutant is unable to

generate turgor pressure to the same extentas wild type and is defective in penetration

and reduced in plant colonization (26). The

trehalose synthase mutant was dramaticallyaffected in the ability to grow on a variety

of carbon sources and the pleiotropy of themutant makes interpretation of its role dif-

ficult. Trehalose may play a regulatory rolein metabolism that is required for proper

carbon metabolism [such as gluconeogenesis(92)] that may be important for accumulation

of glycerol in the appressorium. This view issupported by the finding that mutation of the

genes responsible for the bulk of trehalase ac-tivity did not alter appressorium function, al-

though subsequent colonization was reduced

in one of the trehalase mutants (26).Glycerol is generated by the action of both

NADH-dependent glycerol-3-phosphate de-hydrogenase and NADPH-dependent dihy-

droxyacetone and glyceraldehyde reductases(84). Glycogen and lipid are potential carbon

sources for the production of glycerol. Glyco-gen is present in conidia and is rapidly bro-

ken down during germination. The glycogenreappears in newly formed appressoria con-

comitant with melanin formation and shortlythereafter is broken down (84). Thus, glyco-

gen breakdown could serve directly as one

route for generating glycerol. Lipids are a sec-ond likely source of glycerol, and triacylglyc-

erol lipase activity is abundant in developingappressoria. As with glycogen, lipid droplets

are present in conidia and disappear follow-ing germination. The lipid droplets reappear

in developing appressoria and are degradedduring appressorium maturation (84). The ki-

netics of lipid droplet degradation is slightlydelayed relative to glycogen degradation

Breakdown of lipid could lead to two routesfor glycerol production. First, glycerol is a di-

rect product of hydrolysis of fatty acids fromtriacylglycerol. Second, fatty acid breakdown

would yield acetyl-CoA that could generate

glycerol from the glyoxylate cycle and gluco-neogenesis. However, it appears more likely

that acetyl-CoA metabolism is important as asubstrate for gluconeogenesis to generate cell

wall precursors rather than glycerol (7, 92) Thus, both glycogen and lipid may contribute

444 Ebbole



to generation of turgor pressure, but their

contribution to turgor pressure generationrelative to other pathways needed for success-

ful penetration and invasive growth requiresfurther analysis. Wang et al. (91) recently pub-

lished a detailed review of metabolism duringappressorium development.

Actin is found at the site of penetration pegformation, and establishment of actin local-

ization is likely a key step in penetration pegformation. The punchless ( PLS1) gene encodes

a tetraspanin-like protein that is required for

penetration peg development (12). In animalcells, tetraspanins are involved in a number

of cellular processes including reorganizationof the cytoskeleton. This observation suggests

that punchless mutants may be defective inorganizing actin to initiate formation of the

penetration peg. The appressoria formed inthe pls1 strain attain full turgor pressure, con-

sistent with the view that the defect is in pene-tration peg formation and not morphogenesis

of the appressorium. Although appressoriumformation is not required for cell-to-cell colo-

nization, pls1 mutantsareunabletocolonizeat

wound sites. This suggests that Pls1 is neededin some specialized polar growth needed to

penetrate from cell to cell.

Signaling Events in Germination and Appressorium Development

Understanding the roles of signal transduc-

tion pathways in appressorium formation isan active research area (17, 79, 96). Appres-

sorium formation occurs only on hard sur-

faces. Two major signals, surface adhesionquality and host chemical cues, control induc-

tion of appressoria formation. However, envi-ronmental factors (light, nutrients) can affect

appressorium development (42). Hydropho-bicity is often associated with inductive sur-

faces (58), although some hydrophilic surfacescan also stimulate appressorium development

(6). Strength of adhesion to the substrate may activate mechanosensitive channels that affect

calcium flux across the cell membrane (17). The PTH11 gene was identified as an inser-

tion mutant that did not form appressoria on

inductive surfaces (19). It has been proposedthat Pth11 is a receptor that senses surface ad-

hesion and may act as part of a mechanosens-ing system.

