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Oomycetes and fungi: similar weaponry to attack plants

Maita Latijnhouwers1,2, Pierre J.G.M. de Wit1 and Francine Govers1
1 2

Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, Netherlands Current address: Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Fungi and Oomycetes are the two most important groups of eukaryotic plant pathogens. Fungi form a separate kingdom and are evolutionarily related to animals. Oomycetes are classied in the kingdom Protoctista and are related to heterokont, biagellate, golden-brown algae. Fundamental differences in physiology, biochemistry and genetics between fungi and Oomycetes have been described previously. These differences are also reected in the large variations observed in sensitivity to conventional fungicides. Recently, more pronounced differences have been revealed by genomics approaches. However, in this review we compare the mode of colonization of the two taxonomically distinct groups and show that their strategies have much in common. Together, fungi and Oomycetes cover the majority of eukaryotic plant pathogens. The modes of colonization observed among fungal and Oomycete species are described in Box 1. At rst glance the growth patterns of these two groups of pathogens are similar. For this reason Oomycetes were long considered a class within the kingdom Fungi. Both fungi and Oomycetes show lamentous growth in their vegetative stage, produce mycelia and form spores for asexual and sexual reproduction. However, taxonomic analyses of phenotypic characteristics and sequence comparisons have unambiguously shown that the two groups are placed on different eukaryotic branches of phylogenetic trees. Based on comparisons of genes encoding, among others, the small ribosomal subunit, actin and tubulin, it was concluded that fungi share a common ancestor with animals. By contrast, the Oomycetes closest relatives are the heterokont golden-brown algae [1]. Although the reclassication of the Oomycetes is still under construction, it is no longer disputed that they are taxonomically unrelated to fungi (Figure 1). Several important differences between fungi and Oomycetes have previously been described (Box 2). Among these are the differences observed in sensitivities to conventional fungicides. Azole fungicides, for example, act by inhibition of the ergosterol biosynthesis pathway. However, Oomycetes do not synthesize ergosterol and are therefore insensitive to this important group of fungicides [2]. Because chitin is only a minor component of Oomycete cell walls [3,4], chitin synthase inhibitors, such as
Corresponding author: Francine Govers (Francine.Govers@wur.nl).

Nikkomycin and Polyoxin D, also have no inhibitory effects. Conversely, phenylamides that interfere with RNA polymerases, such as metalaxyl, are highly selective against Oomycetes [5]. As their vegetative stage is diploid, and homologous recombination has not been found to occur, Oomycetes are far less tractable to genetic manipulation than many fungi. Of all Oomycetes, Phytophthora is the best-studied genus, causing diseases not only of the economically important staple crops such as potato, cocoa and soybean, but also of valuable forest trees in California and Australia. Useful resources including genetic linkage maps, bacterial articial chromosome (BAC) libraries and expressed sequence tags (ESTs) of numerous different developmental stages have been generated in the last few years [6]. In addition to the conventional polyethyleneglycol (PEG)mediated protoplast transformation, three new DNA transformation methods have recently been developed. These are based on zoospore electroporation (B. Tyler and

Box 1. Different lifestyles among fungi and Oomycetes [12] Biotrophs

Biotrophs grow and reproduce in living plant tissue. They obtain nutrients through intimate interactions with living plant cells. Examples of plant-pathogenic fungi with a biotrophic lifestyle are Cladosporium fulvum, the causal agent of tomato leaf mold, and the species causing smut diseases, such as Ustilago maydis. Some biotrophs, for example, the species causing rusts and powdery mildews, cannot be cultured on articial media and are therefore called obligate biotrophs. Obligate biotrophic Oomycetes are found within the families Peronosporaceae and Albuginaceae, and cause diseases called downy mildews and white rusts. Obligate biotrophs usually form haustoria for retrieval of nutrients from plants, whereas biotrophs such as C. fulvum and U. maydis behave like endophytes and do not produce haustoria.

Necrotrophs
Necrotrophic species feed on dead plant cells. They kill host tissue before colonizing it. Cochliobolus and Botrytis species are examples of fungal necrotrophs, whereas Oomycete necrotrophs are found among the genera Pythium and Aphanomyces.

