mechanisms and evolution of virulence in oomycetes

PY50CH15-Tyler ARI 4 July 2012 14:8

Mechanisms and Evolutionof Virulence in OomycetesRays H.Y. Jiang1 and Brett M. Tyler2

1The Broad Institute of the Massachusetts Institute of Technology and Harvard,Cambridge, Massachusetts 02142; email: [email protected] for Genome Research and Biocomputing and Department of Botany and PlantPathology, Oregon State University, Corvallis, Oregon, 97331;email: [email protected]

Annu. Rev. Phytopathol. 2012. 50:295–318

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

This article’s doi:10.1146/annurev-phyto-081211-172912

Copyright c© 2012 by Annual Reviews.All rights reserved

0066-4286/12/0908-0295$20.00

Keywords

genome evolution, effectors, transposable elements, host-pathogeninteractions, host specificity

Abstract

Many destructive diseases of plants and animals are caused byoomycetes, a group of eukaryotic pathogens important to agricultural,ornamental, and natural ecosystems. Understanding the mechanismsunderlying oomycete virulence and the genomic processes by whichthose mechanisms rapidly evolve is essential to developing effectivelong-term control measures for oomycete diseases. Several commonmechanisms underlying oomycete virulence, including protein toxinsand cell-entering effectors, have emerged from comparing oomyceteswith different genome characteristics, parasitic lifestyles, and hostranges. Oomycete genomes display a strongly bipartite organizationin which conserved housekeeping genes are concentrated in syntenicgene-rich blocks, whereas virulence genes are dispersed into highly dy-namic, repeat-rich regions. There is also evidence that key virulencegenes have been acquired by horizontal transfer from other eukaryoticand prokaryotic species.

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PLANT OOMYCETE PATHOGENS

Plant oomycete pathogens cause a vast arrayof destructive diseases of plants importantto agriculture, forestry, ornamental andrecreational plantings, and natural ecosystems(both terrestrial and aquatic) (1, 27, 53). Themost destructive pathogens occur within theclass Peronosporomycetidae, in the ordersPeronosporales (Phytophthora species anddowny mildews), Pythiales (Pythium species),and Albuginales (Albugo and other white rusts)(Figure 1). Plant pathogenic species in thisclass likely have a common evolutionary origin.Some plant pathogens occur in the class Sapro-legniomycetidae (32), which otherwise consistsmainly of animal pathogens and saprophytes,

Phytophthoracapsici

Phytophthoraramorum

Phytophthora sojae

Phytophthora infestans

Phytophthora phaseoli

Hyaloperonosporaarabidopsidis

Plasmopara

Pythium ultimum

Pythium insidiosumAlbugo candida

Albugo laibachii Leptomitus

Brevilegnia

Aphanomyces

Achlya

Saprolegniaparasitica

Eurychasma

Aquaticenvironment

Plant pathogen

Animal pathogen

Algal pathogen

Evolution of pathogenicity

Evolution of obligate biotrophy

Figure 1Distribution of plant and animal pathogenicity across the oomycetes. Inferred independent instances of theevolution of pathogenicity and obligate biotrophy are indicated. The pathogens with whole-genomesequences are in red. Color gradients generally indicate pathogenic specialization to plants ( green) or animals(orange).

and among basal oomycetes such as Eurychasma(90) (Figure 1). Plant pathogenicity has likelyevolved independently in these lineages withinthe oomycetes.

Oomycetes have evolved a wide diversityof infectious lifestyles. Necrotrophs such asPythium typically attack plant tissues with com-promised immunity, such as seedlings, fruit,and stressed plants, and typically have verybroad host ranges (1). Hemibiotrophs, such asmost Phytophthora species, biotrophically initi-ate infection, with little damage to host tissues,then switch to necrotrophic growth once colo-nization has been established (1). However, theperiod of biotrophy may range from as shortas a few hours in the cases of Phytophthora sojae

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(112) and Phytophthora parasitica to several daysin the cases of Phytophthora infestans (30) andits close relatives. Obligate biotrophy, in whichthe pathogen is fully dependent on the host, hasarisen at least twice within the oomycetes, oncewithin the downy mildews and once within thewhite rusts (101, 121).

Although oomycetes and fungi have evolvedplant pathogenicity independently withindistinct kingdoms of life (Stramenopila andMycota, respectively), these organisms displaymany morphological and physiological traits incommon. Common traits, such as osmotrophicnutrition, filamentous hyphae, and airborneand waterborne spores, are also found amongsaprotrophic species in each kingdom andspeculatively may have laid the foundationfor the evolution of pathogenicity in eachkingdom. Traits common to pathogens in eachkingdom, such as haustoria and other forms ofspecialized infection hyphae, effector proteinsthat enter host cells, and expanded familiesof hydrolytic enzymes, are likely indicative oftraits essential for a phytopathogenic lifestyle.

Much of our current understanding ofthe mechanisms and evolution of virulencein oomycetes has built on expressed sequencetag (EST) and genome sequencing completedover the past 10 years (52). Genome sequencesinclude those of P. sojae (95 Mb) (116), suddenoak death pathogen Phytophthora ramorum(65 Mb) (116), the late blight pathogen P. infes-tans (240 Mb) (39) and four close relatives (79),the necrotroph Pythium ultimum (42.8 Mb)(55), and four biotrophs: Hyaloperonosporaarabidopsidis (100 Mb) (6), Albugo laibachii(37 Mb) (50), Albugo candida (53 Mb) (57), andPseudoperonospora cubensis (105). The expressedsequences of oomycete pathogens of mammalsand marine organisms have also become avail-able, including the human pathogen Pythiuminsidiosum (51), the fish pathogen Saprolegniaparasitica (110), and the marine algae pathogenEurychasma dicksonii (37).

Here, we review current understanding ofthe molecular and genetic mechanisms of vir-ulence in oomycete plant pathogens and howthey have evolved, including the roles of

transposons, genome partitioning, and hori-zontal gene transfer (HGT) in accelerating theemergence and diversification of these notori-ously adaptable pathogens.

MECHANISMS OF VIRULENCE

The principal theme to emerge from the ge-nomic studies is that virulence in oomycetesdepends extensively, possibly entirely, on large,rapidly diversifying protein families (39, 115,116). These families include extracellular tox-ins, hydrolytic enzymes and inhibitors, and ef-fector proteins that can enter the cytoplasmof plant cells (113). This contrasts with fun-gal pathogens in which secondary metabolitetoxins play a strong role, and families of viru-lence proteins are less extensively expanded anddiversified (115).