Additional evidence demonstrates that cal-cium signaling plays a role in germination

and the induction of appressorium develop-ment (57, 62). Calmodulin gene expression is

dependent on spore attachment to the hostsurface and is induced concomitantly with

appressorium formation. Diacylglycerol also

serves as an inducer of appressorium forma-tion, implicating a role for calcium and pro-

tein kinase C as part of a surface sensingmechanism (83). Still, we have very limited

understanding of how calcium signaling is in-tegrated into pathways for germination and

appressorium development.Surface cues are sensed by a heterotrimeric

G-protein alpha subunit, MagB (61). MAGB

has sequence features of the Gi subfamily of

G-protein that inhibit cAMP synthesis in an-imal systems. The fact that (a) magB dele-

tion mutants fail to form appressoria (or coni-

dia), (b) a dominant active form of MAGB

forms appressoria on noninductive hard sur-

faces, and (c ) cAMP activates appressoriumformationina magB deletionmutantledtothe

view that MagB stimulates rather than inhibitscAMP synthesis. Appressoria are also induced

to form in the presence of cutin monomers,such as 1,16-hexadecanediol (32). Induction

of appressorium formation by plant chemicalsignals occurs in the magB mutant, demon-

strating the existence of a magB-independentroute for stimulating cAMP synthesis (61).

The interplay of cAMP signaling and MAPkinase signaling controls the induction, mor-

phogenesis, and maturation of the appresso-

rium and has been reviewed extensively (18,79, 96).

As mentioned above, the RGS1 gene is im-portant for MagB-mediated control of coni-

diation. Rgs1 interacts with all three G-alphasubunits(MagA,MagB,andMagC),and

presumably negatively regulates their activ-ities (60). rgs1 mutants are derepressed for




appressorium development on hydrophilic

surfaces, although a hard surface is still re-quired (60). Thus it appears that Rgs1 neg-

atively regulates MagB and MagB is neededto activate both conidiation and appressorium

formation. Since magA deletion mutants areable to produce appressoria, it was thought

that magA does not play a significant rolein appressorium development (61). However,

the finding that a dominant activated allele of

MAGA is induced for appressorium formation

on noninductive surfaces raises the possibility

that both magA and magB contribute to sig-naling induction of appressorium formation

by controlling cAMP levels (60). This raisesthe possibility that magA may be responsible

for induction of appressorium formation inresponse to plant chemical cues. Given this

new finding, discerning the relative roles of magA and magB in appressorium development

requires closer inspection. Appressorium formation is induced by ex-

ogenously applied cAMP. There is evidencethat a critical time following germination ex-

ists when this cAMP signal must be delivered.

Germlings can respond to cAMP during thefirst eight hours following germination. Af-

ter that time, appressoria do not form in re-sponse to cAMP (6). This loss of developmen-

tal competence may be related to the recentobservation that the timing of nuclear divi-

sionin nascent germlingsis critical for appres-sorium development. Under inducing condi-

tions, a nucleus migrates into the germ tubeat 4 to 6 hours following germination and un-

dergoes mitosis. One daughter nucleus mi-grates back into the conidium and the other

moves into the incipient appressorium (89).

Using a temperature-sensitive MgNimAts al-lele to block mitosis, it was shown that this first

mitosis in the germ tube is required for subse-quent appressorium development. However,

MgNimAts did not interfere with appresso-rium formation if the temperature shift was

given following mitosis (89). The relationship between cytological

events and the activity of signal transductionpathways is now being explored. The Pmk1

MAP kinase pathway responds to cAMP sig-

naling and is required for appressorium mor-phogenesis (95). Autophagy of the contents

of the conidium is observed during appres-sorium development and is required for pro-

ducing functional appressoria. The role ofautophagy may be to transfer sufficient car-

bon to the appressorium to develop the tur-gor pressure required for penetration of the

host cuticle. Autophagy does not occur inthe pmk1 mutant strain, nor if the first mito-

sis is blocked via MgNimAts or chemical in-

hibitors (89). This indicates a coordinationbetween cytological events, cAMP, and MAP

kinase signaling. As discussed above, appres-sorium maturation requires active lipid and

glycogen metabolism, and carbon mobiliza-tion is affected by both the cAMP and Pmk1

pathways (84). An integrated understandingof metabolism, cell cycle control, autophagy

and the level of flux through signaling pathways leading from conidial germination to

appressorium formation is within reach.