Hemibiotrophs
Hemibiotrophs, such as the rice blast fungus Magnaporthe grisea and several fungal species belonging to the genera Colletotrichum and Venturia, have intermediate lifestyles. They initially establish a biotrophic relationship with their host but subsequently, the host cells die as the infection proceeds. A similar lifestyle is observed in species of the Oomycete genera Phytophthora and Pythium.

http://www.trends.com 0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2003.08.002

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green algae land plants heterokont algae oomycetes ciliates dinoflagellates red algae animals basidiomycete fungi ascomycete fungi
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Figure 1. Phylogenetic tree showing the evolutionary relationships between the major eukaryotic groups. The Oomycetes and the (ascomycetous and basidiomycetous) fungi are highlighted in green. Note the evolutionary distance between the Oomycetes and the fungi. Reproduced from [71] and adapted from [70].

F. Govers, unpublished), microprojectile bombardment [7] and Agrobacterium tumefaciens-mediated transformation [8]. To circumvent the need for homologous recombination to obtain gene-knockout strains, gene silencing has been successfully used as a method to accomplish targeted knockdowns of some genes [9 11], but this still requires further optimization. Because of the development of these new methods and resources, signicant progress has been made in elucidating infection strategies of Oomycetes. In this review, important aspects of fungal and Oomycete pathology are compared, including histopathology of infection structures, the production of pathogenicity factors, and the role of conserved signaling pathways in development and pathogenicity. Spores and infection structures Efcient spore production and dispersal is a prerequisite for successful infection. Both Oomycete and fungal asexual spores can be dispersed by wind or rain. The asexual spores of most Oomycetes (sporangia) can undergo cytoplasmic cleavage, resulting in the formation of

Box 2. Important morphological and physiological differences between Oomycetes and fungi [13]
Fungi are haploid or dikaryotic during the major part of their lifecycle, whereas Oomycetes are diploid. Fungal hyphae are septate, whereas Oomycete hyphae are nonseptate. Many Oomycetes are (partial) sterol auxotrophs. Their membranes contain lipids with unusual structures and long-chain fatty acids that presumably replace sterols in mycelial membranes. Fungi and Oomycetes synthesize lysine by different pathways. The Oomycetes use the a,1-diaminopimelic acid pathway, whereas fungi synthesize this amino acid by the so-called a-aminoadipic acid pathway. Cell walls of most Oomycetes consist mainly of 1,3-b-glucans, some 1,6-b-glucans and 1,4-b glucans (cellulose). Chitin, which is a major constituent of fungal cell walls, has been detected in small amounts in only a few Oomycetes.

zoospores: wall-less, uninucleate cells possessing two agella of unequal length. Zoospores are dispersed in water drops, water lms or through water-rich soils. The mechanism by which these spores are attracted to the host is not fully understood, but there are indications that chemotaxis, electrotaxis and autoaggregation play a role [14,15]. Once zoospores have reached the host, they retract or shed their agella and become immobile cysts. Fungal and Oomycete sexual spores can usually survive under extreme environmental conditions (cold, heat, or drought) encountered during overwintering or oversummering on leaf debris or in the soil. Fungal spores generally use a combination of waterinsoluble glycoproteins, lipids and polysaccharides for host surface-attachment [16,17]. Magnaporthe grisea secretes pre-formed adhesive material from the spore tip, whereas in other fungi, spore attachment requires de novo protein synthesis. Adhesive substances of the fungi Blumeria graminis and Uromyces viciae-fabae include enzymes that can modify the surface of the host plant to improve spore attachment [16]. Germinating sporangia of the Oomycete Peronospora parasitica have been shown to secrete an adhesive layer containing glycoproteins and b-1,3-glucans that might contribute to germling attachment [18]. The Oomycetes Pythium and Phytophthora store adhesive material in small vesicles inside zoospores, which is released upon encystment [19]. In addition, germinating cysts of Phytophthora infestans produce proteins that contain many repeats that have homology with human mucins, and are thought to play a role in the protection of germlings from desiccation or physical damage [20]. Some fungi, such as the tomato leaf mould fungus Cladosporium fulvum, avoid breaching the cell wall by entering the plant tissue exclusively through stomata and remain conned to the apoplastic space. Other fungi, and many Oomycetes, form specialized infection structures named appressoria to enter the plant tissue (Figure 2a c). An appressorium is a swelling of the tip of a germ tube from which a penetration peg emerges that serves to pierce the epidermal cell wall or to enter the epidermis through stomatal apertures [16,17]. Many fungi require a hard, hydrophobic surface for the induction of appressorium formation and the topology of the surface, such as the presence of ridges, can be an important trigger (thigmotropism) [21]. Fungal appressoria are usually separated from the spore by a septum. Appressoria of M. grisea accumulate high concentrations of glycerol causing a turgor pressure of up to 8 MPa [16]. The presence of melanin in appressorial cell walls is also required for turgor pressure to buildup [16]. During the past ve years, many genes have been identied that are required for appressorium formation, turgor generation or for appressorial penetration in M. grisea and an overall picture of appressorium function is beginning to emerge [16,22 24]. Although many Oomycetes produce appressoria, those of Phytophthora, Pythium and Peronospora species are generally small compared with those of the fungi [25,26]. They are not melanized or pigmented, and the appressoria are only separated from germ tubes by so-called false septa, which do not actually divide the two cells [18,25,27]. As in fungi, topological features on the surface of host