Plants protect themselves from infection byoomycetes and other pathogens through di-verse sets of constitutive and induced defensesthat include physical and chemical barriers,antimicrobial enzymes and peptides, antimicro-bial chemicals [phytoanticipins, phytoalexins,and reactive oxygen species (ROS)], and inthe case of biotrophic and hemibiotrophicpathogens, programmed cell death (PCD) (45).Successful pathogens, including oomycetes,must avoid, suppress, or tolerate these de-fenses, as well as gain nutrition from the host(108). Inducible plant defenses consist of twooverlapping modules identified by the micro-bial molecules that induce them (45, 102).Commonly occurring microbial molecules thattrigger defenses in a wide diversity of plantsare referred to as microbe- (or pathogen-)associated molecular patterns (MAMPs orPAMPs) and the responses triggered by themas MAMP- (or PAMP-) triggered immunity(MTI or PTI). Cell surface receptor-like ki-nases (RLKs) commonly mediate the detectionof MAMPs and the induction of PTI (45, 67).Plants also produce intracellular receptors thatcan directly or indirectly detect the presenceof intracellular pathogen effectors, resulting ineffector-triggered immunity (ETI) (45). ETI isgenerally a more rapid and vigorous response

www.annualreviews.org • Virulence in Oomycetes 297

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Celldeath

RONS

Oomycete

IE

NB-LRR

Plant cell

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Intracellulareffector

Protease inhibitor

Protease

Pattern recognitionreceptor

Nucleotide-binding leucine-rich-repeat resistance protein

Otherresponses

Otherresponses

PTI ETI

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Figure 2Suppression of plant immunity by oomycete effectors. Oomycete pathogenssecrete intracellular effectors (IEs) that � enter the host cytoplasm. Plants maysecrete proteases (Pr) that � can degrade intracellular or extracellular effectorsin the apoplast, but pathogens � may secrete protease inhibitors (PIs) thatblock those proteases, or else produce effectors that block secretion �.Recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) � produces signaling events that activatePAMP-triggered immune responses (PTI). Recognition of intracellulareffectors by nucleotide-binding site leucine-rich repeat receptors (NBS-LRRs)� leads to effector-triggered immune responses (ETI). Signaling events forboth PTI and ETI may be inhibited by intracellular effectors �. Both PTI andETI can produce programmed cell death , and effectors may inhibit thetriggering of cell death or the cell death machinery itself. PTI and ETI bothinvolve transcriptional changes , and nuclear-targeted effectors may directlyinterfere with signaling within the nucleus or transcriptional events. PTI andETI involve numerous other responses �, including the production of reactiveoxygen and nitrogen species (RONS), and effectors may also interfere withthose responses.

than PTI and more often includes PCD. Themajority of plant intracellular receptors arenucleotide-binding site leucine-rich repeat(NBS-LRR) proteins (45). NBS-LRR proteinsthat can detect effectors and trigger effectiveresistance are called resistance (R) proteins,and the effectors they detect have historicallybeen called avirulence (Avr) proteins (45).

Most plant-associated symbionts, includingpathogens, have evolved protein or chemicaleffectors that suppress or reprogram PTI andETI (109) (Figure 2). These effectors mayact outside the plant cell, in the apoplast, orat the plant plasma membrane, or they mayenter into the cytoplasm of the plant cell toreprogram its physiology (109). Effectors maybe secreted directly into the apoplast from thepathogen hyphae, or in the case of biotrophicand hemibiotrophic oomycetes, may be se-creted from specialized intracellular hyphae,including haustoria (64). These specializedhyphae penetrate the plant cell wall but remainencased in the host plasma membrane (64).The haustorial cell wall, extrahaustorial space,and extrahaustorial membrane may becomesignificantly differentiated to facilitate deliveryof effectors and acquisition of nutrients (64).

Extracellular Toxins

Several large families of proteins that triggercell death in host plants have been foundin oomycete genomes, including the NLPs(necrosis and ethylene inducing peptide-likeproteins) (76), the PcF family (69, 70), and theScr family (59). NLPs trigger cell death in awide range of dicotyledonous hosts, producinga response with many similarities to PTI (33,78). NLP families are dramatically expandedin Phytophthora species (40 to 80 copies) (39,116), although not in the genomes of Py.ultimum (necrotrophic) (55) or H. arabidopsidis(obligately biotrophic) (6, 15). Many of theexpansion events in Phytophthora specieshave occurred subsequent to speciation (39,116), supporting a key role for NLPs in theinteraction with the plant host. An increasein gene expression coincident with the switch

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to necrotrophy has led to the hypothesisthat NLPs are responsible for this switchin infection strategy (75). NLPs may haveroles other than triggering cell death, asthe small number of NLPs encoded in theH. arabidopsidis genome do not trigger celldeath (6). Two white rust genomes (A. laibachiiand A. candida) lacked NLP genes altogether(50, 57). NLP genes are also widely distributedin fungal pathogens and in a few bacterial plantpathogens, and it has been hypothesized thathorizontal transfer of NLP genes from fungito oomycetes was a key event in the emergenceof oomycetes as plant pathogens (84).

The related PcF (Phytophthora cactorum fac-tor) and Scr (secreted cysteine-rich) toxin fam-ilies are specific to oomycetes and show muchgreater heterogeneity among species (39, 59,69, 116). The numbers of PcF/Scr family genesin Phytophthora and Pythium genomes are highlyheterogeneous (39, 55, 69, 116). The PcF sub-family is expanded in P. sojae (16 genes), andthe Scr family is expanded in P. infestans (11genes), whereas P. ramorum and Py. ultimumcontain only one and three genes, respectively(39, 55, 116). The absence of the genes from thegenomes of obligate biotrophs H. arabidopsidis(6), A. laibachii (50), and A. candida (57) is con-sistent with the proteins triggering cell deathduring infection.

Hydrolytic Enzymes

Plant tissues, especially the intercellularapoplastic spaces, are rich in complex carbo-hydrates. Accordingly, oomycete genomes,like those of other plant pathogens, are rich ingenes encoding a wide array of carbohydrate-degrading enzymes, including pectin esterases,pectate lyases, glucanases, and cellulases (39,116). In addition, a rich abundance of lipasesand proteases is also secreted. Approximately30 to 60 proteins in each class were predictedto be secreted by each Phytophthora species(39, 116). Interestingly, although classifiedas a necrotroph, Py. ultimum contains fewerglycoside hydrolase genes and a more limitedability to degrade complex carbohydrates,

such as cellulose and xylans, than Phytophthoraspecies; it was speculated that the differencereflects that Pythium species may quickly infectimmature tissue; degrade readily accessiblecarbohydrates, such as pectins, starch, andsucrose; and then focus on reproduction (55).H. arabidopsidis, A. laibachii, and A. candida allhave greatly reduced numbers of genes encod-ing hydrolytic enzymes; because carbohydratefragments produced by these enzymes arepotent triggers of PTI, their depletion in theseobligate biotrophs suggests adaptation forstealth (6, 50, 57). Similar genomic adaptationfor stealth has been observed in fungal obligatebiotrophs and mutualists (25, 61, 62, 96).

Enzyme Inhibitors

Because oomycetes secrete a large battery ofvirulence proteins, a natural defense for plantsis the production of proteases to degrade thevirulence proteins. Accordingly, oomycetes arepredicted to secrete proteinase inhibitors tar-geted against the plant proteins. The sequencedPhytophthora and Pythium genomes contain 18to 38 genes encoding secreted inhibitors of ser-ine and cysteine proteases (39, 55, 116). InP. infestans, two serine protease inhibitors, EPI1and EPI10, can bind to and inhibit a tomatoprotease P69B that was responsible for 27%of the pathogen-induced protease activity inthe apoplast (103, 104). Furthermore, two P.infestans cysteine protease inhibitors, EPIC1and EPIC2b, can bind to and inhibit the cys-teine proteases C14, Pip1, and Rcr3 (95, 106).Mutations in the tomato Rcr3 gene or silenc-ing of C14 resulted in increased susceptibilityto P. infestans (95). Cysteine protease inhibitorsalso play a demonstrated role in infection bybiotrophic fungi such as Cladosporium fulvum(95).