Secondary Metabolites and Host Colonization

M. oryzae produces a number of phytotoxiccompounds and some of these have known

structures, including many apparent polyke-tide derivitives similar to pyriculol and pyric-

ulariol. Other phytotoxic compounds havebeen characterized as well; however, most

work on toxin synthesis and characterizationhas not been translated into English and has

notreceived the attention it deserves. It is esti-

matedthat23polyketidesynthasesarepresenin the genome sequence of M. orzyae, along

with 6 nonribosomal peptide synthetaseand 8 nonribosomal peptide synthetase/

polyketide synthetase hybrid genes (18) Thus, a great many metabolites of unknown

structure and function likely are involved inparasitism. Thus far, however, only very lim-

ited information is available concerning therole of specific compounds in disease. One

of the best characterized metabolites is ten-uazonic acid, a photosystem II inhibitor (10)

446 Ebbole



Tenuazonic acid synthesis is thought to in-

volvecondensation of isoleucine with acetoac-etate, and it could be speculated that its syn-

thesis involves the activity of both a nonri-bosomal peptide synthetase and a polyketide

synthase to bind the substrates for conden-sation. Strains producing low levels of tenua-

zonic acid have smaller lesions than those withhigh levels of the phytotoxin (54, 55). How-

ever, the relative contribution of tenuazonicacid in host colonization awaits identification

of the genes involved in its synthesis and in

the creation of isogenic mutant strains. Another illustration of a possible role for

a secondary metabolite comes from a recentstudy on the Abc3 transporter that may de-

liver a small molecule metabolite to the hostplant (77). The abc3 mutant is unable to col-

onize rice and also has a reduced growth ratein vitro. The mutant produces functional ap-

pressoria in vitro, but fails to infect plant cellsor cause an obvious host defense response.

Microscopyrevealed that penetration peg for-mation was roughly 1% of the wild-type rate.

The key finding was that the abc3 mutants

were hypersensitive to oxidative stress in vitro,supporting the idea that the mutant is un-

able to export a compound that sensitizes thestrain. This was tested by applying extracts

from abc3 appressoria to wild-type cells, re-sulting in a reduction in penetration peg for-

mation during attempted plant infection. If this metabolite is delivered into the host cell,

it would presumably promote fungal invasionby affecting host sensitivity to redox status.

Identification of the metabolite and its activ-ity will provide great insight into the roles

of such metabolites. The genome sequence

should now permit workers to address thequestion of the roles of the obvious secondary

metabolite-producing genes. The ABC3 geneis flanked by two polyketide synthases, one of

which (MGG 04774.5) ( Table 1) is inducedduring appressorium formation (18).

In addition to delivery of secondary metabolites, M. oryzae must survive secondary

metabolite defense compounds produced by the host plant. The ABC1 gene encodes a

transporter that most likely serves to defend

the fungus from host-produced metabolites. This view is supported by the finding that

growth and pathogenic development of theabc1 mutant appears to be completely normal

in vitro, although growth is arrested shortly after penetration of plant cells (88). Addi-

tional analyses of the roles of transporters in M. oryzae/Oryza sativa interactions are clearly

needed.

Protein Effectors and Host Colonization

A limited number of examples of proteins af-fecting colonization are available. At present,

the best example is provided by two relatedproteins, Gas1 (also called Mas3) and Gas2

(Mas1). The genes were found to be expressedspecifically during appressorium formation.

This expression required the Pmk1 MAP ki-nase. GAS1 and GAS2 are two of a seven-

member gene family dispersed throughout

the genome of M. oryzae. Functional redun-dancy does not appear to exist since mutation

of either gene leads to reduced appressorialpenetration of plant tissue and reduced lesion

sizes (97). Homologs of these genes are foundin a number of filamentous ascomycetes. GFP

fusion proteins were produced to reveal thatboth Gas1 and Gas2 were localized to the cy-

toplasm of appressoria. Gas2 is induced earlierin appressorium development than Gas1, sug-

gesting that they may play specific roles at dif-ferent times early in plant infection. However,

the GFP fusion proteins were not detected

in infection hyphae (97). In any case, the ob-servation that lesion sizes are reduced in the

mutants and the fact that both proteins havepredicted signal peptides and, thus, would be

expected to be secreted, suggests that they travel from the appressorium to the infection

hyphae or are delivered into the plant cell tocarry out their activities.

A number of other genes may define ef-fector proteins involved in host colonization,

but these genes are not as well characterizedas Gas1 and Gas2 (90, 96). Identification of




additional effector genes is ongoing, guided

in part by examination of secreted proteinspredicted from the genome sequence and the

structures of known effectors from M. oryzae

and other systems (18). These approaches are

aided by transcriptome analysis to identify genes expressed under different growth con-

ditions or in planta (21, 33, 75).

Growth within Host Cells

The interaction of rice blast with its sus-

ceptible host is largely biotrophic, and animportant recent study by KanKanala et al.