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(f)

the case of infection by the rust fungus Puccinia hemerocallidis, the extrahaustorial membrane was recently reported to form tubular extensions into the plant cytoplasm, possibly to enlarge the interacting surface [31]. Genes that encode hexose and amino acid transporters and thiamine biosynthesis genes are highly expressed in haustoria of the rust fungus U. viciae-fabae, adding credence to the idea that haustoria play a central role in primary metabolism and the importing of nutrients [32,33]. Uptake of nutrients was proposed to be driven by a proton gradient generated by H-ATPases [34]. Haustoria of biotrophic Oomycetes such as Pe. parasitica are similar in size to those of rust fungi, but the organization, structure and composition of the electron-dense neckband are different [35]. By contrast, hemibiotrophic Oomycete species belonging to the genera Phytophthora and Pythium form small nger- or digit-like haustoria during the biotrophic phase [27,36] (Figure 2g). Cytological examinations suggest that many of the events following spore and cyst germination are highly similar in fungi and Oomycetes. However, more in-depth molecular and biochemical analyses will be required, especially in Oomycetes, to enable detailed comparisons of the composition of appressoria and extrahaustorial matrices and the identication of transport proteins and mechanisms in haustoria.
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Figure 2. Infection structures of plant-pathogenic fungi (a,d,f) and Oomycetes (b,c,e,g). (a) Cryoscanning electron micrograph of mature appressorium (indicated by an asterisk) of Magnaporthe grisea (micrograph kindly provided by Richard J. Howard, DuPont Crop Genetics). (b) Light microscopy showing a cyst of Phytophthora infestans germinated on an articial surface. At the tip of the germ tube an appressorium is formed (indicated by an asterisk). (c) Cryoscanning electron micrograph of P. infestans cyst (left) with the germ tube forming an appressorium (indicated by an asterisk). Site of penetration is adjacent to a stoma [72]. (d) Haustorium of Erisyphe graminis f. sp. hordei with lobed structure in epidermal host cell of Hordeum vulgare, stained with DiOC7, as viewed by differential interference microscopy [73]. (e) Micrograph showing uorescent trypan blue-stained tissue containing branching hyphae of P. infestans with haustoria in the mesophyll cells of the potato cultivar Bintje [74]. (f) Transmission electron micrograph showing haustorium of E. graminis f. sp. hordei in H. vulgare [75]. (g) Transmission electron micrograph showing haustorium of P. infestans in the potato cultivar Majestic [36]. Note digit-like shape of P. infestans haustoria in (e) and (g). Scale bars (a) 5 mm, (c) 8 mm, (d) 20 mm, (e) 35 mm, (f) 1 mm, (g) 2 mm.