Cell-Entering RxLR Effectors

One of the best-studied classes of virulenceproteins to emerge from the genome sequenc-ing of oomycete pathogens is RxLR effectors,named for a conserved N-terminal amino acid


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sequence motif (arginine, any amino acid,leucine, arginine) (47, 114). The sequencedPhytophthora genomes contain approximately350 to 550 genes that encode these rapidlyevolving small secreted proteins (39, 44, 116).Many, but not all, of the proteins carry a morevariable second motif, dEER [aspartate (lesswell conserved), glutamate, glutamate, argi-nine] at varying distances C-terminal to theRxLR motif (44). Approximately half of theencoded RxLR effectors carry additional con-served motifs in the C terminus, called W, Y,and L motifs, often in repeating blocks of con-secutive W, Y, and L motifs (44).

RxLR effectors include the products ofnearly all 18 oomycete avirulence genes thathave been cloned to date. Those genes in-clude PsAvr1a (77), PsAvr1b (92), PsAvr1k (23,46), PsAvr3a/5 (21, 77), PsAvr3b (20), PsAvr3c(19), and PsAvr4/6 (22) from P. sojae; PiAvr1(119), PiAvr2 (34), PiAvr3a (3), PiAvr3b (56),PiAvrBlb1 (73, 120), PiAvrBlb2 (68), andPiAvrVnt1 (119) from P. infestans; and HaATR1(82), HaATR13 (2), and HaATR39 (35) fromH. arabidopsidis. HaATR5 from H. arabidopsidis(4) resembles other RxLR effectors but lacksa canonical RxLR motif. Where known, the Rgenes matching RxLR avirulence effectors en-code intracellular NBS-LRR proteins (5, 12,29, 31, 35, 40, 56, 60, 71, 72, 86, 94, 117, 118).Hence, these effectors must have a mechanismto enter into host cells. The RxLR and dEERmotifs are required for entry of RxLR effectorsinto host plant cells, and the N-terminal do-main carrying the motifs, plus approximately10 amino acid residues on each side of the mo-tifs, appear to be sufficient for cell entry (24,38, 46, 47, 125). The mechanism of entry me-diated by the RxLR-dEER domain is still anarea of active research. Current data suggestthat RxLR effectors (and some fungal RxLR-like effectors) secreted into the apoplast can en-ter host plant cells in the absence of pathogen-encoded machinery, such as the bacterial typeIII secretion system or the Plasmodium Pexeltranslocon (46, 47, 74, 80). Further data sug-gest that RxLR effectors bind to cell-surfacephosphatidylinositol-3-phosphate (PI3P) be-

fore entering host cells by receptor-mediatedendocytosis (46, 74, 97), although the litera-ture is not yet fully consistent on this point (26,128). In a fascinating parallel, PI3P binding byRxLR-like Pexel motifs is involved in the deliv-ery of effectors in erythrocytes by the malariaparasite Plasmodium (7, 8). Fungal effectors alsoappear to enter via a pathogen-independentmechanism that may involve RxLR-like motifsand binding to PI3P (46, 74, 80). On the otherhand, an RxLR-like effector from the oomycetefish pathogen Saprolegnia parasitica appears toenter via a pathogen-independent mechanismbut utilizes binding to tyrosine sulfate ratherthan PI3P (124). Further careful work is neededto clarify the situation.

Recent structural studies of six oomyceteRxLR effectors [PcAvr3a4 (13), PcAvr3a11(128), PiRD2 (13), PsAvh5 (97), HaATR1 (18),and HaATR13 (54)] have provided additionalinsights into the structure and evolution ofthese proteins. Crystal structures were gen-erated for PcAvr3a4, PiRD2, and HaATR1,whereas nuclear magnetic resonance (NMR)data were collected for PcAvr3a11, PsAvh5, andHaATR13. PcAvr3a4, PcAvr3a11, and PsAvh5are members of a closely related subfamily ofRxLR effectors (the 1b3a subfamily; 97) thatalso includes PsAvr1b and PiAvr3a, whereasPiRD2, HaATR1, and HaATR13 are dis-tinct. Nevertheless, the C-terminal domainsof PcAvr3a4, PcAvr3a11, PsAvh5, PiRD2, andHaATR1 exhibited a common novel fold com-prising three helices that span the conservedC-terminal W and Y motifs, called the WYfold (97, 126). In this fold, the highly conservedtryptophan and tyrosine residues that give theW and Y motifs their name contact each otherto form the hydrophobic core of the fold (97,126). In PcAvr3a4, PcAvr3a11, and PsAvh5,a fourth helix corresponding to a positivelycharged K motif is present, forming a four-helix bundle (13, 97, 128). In PiRD2, a dimer isformed between two three-helix WY domains(13). HaATR1 contains two WY domains ar-ranged in a helical spiral (18). HaATR13 has adistinct structure consisting of three helices anda C-terminal disordered loop (54). Win et al.

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(126) speculated that the WY fold formed a flex-ible scaffold that supported rapid changes in theprimary sequence and structural architecture ofRxLR effectors driven by the host-pathogen co-evolutionary conflict.

In all six structural studies, the RxLR do-mains were found or inferred to be disordered,raising questions about how they could bindPI3P and how they promote cell entry. Yaenoet al. (128) showed that a positively chargedC-terminal patch corresponding to the K mo-tif contributed strongly to PI3P binding byPcAvr3a11, PsAvr1b, and PiAvr3a but couldnot detect binding of the RxLR domains toPI3P using lipid blots. Sun et al. (97) used NMRand surface plasmon resonance to demonstratethat residues in both the C-terminal domainand in the RXLR motif of PsAvh5 contactedPI3P and were required both for efficient PI3Pbinding and for efficient host-cell entry. Fur-ther detailed structural and functional studiesof the interaction of RxLR effectors with PI3Pand other elements of the plasma membrane,including effectors that lack positively chargedC-terminal motifs, are required to understandin more detail how RxLR effectors utilize PI3P,RxLR, and dEER motifs to achieve cell entry.

The functions of RxLR effectors in ma-nipulating host physiology are beginning toemerge. The common theme in each case is thesuppression of PAMP- and effector-triggeredimmune responses, especially suppression ofcell death associated with these responses(108, 109). PiAvr3a could suppress cell deathtriggered by the PAMP INF1 (11). Thisprocess appears to involve binding of PiAvr3ato a positive regulator of defense-related celldeath, CMPG1 (10). PsAvr1b appears to havethe ability to suppress cell death generally,even in yeast and even when triggered by themouse proapoptotic protein BAX, suggestingthat it may target conserved elements of theeukaryotic cell death pathway (23). PiSNE1is another effector with a powerful abilityto suppress plant cell death, even cell deathtriggered by NLP toxins (49). HaATR1 andHaATR13 could suppress PAMP-triggeredresponses in Arabidopsis, including deposition

of callose and accumulation of ROS (93).PsAvr3b, which is a nudix hydrolase that candestroy NADP and ADP-ribose, also is a pow-erful suppressor of ROS accumulation (20).PiAvrBlb2 appears to suppress plant defensesvia a different mechanism; it accumulates onthe inner face of the host plasma membrane andthe plasma membrane–derived extrahaustorialmembrane, where it inhibits secretion of hostdefense proteins, such as the cysteine proteaseC14 (14). PiAvrBlb1 (also called ipiO1) mayinterfere with defense by disrupting RGD-motif-mediated adhesions between the plantcell wall and the plasma membrane (36, 91).