(47) provides new insights into the processof host colonization. Rice cells are alive dur-

ing growth of the fungus until well after thefungus has moved from one living cell to the

next. It appears that it is only after the hostcell is full of fungal hyphae that it dies. As the

fungus penetrates into the first cell from theappressorium or moves from cell to cell, the

plant plasma membrane invaginates to sur-

round the growing invasive hyphae. This hasbeen demonstrated by staining of membranes

with the membrane dye FM4-64 that is in-corporated into membranes but is only inter-

nalized by endocytosis (47). The plant plasmamembrane canbe seenstained by FM4-64and

surrounding the fungal cell wall. However,the fungal plasma membrane is not stained

by FM4-64 since the dye cannot gain accessto the fungal membrane.

Fungal hyphae appear to penetrate theplant cell wall at very specific sites when mov-

ing from cell to cell (47). Live cell imagingshows hyphae “searching” for preferred sites

of penetration. The sites of cell-to-cell move-

ment colocalize with plasmadesmata. Rice cell walls have clusters of plasmadesmata, and it

is at these pit fields (Figure 1e) where pene-tration occurs. The hyphal diameter narrows

remarkably (to as little as 0.1 to 0.2 microns)as the fungus moves through the plant cell

wall (47). The cell wall may have less strengthat sites where groups of plasmadesmata are

found, or, conceivably, the fungus may mod-

ify the plasmadesmata to cause it to expand

tremendously to accommodate the thin seg-ment of hypha. These thin hyphae are remi-

niscent of the penetration peg of theappresso-rium. Thus, although functional appressoria

are not required for cell-to-cell movement, asmentioned above, PLS1 is likely required for

the highly focused growth observed for inter-cellular penetration.

Fromthesamestudy(47),itwasdiscoveredthat the plant plasma membrane is extremely

active during fungal colonization. Strandstubes, and other membranous forms connect

the plasma membrane at the plant cell wall to

the plant plasma membrane surrounding thefungal hyphae. Membrane lamellae form at

the tips of hyphae penetrating into plant cellsHyphae growing intracellularly do not pos-

sess these membrane caps (Figure 1e

). Theplant-derived plasma membrane surrounding

the fungal cell wall extends around the out-side of the membrane cap (47). It is appar-

ent from the transmission electron micro-graphs of these membrane caps that proteins

produced by the fungus could enter into thelamellar structure of the membrane cap and

that subsequent movement of proteins out of

the distal regions of the membrane caps rep-resents a plausible mechanism for delivery of

fungal proteins into the cytoplasm of the liv-ing host cell.

Other genes affecting colonization areknown; for example, the PDE1 gene has ho-

mology to aminophospholipid translocasesthat affect the maintenance of membrane

asymmetry and mutants are affected in golgfunction and/or vesicle trafficking (4). Dele-

tion of PDE1 affects the ability of the mutantto penetrate rice, but the major phenotype

is an inability to properly colonize the host

There are four members of the aminophos-pholipid translocase family in M. oryzae. A

second member, MgApt2, was mutated andfound to have no growth or sporulation de-

fect in vitro, yet was severely compromised inits ability to parasitize rice (31). The MgApt2

mutant is impaired in secretion of some, but

448 Ebbole



not all, proteins and is unable to induce a

strong host response. The current model isthat these proteins affect vesicle transport

(endo- and exocytosis) such that effector pro-teins are not efficiently delivered to the host

plant and thus lack the ability to colonize theplant. At the same time, they do not deliver

proteins that evoke strong defense responses.In some ways, these phenotypes are analogous

to that observed for HRP (Hypersensitive

Response andPathogenicity) genesof bacteriathat are required for delivery of bacterial pro-

teins into the host cell (2). It has not yet beenestablished if membrane caps are composed

solely of plant-derived membranes or if thefungus might contribute lipids to the mem-

branes. It will be interesting to determine if these aminophospholipid translocases are in-

volved in membrane cap formation.

Avirulence Genes

Avirulence genes encode factors that can be

recognized by specific genotypes (cultivars) of the host species that contain corresponding

resistance genes. Three particularly instruc-tive examples of avirulence genes have been

discovered in the rice blast system. One in-teresting case is the pwl gene family (46, 78).

These are present as a rapidly evolving genefamily of small, glycine-rich, secreted pro-

teins. One or more members of this family are generally found in rice pathogens. The

genome sequence of M. oryzae 70–15 con-tains three members of the pwl family. One

sequence is identical to pwl2, whereas the

other two are duplicate copies of the pwl3

gene ( Table 1). The pwl genes do not encode

avirulence factors toward any known rice cul-tivar, although functional copies of pwl are

recognized by weeping lovegrass ( Eragrostis

curvula). Rice pathogens that have lost the pwl

genes typically gain the ability to parasitize weeping lovegrass. Thus, the pwl genes ap-

pear to act as avirulence factors at the hostspecies level, rather than at the cultivar level.