tissues, such as ridges and irregularities, can induce formation of appressoria in Pe. parasitica, P. infestans, Phytophthora palmivora and Phytophthora sojae [14,25,28,29]. In contrast to fungi, turgor pressure in Oomycete appressoria has not been quantied, nor has the chemical composition of the low molecular weight solutes in appressoria been analyzed. Intercellular hyphae of biotrophic fungi, as well as Oomycetes, form spherical or lobed structures named haustoria, which penetrate the adjacent plant cells (Figure 2d g). Haustoria are structures specialized in retrieving nutrients from the host plant [30]. At the site of haustorium formation, the plant plasma membrane invaginates and surrounds the pathogens cell wall. The electron-dense material between the pathogen cell wall and the plant plasma membrane is known as the extrahaustorial matrix. A so-called neckband separates the extrahaustorial matrix from the plants intercellular space to prevent leakage of the material in the matrix. In
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Pathogenicity factors Compounds synthesized by pathogens are designated pathogenicity factors when they are essential for the successful infection and colonization, and are called aggressiveness factors when they contribute to the efciency of the infection process. Whether or not a compound is a pathogenicity or aggressiveness factor can be established by gene disruption or gene silencing, which should result in non-pathogenic mutants or less pathogenic mutants, respectively. Cell-wall-degrading enzymes (CWDEs), proteins involved in protection against plant defense compounds (counter-defense), and toxins are examples of pathogenicity or aggressiveness factors. Pathogen-derived compounds can also induce the arrest of pathogen infection. Many of these so-called elicitors, or avirulence factors, were discovered owing to their ability to elicit numerous defense responses that culminate in host plant resistance to pathogen attack [37,38]. Some of these compounds might in fact have intrinsic functions as pathogenicity or aggressiveness factors, but, as plants have evolved the ability to recognize them, they have become the telltale signals of the pathogens presence (Box 3) [39]. For this reason, they were initially called avirulence factors but gradually the terminology is changing; the word effector is taking root. Cell-wall-degrading enzymes CWDEs are often expressed in germ tubes and infection hyphae, which suggests that they are used to loosen the plant cell wall, facilitating successful penetration [40]. By contrast, necrotrophic fungi are thought to use CWDEs to break down the plant cell wall and feed on the released nutrients. A large number of fungal CWDE genes that encode polygalacturonases, pectin-lyases, pectinases,

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Box 3. Elicitors
Fungal and Oomycete elicitors were thoroughly reviewed recently [38,55]. Elicitors are pathogen-derived molecules, mostly secreted proteins, that are specically recognized by plants. The recognition event results in the triggering of efcient defense responses, often including a hypersensitive response (HR). It is assumed at present that many elicitors are pathogenicity or aggressiveness factors, and that plants have evolved the ability to recognize such factors at a later stage of (co)evolution with their pathogens [39]. This would explain why elicitors form such a heterogeneous group of molecules. However, as a consequence of this heterogeneity, searching for Oomycete elicitors based on homology with those of fungi, or vice versa, is not expected to be a successful approach. To date there is only one example of a fungal elicitor that has counterparts in Oomycetes. However, it also has a counterpart in bacteria, which suggests a horizontal gene transfer event across kingdoms. This elicitor was initially identied as a necrosisinducing protein in Fusarium oxysporum [56]. The Oomycete counterpart was discovered when a secreted Phytophthora protein with elicitor activity was sequenced [57]. The gene encoding this protein showed up independently in a computational analysis that selected for those genes that encode proteins secreted by Phytophthora species. This also demonstrates that such an approach can be used successfully to identify putative elicitors [58 60]. Unraveling the elicitors intrinsic functions and their roles in pathogenicity can be very informative to understand the infection strategies of pathogens. Table 1 gives an overview of: (i) putative functions of the few elicitors known to date that are homologous to known proteins in the databases; and (ii) the elicitors that have a role in pathogenicity or aggressiveness as shown by the disruption or silencing of the encoding gene.