High-throughput screens of the functionsof the RxLR effectors encoded in oomycetegenomes are beginning to fill in the broaderpicture of how these innumerable effectorscontribute jointly to infection. Oh et al. (68)conducted a survey of 16 effectors encodedin the P. infestans genome, finding that twosuppressed cell death triggered by the PAMPINF1, two triggered R gene–mediated celldeath, and one triggered R gene–independentcell death. Wang et al. (122) conducted a muchlarger survey of 169 predicted P. sojae effectors,finding that 63% (107/169) suppressed celldeath, and 11 triggered R gene–independentcell death. Of 49 that were screened in more de-tail, 40 suppressed effector-triggered cell death,and 20 suppressed PAMP (INF1)-triggered celldeath. Thus, P. sojae effectors appear to be heav-ily targeted to suppression of cell death. Severalsurveys of the functions of H. arabidopsidis ef-fectors have been conducted, including a yeasttwo-hybrid screen for protein-protein interac-tions (66), a screen of the intracellular locationsof the effectors (17), and a screen of the abilityto suppress immunity against bacteria (28). Theyeast two-hybrid screen, involving 131 proteinscorresponding to alleles of 99 genes, revealedmany effectors interacting with large numbersof host proteins (66). For example, HaATR1interacted with 19 targets, and HaATR13interacted with 24 targets. The majority of thetargets were proteins that interacted with re-ceptors involved in PTI or ETI, suggesting thatH. arabidopsidis effectors are heavily targeted to


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suppression of the plant immune system (66).This conclusion is supported by the observationthat of 64 H. arabidopsidis RxLR effectors tested,approximately 70% promote the growth ofbacteria in Arabidopsis (28). The majority (66%)of 49 H. arabidopsidis effectors were targetedpartially or entirely to the host nucleus, sug-gesting that interference with nuclear signalingand/or transcription is a common mechanismfor suppressing host immunity (17). A minorityof H. arabidopsidis effectors were targeted tothe plasma membrane or tonoplast (17).

An unexpected finding from transcriptomicanalysis of RxLR effectors is that a relativelysmall percentage (approximately 10% to 15%)of the effector genes are moderately to highlytranscribed during infection (16, 39, 122). Fur-thermore, resequencing surveys revealed thatonly approximately 10% to 15% of predictedP. sojae RxLR effectors show evidence of sig-nificant positive selection (122). These resultssuggest that these pathogens rely heavily on asmall number of elite effectors. Gene-silencingexperiments have begun to confirm that anumber of these elite effectors are individuallyessential for full virulence. These includePiAvr3a (10), PsAvh172 (122), PsAvh238(122), and PsAvr3b (20). The transcriptomeanalysis of P. sojae effectors also identified twooverall patterns of expression: immediate-earlyeffectors were strongly expressed prior to in-fection and moderately induced upon infection(two- to tenfold), whereas early effectors werevery weakly expressed prior to infection, butstrongly induced (10- to 120-fold) during thefirst 12 h of infection (122). The ability to sup-press ETI was concentrated among immediate-early effectors, whereas the ability to suppressPTI was concentrated among early effectors,suggesting that P. sojae utilizes a preemptivestrategy to suppress ETI prior to the expressionof effectors for the suppression of PTI (122).Given the ability of RxLR effectors to travelthrough the apoplast and enter host cells in theabsence of the pathogen (46, 92), this obser-vation suggests that immediate-early effectorsmay travel ahead of the pathogen to preemp-tively suppress ETI, whereas early effectors

might be delivered from haustoria, where theywould suppress both PTI and ETI (Figure 3).

Cell-Entering Crinkler Effectors

Oomycete pathogens produce a second majorclass of small secreted effector proteins calledcrinklers, named for their ability to producecrinkling and necrosis when overexpressed intransient expression assays (107). Like RxLReffectors, crinklers are rapidly evolving, highlydiverse, and modular (39). They consist ofa well-conserved N-terminal domain requiredfor host-cell entry (the crinkler domain) (89)connected to a very diverse collection of C-terminal domains (39). Crinkler effectors areamong the most highly expressed pathogengenes both prior to and during infection (39,81, 111). The highly diverse C-terminal do-mains of these effectors suggest that they mayhave a wide diversity of functions, possibly cre-ated by opportunistic fusions of an N-terminaldomain consisting of secretion and host-cellentry signals to a C-terminal domain derivedfrom an intracellular protein. Several crinklersexamined to date appear targeted to the hostnucleus (58, 89), and one crinkler has the abil-ity to suppress cell death (58). Several crinklersare essential for virulence (58). An intriguingaspect of crinkler evolution is that these pro-teins are much more widely distributed amongoomycete pathogens than RxLR effectors (32).They have been found in the orders Saproleg-niales (32), Pythiales (55), and Albuginales (50,57) as well as Peronosporales (127), whereasRxLR effectors have been found only in thelatter two orders. Thus, they may be very an-cient to pathogenic oomycetes (89) and/or havebeen disseminated by HGT. Even more in-triguingly, there are hints that crinkler-like ef-fectors may be present in the chytrid fungusBatrachochytrium dendrobatidis, possibly as a re-sult of HGT (98).

Other Classes ofCell-Entering Effectors

Given the opportunistic nature of pathogenevolution and adaptation, it is plausible that

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Figure 3Programmed expression of RxLR effectors in Phytophthora sojae. (Upper left) Aggregate transcript levels ofRxLR effector genes (log2 microarray signal) during the first 6 h post-infection (adapted from 122). Genesexpressed prior to contact with the pathogen (immediate-early genes) encode effectors that mostly suppresseffector-triggered immunity (ETI), whereas genes that are predominantly expressed following infection(early genes) encode effectors that suppress both PAMP-triggered immunity (PTI) and ETI. (Lower right)Speculative model in which immediate-early effectors diffuse through the apoplast ahead of the pathogen,directly entering host cells to preemptively suppress ETI, whereas early effectors enter from pathogenhaustoria. Lightning symbols indicate signaling events.

new mechanisms of host-cell entry have arisenthrough chance binding of a secreted pathogenprotein to a host-cell surface molecule.Computational and experimental analyses ofcandidate effector proteins in Albugo laibachiihave uncovered a novel motif, CHXC (cys-teine, histidine, anything, cysteine) associatedwith host-cell entry (50). Novel motifs remi-niscent of, but distinct from, RxLR motifs werefound among candidate Pythium effectors alsopresent in Phytophthora species, although thefunctionality of these has not been determinedyet (55).

GENOME CHARACTERISTICS

The whole-genome sequences of majorpathogens have revealed common features of

oomycete genomes as well as unique aspects ofpathogen evolution. Several patterns relatedto pathogenesis, such as genome reductionassociated with biotrophy and repeat-drivenvirulence change, have emerged from genomeanalysis and comparisons.

Core Proteome andLineage-Specific Genes

The currently sequenced oomycete species dis-play divergent lifestyles. A large fraction oftheir genes are unique to each pathogen lineage(6, 39, 55, 116). Nonetheless, a common pro-teome can be identified by ortholog analysisacross phylogenetically divergent species (116).The Phytophthora species P. infestans, P. sojae,


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and P. ramorum share a core set of 8,492 or-tholog clusters (39). The species of Albugo andPhytophthora belong to different phylogeneticgroups in oomycetes, and they share from 4,826(A. laibachii versus H. arabidopsidis) to 5,722(A. laibachii versus P. infestans) orthologousgenes (50). Taken together, a set of con-served 5,000 to 6,000 orthologs represents thecore cellular processes of oomycetes, includ-ing DNA replication, transcription, and proteintranslation.