The importance of the pwl genes to parasitism

is unclear, and it would be of interest to as-

sess the relative aggressiveness of isogenic ricepathogens lacking the pwl genes. If pwl genes

do contribute to aggressiveness in some of therice pathogen lineages, it would be an inter-

esting test case to determine if the resistancegene(s) from weeping lovegrass recognizing

the pwl genes could be moved into rice andprovide useful resistance.

The ACE1 avirulence gene is unusualin that it encodes a polyketide synthase/

nonribosomal peptide synthetase fusion pro-tein expected to produce a secondary metabo-

lite (8). The current view is that the secondary

metabolite product is recognized by the Pi33resistance protein of rice. ACE1 is expressed

specifically in mature appressoria, but curi-ously, expression is observed in appressoria

formed on rice or cellophane but not on ar-tificial plastic sheets (28). In addition, ACE1

was expressed in the pls1 penetration defectivemutant but not in mutants that were blocked

in melanin formation, nor in wild-type cellstreated with a chemical inhibitor of melanin

synthesis. This is remarkable since the only defect expected for a melanin mutant is the in-

ability to accumulate turgor pressure. To test

the idea of turgor pressure–regulated expres-sion, extracellularsolutes were added to devel-

oping melanin-less appressoria to mimic theturgor pressure found within appressoria. In-

deed, appropriate concentrations of sodiumchloride, sorbitol, or sucrose restored ACE1

expression in appressoria, butnotin hyphaeorother cell types. This form of regulation guar-

anteesproductionofthesecondarymetaboliteexactly at the time the appressorium is mature

and ready to initiate penetration of the hostcell.

The avr-Pita gene was cloned using map-

based methods. The gene is located nearthe end of a chromosome, and loss of the

chromosome tip is one mechanism for gain-of-virulence variants. The avr-Pita gene en-

codes a secreted metalloprotease homolog ex-pressed during infection and colonization of

rice (9, 67). The active site of the protease is




required for avirulence activity, initially sug-

gesting that the activity of the protease wasimportant for generating the signal recog-

nized by the host. However, the protease in-teracts directly with the resistance protein Pi-

ta, demonstrating that the conformation of the protein with an intact active site is impor-

tant for the interaction necessary for recogni-tion. Subsequently, we are left with the view

that the protease activity may be involved inpromoting plant colonization. Pi-ta is consti-

tutively expressed and encodes a cytoplasmic

resistance protein. The demonstration of di-rect interaction was accomplished in vitro by

direct binding studies between the Avr-Pitaand Pi-ta and the interaction was further ex-

plored by the yeast two-hybrid system (44). That the interaction betweenthe two proteins

occurred within the plant cell was shown by transient expression of avr-Pita by delivering

the avirulence gene into rice cells by particlebombardment. The avr-pita /Pi-ta interaction

provides the first demonstration of direct in-tracellular Avr/resistance protein interaction

for a fungal avirulence protein.

CONCLUSION

Large-scalefunctionalgenomics projects with

M. oryzae are beginning to generate signif-icant resources for the research community,

such as mutant collections and transcriptomedata (33, 43, 96). Obtaining the genome se-

quence of M. oryzae has been instructive

defining the gene content of the organism sothat the community can prioritize and collab-

orate in a way that was not possible previously(18, 75, 76). Research on M. oryzae will also

benefit from the determination of additionalfungal genomes as workers increasingly em-

brace comparative functional analysis to ex-amine the roles of genes across fungal species

As illustrated for the rgs1/flbA and magB/fadA

examples given for conidiation, conservation

of biochemical function does not equate to an

identical role in determining the analogousphenotype. The novel genes only found in

the species of interest represent high-prioritytargets for functional analysis. Many of the

genes for secondary metabolism and a signifi-cant fraction of secreted proteins fall into this

category.

SUMMARY POINTS

1. M. oryzae serves as an instructive andintegrated model for fungal pathogen population

biology and evolution, and for the molecular genetics governing pathogenesis of

biotrophic/hemibiotrophic interactions with host plants.

2. Studies of carbon metabolism, cell cycle, autophagy, and signal transduction pathwaysareleading to a holistic view of thebiology of appressorium development andfunction.

3. M. orzyae provides examples of theroles of secondary metabolites andsecreted effectorproteins as quantitative determinants of disease.