Table 1. Putative functions of elicitors with homology to known proteins and elicitors of which disruption or silencing of the encoding gene revealed a role in pathogenicity or aggressivenessa
Elicitor Fungi AvrPita ECP1 ECP2 AVR4 NIP1 ACE1c Oomycetes INF1d GPE1 CBEL
a b c

Organism

Putative function

Gene disrupted or silenced

Role in pathogenicity or aggressiveness?b

Refs

Magnaporthe grisea Cladosporium fulvum Cladosporium fulvum Cladosporium fulvum Rhynchosporium secalis Magnaporthe grisea

zinc metallo protease Unknown Unknown chitin binding or protection against chitinases unknown polyketide synthese or nonribosomal peptide synthetase sterol binding transglutaminase; crosslinking of cell wall proteins cellulose binding glycoprotein

No Yes Yes Yes Yes No

Yes Yes No Yes

[48]

Phytophthora infestans Phytophthora sojae Phytophthora parasitica

Yes No Yes

No

No

[10]

references [38,55] unless otherwise indicated. only relevant for the genes of which the function in pathogenicity was investigated by gene disruption or silencing. it is hypothesized that ACE1 is an enzyme involved in the synthesis of a so far unknown secondary metabolite elicitor (I. Fudal et al. XXII Fungal Genetics Conference, Asilomar, CA, 2003). d INF1 belongs to a class of proteins named elicitins. The sterol binding property of this class of proteins was initially shown for elicitins from other Phytophthora species [61].

cellulases, xylanases and glucanases have been cloned [41]. Targeted disruption of many of these genes clearly shows their role in fungal pathogenicity [41]. However, there are several examples where the disruption or replacement of genes encoding CWDEs does not alter pathogenicity or aggressiveness, possibly because these genes are redundant and other isozymes encoded by paralogous genes are able to take over the function of the defective enzyme [41]. Oomycete species of the genera Phytophthora and Pythium are known to produce and secrete CWDEs in liquid cultures and on solid media [26,42]. Recently, a gene that encodes an extracellular endopolygalacturonase was identied in P. infestans. Polygalacturonases (PGs) are pectinases that hydrolyze the homogalacturonan backbone of pectin [41]. The P. infestans PG gene Pipg1 is expressed in germinating cysts, suggesting that this PG plays a role in the initial stage of infection. However, the gene is also expressed during all stages of growth in tomato. Therefore, the same enzyme could be involved in inducing or maintaining necrotrophic growth [43]. A remarkably large PG gene family that has 19 members was identied in Phytophthora cinnamomi [44]. In
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addition, ve genes that encode exo- and endo-1,3(1,4)-bglucanases were cloned from P. infestans, and lowstringency hybridization studies indicated the presence of additional glucanase genes in the P. infestans genome [45]. Phytophthora PGs and glucanases are more similar to the corresponding fungal and insect enzymes than to their plant counterparts, which seems to contradict the previously described evolutionary distance between Oomycetes and fungi. However, the enzymes probably perform similar functions and encounter a similar selection pressure. It has therefore been suggested that sequence convergence could explain the similarities between the Phytophthora and fungal CWDEs [43 45]. Plants produce CWDEs directed against fungal and Oomycete cell walls. These CWDEs presumably function by affecting the invaders cell wall integrity [41]. In addition, CWDE-mediated release of oligosaccharides from the pathogens cell wall is known to elicit defense responses in plants [46]. To escape from damage by plant glucanases, the Oomycete P. sojae secretes proteins that can inhibit those CWDEs. One of the glucanase inhibiting proteins, GIP1, specically inhibits EgaseA, the soybean endo-b-1,3-glucanase that is responsible for the release of