In the core proteome, genes involved incellular processes related to pathogenesis areunderrepresented. In contrast, gene familiesexpanded in specific lineages are enriched infunctions associated with pathogenesis. Theselineage-specific genes are probably responsiblefor different pathogenic traits among oomycetespecies, such as adaption to different envi-ronments and modulation of host physiology.Families showing lineage-specific expansionsare very fluid, and distinct subfamilies areexpanded (or lost) in individual lineages.For example, the members of the RxLR andcrinkler effector families appear largely specificto particular Phytophthora or Hyaloperonosporaspecies (6, 39, 116). In Albugo, the novel classof CHXC effectors exhibits lineage-specificexpansion in the two sequenced Albugo species(50, 57). Similarly, for the biotrophic oomycetePseudoperonospora cubensis that causes downymildew of cucurbits, a family of PcQNEeffectors has been identified from a partiallysequenced genome (105). Massive duplicationof this family in a lineage-specific fashionsuggests its important role in pathogenesis.Py. ultimum possesses several distinct familiesof lineage-specific genes associated with rootparasitism and opportunistic pathogenesis (55).

The signature of lineage-specific expansionis also characteristic of pathogenesis-relatedgenes in species other than oomycete plantpathogens. Different species of the malariaparasite Plasmodium have distinct massivelyexpanded families that play important roles,such as host modification and antigenic vari-ation, in pathogenesis (99, 100, 123). Thefish oomycete pathogen Saprolegnia parasit-

ica has many lineage-specific expanded pro-tease and lectin families that are likely associ-ated with animal parasitism (110; R.H.Y. Jiang,unpublished data).

From Compact Genome to MassiveRepeat Expansion

Some oomycetes have a compact genome(<50 Mb) (55, 57), whereas others, such as P. in-festans and its nearest relatives, have the largestand most complex genomes (>200 Mb) se-quenced so far in the kingdom of Stramenopiles(39, 79). All sequenced oomycete genomes en-code similar numbers of genes, ranging from14,000 to 19,000; it is the large difference inthe repeat content that accounts for the genomesize differences. More than 75% of A. laibachiiis nonrepeated, whereas approximately 75% ofP. infestans is made up of repeats (Figure 4a)(39, 50). Many of the repetitive regions of theselarge genomes are highly dynamic and proneto evolutionary changes, apparently due to fastbursts of mobile element activities. Below, wediscuss the expansion of genomes by repeatedelements and the resulting genome dynamics.

Genome Reduction inObligate Pathogens

Obligate parasitism has evolved at least threetimes independently in oomycetes: in basalmarine algae parasites, such as Eurychasma; inthe land plant white rust pathogens, such asAlbugo; and in the land plant downy mildewpathogens, such as Peronospora, Bremia, Pseu-doperonospora, and Hyaloperonospora (101).Genome sequences have become availablefrom the two branches of land plant obligateplant pathogens, Albugo (50, 57) and Hyaloper-onospora (6); these sequences provided insightsinto the evolutionary processes leading tohighly specialized parasitism (Figure 4b). Onecommon trend is that the intimate associationwith host cells has resulted in relaxed selectionand loss of several biosynthetic pathways (63).The same trend has been observed in obligatefungal plant pathogens (63) and in intracellular

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0

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capabilities

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Specializationof haustoria

Evolutionary trendsshaping the

oomycete genome

Figure 4Genome evolution of oomycetes. (a) Genome composition of small and large oomycete genomes. DNAtransposons include PiggyBac, Helitron, Crypton, Mutator, hAT, and Mariner. Long terminal repeat (LTR)retrotransposons include LTR-Gypsy and LTR-Copia. (b) Evolutionary processes of obligate pathogens.The degeneration processes are indicated with down-pointing arrows. The expansion processes are indicatedwith up-pointing arrows. (c) Major evolutionary events shaping oomycete genomes. Whole-genomesequences are available for Plasmodium, Albugo, Pythium, Phytophthora, and Hyaloperonospora. Color gradientsindicate progressive pathogenic specialization.

parasites of animals and humans, such as vari-ous Apicomplexa species, including the malariaparasite Plasmodium (123). For example, closeassociation with the host provides obligate

pathogens with a ready source of organicnitrogen. Degeneration of inorganic nitrogen(nitrate) assimilation pathways has been foundin biotrophic fungi [powdery mildew (96)


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and rust fungi (25)], in the malaria parasitePlasmodium (50, 100), and in the oomycete plantparasites Albugo and Hyaloperonospora (6, 50).

Another evolutionarily convergent featureof biotrophs is the reduction in the numberand diversity of hydrolytic enzymes able toattack host components, particularly cell wall–hydrolyzing enzymes, presumably reflecting aninfection strategy of minimizing host damageto avoid eliciting host immune responses (6).Similar reduction has been observed inbiotrophic plant pathogenic fungi (25, 48, 63,88, 96) and mutualistic fungi (61, 62). Theability to produce zoospores for waterbornedissemination has been lost in many lineages ofdowny mildew pathogen, in favor of airborneconidial dispersal. In H. arabidopsidis, manygenes associated with zoospore formationand motility have been lost as compared withsoilborne Phytophthora and Pythium species (6).For example, none of 90 flagella-associatedgenes in Phytophthora could be detected in

H. arabidopsidis, representing a further dramaticexample of genome minimization.

EVOLUTIONARY ORIGINSOF OOMYCETE GENOMES

Pathogenicity has evolved multiple times in-dependently in all major domains of eukary-otes. Oomycetes evolved within the eukary-otic kingdom of Stramenopila, which includessome of the most important photosyntheticmarine algae, such as diatoms and brown al-gae. Three major evolutionary trends shapingoomycete genomes are the loss of photosyn-thetic plastids (116), novel protein domain com-binations, and HGT from bacteria and fungi(Figure 5).

Pathogenesis-Related HorizontalGene Transfer

HGT enables the transfer of genetic materialbetween otherwise reproductively isolated

Fungi

Animals

Plants

Green algae

Red algae

Saprolegnia

Phytophthora

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Secondaryendosymbiosisfrom red algae

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Genes acquiredfrom bacteria

Major flows of horizontal gene transfer

to oomycetes

Figure 5Gene acquisitions from horizontal gene transfer in oomycetes. The three major flows of genes are annotatedin ovals. Only three eukaryotic domains are drawn schematically on the tree. The direction of gene transferis indicated by arrows. Multiple acquisitions have occurred from different fungal species and bacterial species(84, 85, 116).

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lineages. The evolution of pathogenicity ofoomycetes appears to have been greatly facili-tated by extensive cross-kingdom HGT eventsbetween fungi and oomycetes and betweenbacteria and oomycetes (65, 84, 85). A whole-genome analysis revealed an estimated 7.6% ofthe secretome of P. ramorum has been acquiredfrom fungi by HGT (84). A gene-by-genephylogenetic analysis suggested a pattern of 33transfers from fungi to oomycetes. Compar-ative genomics showed that the HGT eventsprobably are associated with the radiation ofplant pathogenic oomycetes because thesegenes were not detected in the animal parasiticoomycete Saprolegnia or the free-living sistertaxon of Hyphochytrium (84). One of the moststriking examples of likely acquisition of keypathogenicity genes from fungi involves theNLP toxins. As detailed above, these toxins canbe found in bacteria, fungi, and oomycetes andcan trigger immune responses in diverse di-cotyledonous plants. The wide distribution ofthese genes in fungi but narrower distributionwithin plant pathogenic oomycetes in the classPeronosporomycetidae (Phytophthora, Pythium,and downy mildews) suggests a major role forthese genes in the emergence of oomycetesas plant pathogens (84). Another possibleexample of HGT from bacteria to oomycetesis the transglutaminases from Phytophthora anddowny mildews that act as PAMPs, activatingdefense responses in plants; homologs of thisprotein are found in a marine Vibrio bacterium(83).