4. Observation of cell-to-cell movement in the susceptible host provides insight intopotential mechanisms of virulence factor delivery into the host and highlights the

biotrophic nature of the M. oryzae /rice interaction.

5. M. oryzae provides several excellent examples of avirulence factors and their recogni-

tion by the host.

FUTURE ISSUES

1. Genome sequences of additional isolates of M. oryzae will help answer questions about

pathogen evolution and aid in the quest to identify high-quality resistance genes.

450 Ebbole



2. The role of calcium signaling and its relationship to the known signal transduction

pathways need to be defined.

3. The mechanism for perception of extracellular cues (e.g., the role of PTH11 in surfacesensing) and the downstream targets of the signal transduction pathways are still

largely undefined.

4. The availability of the genome sequence and transcriptome analysis will facilitate amore systematic analysis of the role of secondary metabolites, cell wall–degradingenzymes, and secreted proteins in host infection and colonization.

5. Comparative functional genomics with other plant-associated fungi will reveal thedifferent strategies and molecular activities that lead us to classify them as biotrophs,

necrotrophs, or endophytes.

DISCLOSURE STATEMENT

The author is not aware of any biases that might be perceived as affecting the objectivity of

this review.

ACKNOWLEDGMENTS

I thank Drs. Heather Wilkinson and Won-Bo Shim for their comments on the manuscript and

support from National Science Foundation Award DBI0115642.

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RELATED RESOURCES

MGOS: Magnaporthe grisea/Oryza sativa interaction database: http://www.mgosdb.org/

Broad Institute Magnaporthe grisea database: http://www.broad.mit.edu/annotation/

genome/magnaporthe grisea/Home.html

456 Ebbole



Annual Review

Phytopathology

Volume 45, 2007Contents

Tell Me Again What It Is That You Do

R. James Cook p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1

Noel T. Keen—Pioneer Leader in Molecular Plant Pathology

Alan Collmer and Scott Gold p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 25

Structure and Function of Resistance Proteins in Solanaceous PlantsGerben van Ooijen, Harrold A. van den Burg, Ben J.C. Cornelissen,

and Frank L. W. Takken p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43

Family Flexiviridae: A Case Study in Virion and Genome Plasticity

Giovanni P. Martelli, Michael J. Adams, Jan F. Kreuze, and Valerian V. Dolja p p p p p p 73

Cell Wall-Associated Mechanisms of Disease Resistance

and Susceptibility

Ralph Hückelhoven p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 101

Genomic Insights into the Contribution of Phytopathogenic Bacterial

Plasmids to the Evolutionary History of Their HostsGeorge W. Sundin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 129

Identifying Microorganisms Involved in Specific Pathogen Suppression

in Soil

James Borneman and J. Ole Becker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153

Safety of Virus-Resistant Transgenic Plants Two Decades After Their

Introduction: Lessons from Realistic Field Risk Assessment Studies

Marc Fuchs and Dennis Gonsalves p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 173

Disease Cycle Approach to Plant Disease Prediction

Erick D. De Wolf and Scott A. Isard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

203

Virus-Induced Disease: Altering Host Physiology One Interaction

at a Time

James N. Culver and Meenu S. Padmanabhan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221

Bacteriophages for Plant Disease Control

J.B. Jones, L E. Jackson, B. Balogh, A. Obradovic, F. B. Iriate,

and M. T. Momol p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 245

v



Reniform in U.S. Cotton: When, Where, Why, and Some Remedies

A. Forest Robinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

Flax Rust Resistance Gene Specificity is Based on Direct

Resistance-Avirulence Protein Interactions

Jeffrey G. Ellis, Peter N. Dodds, and Gregory J. Lawrence p p p p p p p p p p p p p p p p p p p p p p p p p p p p

Microarrays for Rapid Identification of Plant Viruses Neil Boonham, Jenny Tomlinson, and Rick Mumford p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

Transcript Profiling in Host-Pathogen Interactions

Roger P. Wise, Matthew J. Moscou, Adam J. Bogdanove,

and Steven A. Whitham p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

The Epidemiology and Management of Seedborne Bacterial Diseases

Ronald Gitiatis and Ronald Walcott p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

Elicitors, Effectors, and R Genes: The New Paradigm and a Lifetime

Supply of Questions Andrew F. Bent and David Mackey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

Magnaporthe as a Model for Understanding Host-Pathogen

Interactions

Daniel J. Ebbole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

Challenges in Tropical Plant Nematology

Dirk De Waele and Annemie Elsen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

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