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glucan oligosaccharide elicitors from the pathogens cell wall. By secreting this protein into the apoplast, P. sojae prevents the release of oligo-b-glucans and therefore avoids or slows down early recognition by the host [47]. Similarly, P. infestans has evolved mechanisms to inhibit the activity of plant extracellular proteases. This pathogen secretes protease-inhibiting proteins, one of which, EPI1, interacts with and inhibits P69, which is a tomato serine protease involved in plant defense. Homologues of this protein have not been found in fungi (M. Tian and S. Kamoun, XXII Fungal Genetics Conference, Asilomar, CA, 2003). Fungi have also evolved strategies to escape from the action of plant CWDEs, in particular plant chitinases. AVR4 is an extracellular protein produced by C. fulvum while growing in planta. It contains a domain that, by binding to chitin, can protect the chitin in the cell wall of C. fulvum against the deleterious effects of basic tomato chitinases. AVR4 is also an avirulence factor, but its primary function probably involves a passive defense against plant chitinases [48]. AVR4 can also protect other fungi such as Trichoderma viride and Fusarium solani against the lytic effects of basic plant chitinases. Results show that Oomycetes and fungi have both found ways to defend themselves against plant CWDEs and other plant extracellular enzymes involved in defense, some by binding to the enzyme itself, but in the case of AVR4, by binding to chitin and preventing the enzyme binding to and acting on its substrate. Inactivators of plant defense compounds and toxins Plants not only produce CWDEs to inhibit pathogen proliferation, they also synthesize antimicrobial secondary metabolites such as phytoanticipins (pre-existing) and phytoalexins (induced by pathogens) for the same purpose. However, fungi have developed ways to prevent damage by these compounds. Some fungi produce enzymes that detoxify antimicrobial plant secondary metabolites, some of which have been shown to contribute to pathogenicity [49]. Another mechanism of tolerance to antimicrobial plant compounds in plant-pathogenic fungi seems to be based on the active secretion of these compounds. ABCtransporters are membrane pumps that are involved in transporting a wide variety of compounds over the plasma membrane: ABC-transporter knockout strains of Botrytis cinerea and Gibberella pulicaris are more sensitive to secondary metabolites of their respective hosts, which suggests that ABC-transporters actively secrete these compounds. The aggressiveness of these knockout strains, as well as the ABC-transporter disruptants of Mycosphaerella graminicola and M. grisea, is thereby reduced [50]. Often, Oomycete plant pathogens have to cope with the same antimicrobial compounds in their hosts as these fungi, which suggests that they too have evolved mechanisms to withstand them. To discover counter-defensive mechanisms that occur in pathogenic Oomycetes, the mechanisms described for fungi should be fully explored. Toxin biosynthesis also remains unexplored in Oomycetes. Some necrotrophic fungi produce toxins that kill host cells, and the genes involved in their biosynthesis have been studied in detail. Two important groups of fungal phytotoxins are non-ribosomally synthesized linear
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or cyclic peptides and polyketide derivatives [51]. Peptide and polyketide synthetases involved in the synthesis of the respective groups of toxins contain domains that are conserved among different fungal species. A large number of Oomycete pathogens are necrotrophic or have a necrotrophic growth phase. Therefore, the possibility that they too use toxic compounds to kill their host cells should be considered. Plants produce reactive oxygen species (ROS) as an early response to infection. These are frequently associated with the hypersensitive response (HR) [52]: a defense response that involves plant cell death around the site of infection. ROS are thought to be involved in limiting pathogen growth. Enzymes produced by pathogens, such as superoxide dismutases, catalases and peroxidases, are thought to be involved in the breakdown or inactivation of ROS. Several genes encoding ROS-inactivating enzymes have been cloned from fungi. However, their role in pathogenicity or aggressiveness remains uncertain [53]. Sequences derived from ROS-inactivating enzymes were identied in Phytophthora EST databases, indicating that Oomycetes can also defend themselves against damage by host-generated ROS [54]. Signal transduction involved in pathogenesis Recently, signaling pathways that have been conserved throughout evolution and underlie fungal development and pathogenicity have received much attention. These pathways transmit extracellular signals across the plasma membrane to the interior of the cell, allowing the pathogen to respond adequately and timely to changes in its environment [62]. The plant-pathogenic fungi Cryphonectria parasitica, M. grisea and Ustilago maydis have served as models for the dissection of the G-protein, cAMP and MAPK (mitogen-activated protein kinase) signaling pathways. Signaling through heterotrimeric G-proteins controls a wide range of developmental processes, including conidiation, vegetative growth, fertility, dimorphic growth and appressorium development, and plays a role in infectious, invasive growth [62,63]. In addition, a B. cinerea Ga subunit (BCG1) is involved in the secretion of proteases [64]. There is ample evidence that G-proteins feed into the cAMP pathway in several different fungal plant-pathogens; the addition of pathway constituents was able to restore normal development in fungal strains that contained mutations in the G-protein subunits. Also, strains with mutations in the catalytic subunits of cAMP-dependent protein kinase (PKA) or adenylyl cyclase displayed phenotypes similar to those of G-protein deletion mutants [62]. The role of MAP kinases in the mating pathway and pathogenicity of U. maydis has been extensively studied [62,65]. In addition, the MAPK PMK1 of M. grisea plays a key role in the onset of appressorium formation, in the transfer of storage carbohydrate and lipid reserves to the appressorium, and is also required for pathogenicity ([66] and reviewed in [62,67]). Another MAPK of M. grisea, MPS1, is thought to be involved in cell-wall integrity and is indispensable for appressorial penetration. The role of