In Eurychasma dicksonii, a widespreadpathogen of marine brown algae, an EST sur-vey (37) revealed unique pathogenicity fac-tors in Eurychasma, such as alginate lyases, thatcan break down alginates, a key componentof the brown algal cell wall and an abundantbiopolymer in coastal waters (37). Interestingly,the Eurychasma alginate lyase genes have sim-ilarity with those of marine invertebrates andfungi, hinting at possible HGT (37). Phylo-genetic analysis revealed that the oomyceteenzyme shows homology with enzymes fromPseudomonas syringae, Chlorella virus, and algalgastropods (37). Furthermore, alginate lyases

are not present in any of the other sequencedoomycetes.

GENOMICS OF HOSTSPECIFICITY

Genomic Signatures of Host Range

Oomycete pathogens display an enormousspectrum of host ranges, from extremely broadhost-range Pythium species that have hundredsor thousands of hosts to downy mildews thatare often specialized to a single host. Themolecular basis of host range is not yet evi-dent from genome sequencing studies, as notenough examples of each pathogen have beenexamined. Win et al. (127) observed that re-cently duplicated families of RxLR effectorswere less diversified in the broad host-rangepathogen P. ramorum than in the narrow host-range pathogen P. sojae. This suggests that di-versifying selection may be weaker in broadhost-range pathogens, possibly because loss ofa host species due to the emergence of a newR gene may exert weaker selective pressure onthe pathogen population to adapt. Emergenceof a new R gene in the host of a narrow host-range pathogen would, however, exert extremepressure on the pathogen population to adaptthrough diversification of its effector repertoire.

Host Jumps and Specialization

Major mechanisms responsible for the emer-gence of new pathogens include host-rangeexpansion and host jumps. Genome analysis offour closely related Phytophthora species thatappear to have undergone recent host jumpshas shed light on one example of genomeadaptation. The four species infect hosts fromdifferent plant families as follows: P. infestanscauses late blight disease on Solanum species,including potato and tomato (Solanaceae);P. ipomoeae infects morning glory, Ipomoealongipedunculata (Convolvulaceae); P. mirabilisinfects four o’clock, Mirabilis jalapa (Nyctag-inaceae); and P. phaseoli is pathogenic on limabeans, Phaseolus lunatus (Leguminosae). These


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four Phytophthora species belong to a veryclosely related clade of pathogen species (clade1c; 9) that display 99.9% sequence identityamong their ribosomal DNA internal tran-scribed spacer regions. Genomic regions richin repeats show higher rates of structural poly-morphisms among these species (79). Further-more, these dynamic regions frequently harborgenes with signatures of positive selection,which is a hallmark of pathogen host adap-tation. Consistent with these dynamic genesbeing involved in infection, these repeat-richregions are enriched in genes induced in planta.

Genes involved in epigenetic processes, suchas histone methyltransferases, are also enrichedin these dynamic regions (79). Histone modifi-cation is considered to be a key epigenetic reg-ulatory mechanism in most eukaryotes, such asP. infestans. Thus, epigenetic regulation couldmediate rapid expression changes needed forthe host adaptation. Taken together, these ob-servations support the idea that genome com-partments with accelerated gene evolution areplaying important roles in host jumps in thispathogen lineage.

GENOME PARTITIONING ANDMECHANISMS OF PLASTICITY

Successful pathogens often possess plastic anddynamic genomes. The genomes of oomycetesare partitioned into conserved syntenic regionsand highly repetitive species-specific regions(6, 39, 55, 116). The conserved regions mostlyharbor housekeeping genes, and the highlyvariable regions contain genes underlyingpathogenicity and host range. The fungalpathogen Leptosphaeria maculans displayssimilar genome partitioning (87).

The genome architecture of oomycetes isremarkably uneven. In the larger Phytophthoragenomes, especially P. infestans and its sisterspecies, the genome architecture is separatedinto large gene-dense partitions and evenlarger repeat-rich partitions (39, 79, 116). Thestructural partitioning of oomycete genomeshas led to several functional consequences

for these pathogens, such as different rates ofgene evolution, uneven paces of gene familyexpansion, differential gene expression, andpathogen speciation (Figure 6c). As discussedin the previous section, adaptation to new hostplants most likely involves mutation and copynumber changes in hundreds of effector genes,which mostly populate repeat-rich partitions(79). Thus, the highly dynamic partitions fosteremergence of new pathogens.

Syntenic Ortholog-Rich Partitions

In oomycetes, there is a high degree ofsynteny (conserved gene order) among thecore ortholog genes of Phytophthora speciesthat also extends to downy mildews such asH. arabidopsidis (6, 39, 116). The conservation iseroded to a certain degree in comparison withthe Py. ultimum genome (55) and even more soin comparisons with the more divergent Albugospecies (50, 57). Conserved runs of more than100 genes spanning genomic regions of severalmegabases can be observed among Phytophthoraspecies. Many of these runs are also conservedin H. arabidopsidis (6). Conserved synteny overbroad regions of the Py. ultimum and Phytoph-thora genomes can be identified by the orthologcontent; however, the local gene order has beengreatly rearranged (55). As a result, only shortruns of up to 10 orthologs were found to becollinear between Py. ultimum and Phytophthoraspecies. As the evolutionary distance furtherincreases between species, such as betweenPhytophthora and Albugo, only small conservedgenomic regions can be identified (50, 57).

Dynamic Repeat-Rich Partitions

Separating the blocks of conserved syntenyamong oomycete genomes are highly dynamiclineage-specific regions that are enriched intransposable elements (6, 39, 55, 116). Thesedynamic regions are the sites for two major evo-lutionary processes; one is rapid changes in therepertoires of pathogenesis-related genes, andthe other is the loss of biochemical functions or

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a

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Repeat rich / gene sparse

Phytophthora infestanscrinkler gene cluster

Gene cluster of nitrate reductaseand nitrate transporter

Genome territory partitioning in Phytophthora infestans

Phytophthora sojae

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Gene loss

Phytophthora infestans Sc1.25

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Lack of synteny

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Induction at earlyinfection

Elevated SNPs andCNVs associatedwith host jump

Conserved betweenspecies

Suppressedat early

infection

Oomycete ortholog

Repeat sparseGene rich

Conserved synteny

Phytophthora sojae

Figure 6Genome organization of oomycetes. (a) Conserved synteny and expansion of the crinkler gene family inPhytophthora species. The tandemly repeated crinkler genes are represented by red triangles. The crinklergene cluster disrupts the collinearity between Phytophthora infestans scaffold 1.6 and other Phytophthoraspecies. Panel a is adapted from Reference 39. (b) Nitrate and nitrite reductase genes are conserved betweenPhytophthora genomes but have been deleted from the Hyaloperonospora arabidopsidis genome. Panel b isadapted from Reference 6. (c) Genome territory partitioning of P. infestans. The distinct genomeenvironments show different properties in synteny, gene content, expression, and evolutionary rates.Abbreviations: SNP, single nucleotide polymorphism; CNV, copy number variation.

cellular apparatus due to pathogen specializa-tion. Not surprisingly, therefore, effector genestend to localize to repeat-rich regions, wherethey display rapid expansion and diversification.