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orthologues of PMK1 in pathogenicity has been studied in a wide range of other fungal species [67]. P. infestans is the only Oomycete in which the functions of G-protein subunits have been analyzed. Silencing of the Gb subunit gene Pigpb1 resulted in a defect in sporangia formation [11], reminiscent of the sporulation defect observed in the C. parasitica Gb disruptant strain [68]. Mutants decient in the Ga subunit PiGPA1 show a reduction in zoospore release and their aggressiveness is severely impaired. The zoospores of the PiGPA1-decient mutants change direction frequently and fail to aggregate or be attracted to chemotactic compounds. This suggests that PiGPA1 plays a role in the sensing capabilities of zoospores that are required to control motility (M. Latijnhouwers et al., XXII Fungal Genetics Conference, Asilomar, CA, 2003). As the genes that encode the respective kinases are well represented in the Phytophthora EST databases, a role for the cAMP and the MAPK pathway downstream of G-proteins is conceivable, but this has not yet been investigated. It can be concluded that both Oomycetes and fungi rely on a functional G-protein pathway for normal development and pathogenicity, whereas downstream pathways and targets of G-proteins have been studied only in fungi. To better understand how environmental cues affect development in these pathogens, the identication of receptors and ligands deserves more attention in future research. Concluding remarks In spite of the distinct evolutionary origin of the two groups, fungi and Oomycetes use infection strategies that have much in common. Their specialized infection structures (appressoria, infection hyphae and haustoria) share many features. A signaling pathway (the heterotrimeric G-protein pathway) that is a key-regulator in development and pathogenicity of fungi also seems to govern crucial processes in Oomycetes. Moreover, they both use an extensive toolbox of CWDEs to penetrate into and to degrade plant cell walls. This in itself may not be surprising, but the protein sequences of CWDEs of fungi and Oomycetes are more conserved than would be expected from their evolutionary relationship alone. This high degree of similarity in attacking and defensive mechanisms suggests that the in planta conditions are very similar for plant-pathogenic fungi and Oomycetes where they encounter a wide range of preformed and induced antimicrobial compounds. Thus, convergent evolution seems to have forced the development of similar infection strategies in these two types of plant pathogens. Horizontal gene transfer might also have occurred, either between the fungi and Oomycetes, or between these organisms and other species. It is hypothesized that fungi have acquired gene clusters from other organisms [69] and that the occurrence of homologues of the Fusarium oxysporum f.sp. erythroxyli necrosis-inducing protein in many Oomycete species might have been the result of horizontal gene transfer [59]. However, the frequency of occurrence of this phenomenon and its impact on evolution is not clear.
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Important differences between the infection strategies of fungi and Oomycetes involve classes of pathogenicity or aggressiveness factors that occur only in one of the two groups of plant pathogens. For example, antimicrobial plant secondary metabolitedetoxifying enzymes and fungal phytotoxins have not yet been detected in Oomycetes. Nearly complete genomic sequences of plant-pathogenic fungi have recently been released (http://www-genome.wi.mit.edu/seq/fgi/). In addition, funds have been allocated to produce genomic sequences of P. sojae and P. ramorum and recently random shotgun reads of the P. sojae genome corresponding to a depth of 8X have been released (http://www.jgi.doe.org). Complete sequence information for these Oomycetes will be very useful in aiding the discovery of novel pathogenicity and aggressiveness factors, based on either homology with fungi, or in high-throughput functional genomics approaches that employ gene silencing.
Acknowledgements
We thank the Dutch Science Foundation, Council Earth and Life Sciences (NWO-ALW) for nancial support. Thanks to Andre van t Slot and Arjen ten Have for critically reading the manuscript.

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