Host-imposed immune pressure drives therapid evolution of pathogen effector genes(2, 82, 92, 127). These effector genes are fre-quently rearranged, duplicated, or expanded,


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and they are enriched in dynamic genomic re-gions (41–43, 77). For example, most of thehundreds of RxLR effector genes are located inregions without conserved synteny (44). Evenfor the remaining 10% of RxLR genes residingin syntenic genomic regions, frequent gene re-arrangements have occurred (44). The crinklerand NLP gene families have also undergone lin-eage specific expansions in Phytophthora species(39, 116). For example, P. infestans has almost200 CRN genes. Dozens of CRN genes are oftenclustered and are located in the nonsyntenic re-gions (Figure 6a). In P. infestans, helitron trans-posons may have facilitated rapid tandem geneduplications of the CRN families (39).

Genome degeneration associated with obli-gate biotrophy has also led to rearrangementsand deletions. For example, in the obligatebiotroph H. arabidopsidis, which has lost theability to produce motile zoospores, the geneencoding the flagellar inner arm dynein 1 heavychain α is missing. The genomic context of thisgene is conserved even between oomycetes asdistant as Py. ultimum and A. laibachii. In con-trast, the syntenic region in the H. arabidopsidis

genome has become populated by diverse mo-bile elements (50).

CONCLUSIONS

As a group of successful pathogens that haveparasitized diverse host taxa, oomycetes haveevolved many virulence traits to adapt to novelhosts, colonize new tissue types, and copewith host immune responses. In this review,we have summarized recent discoveries fromoomycete comparative genomics regarding vir-ulence mechanisms employed by oomycetesand the genomic mechanisms that underliethe adaptability of oomycete virulence. Un-derstanding these mechanisms of virulenceand adaptability is instrumental for designingmanagement strategies for oomycete diseases.The high level of genome plasticity and ever-expanding reservoirs of effectors will require usto locate the most essential targets for geneticresistance or chemical controls. The uniquemetabolic features of oomycetes and commoneffector translocation pathways may offer keytreatment targets.

SUMMARY POINTS

1. Pathogenicity has independently evolved multiple times within oomycetes. Most terres-trial plant pathogens within the Peronosporomycetidae likely share a single acquisitionof pathogenicity.

2. The pathogenesis mechanisms in oomycetes depend extensively on large protein families.These rapidly diversifying families include extracellular toxins, hydrolytic enzymes andinhibitors, and effector proteins that can enter the cytoplasm of plant cells.

3. Oomycete cell-entering effectors target numerous host cellular processes, commonlyresulting in suppression of PAMP-triggered immunity, effector-triggered immunity, andprogrammed cell death.

4. Genome evolution of oomycetes has been characterized by genome reduction in obligatepathogens and repeat-driven genome expansion in some species with large genomes.

5. Oomycete genomes have a mosaic origin, with genes acquired from secondary endosym-bionts, from fungi, and from bacteria.

6. Genome partitioning of oomycetes into conserved ortholog-rich regions and dynamicrepeat-rich regions has facilitated effector protein family expansion, diversification, andadaptation to new hosts.

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FUTURE ISSUES

1. The potentially powerful role of epigenetics in generating diversity and adaptation at thetranscriptional level is poorly studied.

2. The same family of toxins or effectors may well adopt different roles in pathogenesisin different pathogen species or against different plant host species. More resolution intheir function is needed.

3. The mechanism(s) by which RxLR, crinkler, and other cell-entering effectors reachthe cytoplasm is still poorly understood. The role of PI3P in particular remains highlycontroversial. Effector entry mechanisms are a promising target for novel disease controlstrategies.

4. The role of HGT events in oomycete evolution and adaptation might be underesti-mated; with more genome resources and computational tools, the source and functionsof acquired genetic elements could be further studied.

5. By combining evolutionary and molecular analyses of pathogenicity mechanisms, it maybe possible to identify new targets for disease control that resist pathogen adaptation.

DISCLOSURE STATEMENT

R. Jiang is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review. B. Tyler discloses a patent application(“Methods and Compositions to Protect Plants and Animals against Pathogen Infection by Block-ing Entry of Virulence Proteins” Serial No. 61/128,080), a paid consultancy with Monsanto, Inc.(2011), and the following grants awarded over the past three years:

- NSF BREAD: “Engineering novel resistance against fungal and oomycete pathogens indeveloping country crop plants.” PI: B. Tyler; 3 co-PIs

- NSF: “How do oomycete and fungal effectors enter host plant cells?” PI: B. Tyler; 3 co-PIs- NSF:”Comparative functional genomics of oomycete effector proteins.” PI: J. McDowell;

co-PI: B. Tyler- USDA AFRI: “Integrated management of oomycete diseases of soybean and other crop

plants.” PI: B. Tyler. 18 co-PIs.- USDA AFRI: “Management of the switchgrass rust disease by deploying host resistant genes

and monitoring the dynamics of pathogen populations.” PI: B. Zhao. co-PIs: B. Tyler, et al.- USDA AFRI: “Insect effectors in molecular plant-insect interactions.” PI: J. Stuart co-PIs:

B. Tyler, et al.- USDA AFRI: “Probing oomycete-host interactions using an effector ORFeome and effector

protein microarrays.” PI: D. Kumar; co-PIs: B. Tyler, et al.- USDA AFRI: “Comparative genomics of host range in Phytophthora parasitica.” PI: B. Tyler;

2 co-PIs- USDA AFRI: “Genome sequence of the oomycete aquaculture pathogen Saprolegnia para-

sitica.” PI: B. Tyler; 3 co-PIs- USDA AFRI/NSF: “Phytophthora sojae: A high quality reference sequence for the

oomycetes.” PI: B. Tyler; 6 co-PIs- USDA AFRI: “Function of Phythopthora sojae effector Avr1b in infection.” PI: B. Tyler; 1

co-PI


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ACKNOWLEDGMENTS

We thank Jeannette Copley for manuscript assistance. This work was supported by federalfunds from NIAID grant number HHSN27220090018C and USDA grant 2008–35600-04646 toR.H.Y.J. and by grants 2004–35600-15055, 2007-35600-18530, 2007–35319-18100, and 2011–68004-30104 to B.M.T. from the Agriculture and Food Research Initiative of the USDA NationalInstitute of Food and Agriculture and by grants EF-0412213, MCB-0731969, IOS-0744875, andIOS-0924861 from the U.S. National Science Foundation.

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107. Torto TA, Li S, Styer A, Huitema E, Testa A, et al. 2003. EST mining and functional expression assaysidentify extracellular effector proteins from the plant pathogen Phytophthora. Genome Res. 13:1675–85

108. Torto-Alalibo T, Collmer CW, Gwinn-Giglio M, Lindeberg M, Meng S-W, et al. 2010. Unifying themesin microbial associations with animal and plant hosts described using the gene ontology. Microbiol. Mol.Biol. Rev. 74:479–503

109. Torto-Alalibo T, Collmer CW, Lindeberg M, Bird D, Collmer A, Tyler BM. 2009. Common andcontrasting themes in host cell–targeted effectors from bacterial, fungal, oomycete and nematode plantsymbionts described using the gene ontology. BMC Microbiol. 9(Suppl. 1):S3

110. Torto-Alalibo T, Tian M, Gajendran K, Waugh ME, van West P, Kamoun S. 2005. Expressed se-quence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors.BMC Microbiol. 5:46

111. Torto-Alalibo T, Tripathy S, Smith BM, Arredondo F, Zhou L, et al. 2007. Expressed sequence tagsfrom Phytophthora sojae reveal genes specific to development and infection. Mol. Plant-Microbe Interact.20:781–93

112. Tyler BM. 2007. Phytophthora sojae: root rot pathogen of soybean and model oomycete. Mol. Plant Pathol.8:1–8

113. Tyler BM. 2009. Effectors. In Oomycete Genetics and Genomics: Diversity, Interactions and Research Tools,ed. K Lamour, S Kamoun, pp. 361–85. Hoboken, New Jersey: Wiley-Blackwell

114. Tyler BM. 2009. Entering and breaking: virulence effector proteins of oomycete plant pathogens.Cellular Microbiol. 11:13–20

115. Tyler BM, Rouxel T. 2012. Effectors of fungi and oomycetes: their virulence and avirulence functions,and translocation from pathogen to host cells. In Molecular Plant Immunity, ed. G Sessa. New York:Wiley & Sons

116. Tyler BM, Tripathy S, Zhang X, Dehal P, Jiang RH, et al. 2006. Phytophthora genome sequences uncoverevolutionary origins and mechanisms of pathogenesis. Science 313:1261–66

117. van der Vossen E, Sikkema A, Hekkert BL, Gros J, Stevens P, et al. 2003. An ancient R gene from thewild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans incultivated potato and tomato. Plant J. 36:867–82

118. van der Vossen EAG, Gros J, Sikkema A, Muskens M, Wouters D, et al. 2005. The Rpi-blb2 gene fromSolanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance inpotato. Plant J. 44:208–22

119. Vleeshouwers VG, Raffaele S, Vossen JH, Champouret N, Oliva R, et al. 2011. Understanding andexploiting late blight resistance in the age of effectors. Annu. Rev. Phytopathol. 49:507–31

120. Vleeshouwers VGAA, Rietman H, Krenek P, Champouret N, Young C, et al. 2008. Effector genomicsaccelerates discovery and functional profiling of potato disease resistance and Phytophthora infestans avir-ulence genes. PLoS ONE 3:e2875

121. Voglmayr H. 2008. Progress and challenges in systematics of downy mildews and white blister rusts:new insights from genes and morphology. Eur. J. Plant Pathol. 122:3–18

122. Wang Q, Han C, Ferreira AO, Yu X, Ye W, et al. 2011. Transcriptional programming and functionalinteractions within the Phytophthora sojae RXLR effector repertoire. Plant Cell 23:2064–86

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124. Wawra S, Bain J, Durward E, Bruijn Id, Minor KL, et al. 2012. Host-targeting protein 1 (SpHtp1)from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate–dependent manner. Proc. Natl. Acad. Sci. USA 109(6):2096–101

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PY50-FrontMatter ARI 9 July 2012 19:8

Annual Review ofPhytopathology

Volume 50, 2012Contents

An Ideal JobKurt J. Leonard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Arthur Kelman: Tribute and RemembranceLuis Sequeira � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �15

Stagonospora nodorum: From Pathology to Genomicsand Host ResistanceRichard P. Oliver, Timothy L. Friesen, Justin D. Faris, and Peter S. Solomon � � � � � � � � � �23

Apple Replant Disease: Role of Microbial Ecologyin Cause and ControlMark Mazzola and Luisa M. Manici � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Pathogenomics of the Ralstonia solanacearum Species ComplexStephane Genin and Timothy P. Denny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �67

The Genomics of Obligate (and Nonobligate) BiotrophsPietro D. Spanu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �91

Genome-Enabled Perspectives on the Composition, Evolution, andExpression of Virulence Determinants in Bacterial Plant PathogensMagdalen Lindeberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 111

Suppressive Composts: Microbial Ecology Links Between AbioticEnvironments and Healthy PlantsYitzhak Hadar and Kalliope K. Papadopoulou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 133

Plant Defense Compounds: Systems Approaches to Metabolic AnalysisDaniel J. Kliebenstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 155

Role of Nematode Peptides and Other Small Moleculesin Plant ParasitismMelissa G. Mitchum, Xiaohong Wang, Jianying Wang, and Eric L. Davis � � � � � � � � � � � � 175

New Grower-Friendly Methods for Plant Pathogen MonitoringSolke H. De Boer and Marıa M. Lopez � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 197

Somatic Hybridization in the UredinalesRobert F. Park and Colin R. Wellings � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 219

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PY50-FrontMatter ARI 9 July 2012 19:8

Interrelationships of Food Safety and Plant Pathology: The Life Cycleof Human Pathogens on PlantsJeri D. Barak and Brenda K. Schroeder � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 241

Plant Immunity to NecrotrophsTesfaye Mengiste � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

Mechanisms and Evolution of Virulence in OomycetesRays H.Y. Jiang and Brett M. Tyler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 295

Variation and Selection of Quantitative Traits in Plant PathogensChristian Lannou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 319

Gall Midges (Hessian Flies) as Plant PathogensJeff J. Stuart, Ming-Shun Chen, Richard Shukle, and Marion O. Harris � � � � � � � � � � � � � � 339

Phytophthora Beyond AgricultureEverett M. Hansen, Paul W. Reeser, and Wendy Sutton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 359

Landscape Epidemiology of Emerging Infectious Diseasesin Natural and Human-Altered EcosystemsRoss K. Meentemeyer, Sarah E. Haas, and Tomas Vaclavık � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 379

Diversity and Natural Functions of Antibiotics Produced by Beneficialand Plant Pathogenic BacteriaJos M. Raaijmakers and Mark Mazzola � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

The Role of Secretion Systems and Small Molecules in Soft-RotEnterobacteriaceae PathogenicityAmy Charkowski, Carlos Blanco, Guy Condemine, Dominique Expert, Thierry Franza,

Christopher Hayes, Nicole Hugouvieux-Cotte-Pattat, Emilia Lopez Solanilla,David Low, Lucy Moleleki, Minna Pirhonen, Andrew Pitman, Nicole Perna,Sylvie Reverchon, Pablo Rodrıguez Palenzuela, Michael San Francisco, Ian Toth,Shinji Tsuyumu, Jacquie van der Waals, Jan van der Wolf,Frederique Van Gijsegem, Ching-Hong Yang, and Iris Yedidia � � � � � � � � � � � � � � � � � � � � � � 425

Receptor Kinase Signaling Pathways in Plant-Microbe InteractionsMeritxell Antolın-Llovera, Martina K. Ried, Andreas Binder,

and Martin Parniske � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 451

Fire Blight: Applied Genomic Insights of thePathogen and HostMickael Malnoy, Stefan Martens, John L. Norelli, Marie-Anne Barny,

George W. Sundin, Theo H.M. Smits, and Brion Duffy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 475

Errata

An online log of corrections to Annual Review of Phytopathology articles may be found athttp://phyto.annualreviews.org/

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mechanisms and evolution of virulence in oomycetes

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