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Physiological and Molecular Plant Pathology 62 (2003) 99113 www.elsevier.

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Review

Advances in research on oomycete root pathogens


Pieter van West*, Alex A. Appiah, Neil A.R. Gow
Department of Molecular and Cell Biology, Aberdeen Comycete Group, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland AB25 2ZD, UK Accepted 26 January 2003

Abstract This review discusses recent advances in research into plant pathogenic oomycetes with an emphasis on root-infecting species. We focus on aspects of host targeting, mycoparasitism, and the development of molecular techniques that enable functional dissection of key genes of this economically important group of pathogens, including genomics, proteomics and gene silencing. We have not incorporated aspects relating to host resistance, research carried out into downy mildews, and other phyloplane oomycetes, unless there is also a specic relevance to the root-oomycete research community. The analysis of the asexual life stages of these organisms from zoosporogenesis through zoospore liberation, host targeting, encystment, germination, and the formation of appressoria-like structures in the rhizosphere offer signicant potential in the establishment of new approaches for the treatment of disease in these organisms. The advent of appropriate molecular tools is now enabling the molecular analysis of these developmental stages to begin in earnest and will stimulate research into an economically important but scientically neglected group of organisms. q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Chemotaxis; Electrotaxis; Oomycete; Aphanomyces; Phytophthora; Pythium; Gene silencing; Rhizosphere; Soil borne diseases

1. Impact of oomycete diseases Within the group of oomycetes several genera are found that contain economically important plant pathogens. For example the genera Phytophthora and Pythium include some of the worlds most destructive plant pathogens which account collectively for multibillion dollar losses in world cash crops. Presently, over 60 Phytophthora species (excluding marine species) have been described [29], all of which cause plant disease of crops, shrubs, and trees on a global scale. The importance of this group of organisms has both historic as well as economic impact. Historically, their importance predate the development of the germ theory of disease, and the science of plant pathology has its origins in the analysis of the major disease epidemics of the nineteenth century, the Irish potato famine (1845 1846) caused by the type species Phytophthora infestans [29]. It was estimated that this famine caused starvation and death of over 1 million people in Ireland and a similar number of Irish citizens emigrated to the United States and
* Corresponding author. Tel.: 44-1224-555800/555927; fax: 441224-555844. E-mail address: p.vanwest@abdn.ac.uk (P. van West).

elsewhere [40]. In a contemporary setting, it is estimated that P. infestans still costs the global potato industry $3 billion per annum [3]. Other equally devastating species of the genus include Phytophthora cinnamomi , Phytophthora sojae , Phytophthora palmivora , and Phytophthora megakarya . P. cinnamomi is a major root rotting pathogen with a very broad host range of over 900 species of plants [135], which has caused severe epidemics in the jarrah tree forest in Western Australia [99,100] as well as more recent outbreaks across the world. Both P. palmivora and P. megakarya are tropical species, which cause leaf blight, bark canker, pod rot (black pod) and root rot diseases of cocoa. These diseases are found in all the cocoa growing regions of the world and in particular Africa where P. megakarya is indigenous [14]. Its effects threaten directly the economies of cocoa producing countries [4]. Crop losses attributed to black pod disease caused by Phytophthora species amounts to 15 billion per year on the basis of an estimated 10 30% loss of world cocoa production [31]. Recent estimates of global crop losses due to black pod have increased to 44% [118] due to widespread devastation caused by P. megakarya. Another species, P. sojae (syn. P. megasperma f.sp. glycinea), is a constant

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problem in soybean growing areas around the world especially in the United States [117]. Pythium species such as P. aphanidermatum , P. debaryanum, P. myriotylum, and P. ultimum cause damping of diseases such as root rot, seedling blight and stem rot of many plants, including agronomic and vegetable crops [71]. It is estimated that Pythium diseases are also responsible for billion dollar losses around the world. Diseases caused by Pythium spp. are very common and important in the context of hydroponic cultivation of plants, in particular in the early seedling stages of crop development. There has been increased and widespread concern regarding the impact of oomycete diseases on food and bre production. Only last year, warnings were issued for the potential of a catastrophic potato famine in Russia, the worlds second-largest potato producer [109]. An epidemic of P. ramorum (an airborne pathogen) that causes sudden death of oak trees could be spreading to redwood and other species in California [67]. The Irish potato famine is therefore by no means limited to a historic footnote. Indeed many oomycete-based epidemics are only barely being kept in check by the aggressive use of prophylactic agrochemical oomycetecides.

3. Life cycle of root-infecting oomycetes The major stages in life cycles of most root-infecting oomycete species of Pythium and Phytophthora are similar. The life cycle of a typical Pythium sp. is shown in Fig. 1. This consists of two cycles that are usually stimulated by differing environmental conditions. The asexual cycle is characterised by the production of sporangia. Sporangia may germinate either directly in liquid or on a surface to produce a germ tube (direct germination) or may differentiate by a process of cytoplasmic cleavage to form uninucleate, biagellate zoospores (indirect germination). In root pathogenic oomycete species the zoospores are often retained within a discharge vesicle (Fig. 2(c)) that evaginates from the sporangium pore before being liberated by vesicle disruption. The zoosporangia of Phytophthora species are formed in an aqueous medium, often when the ambient temperature falls. The released zoospores swim in water in search of host tissues (seeds, roots, stems or leaves) where they settle and encyst. This rapid process takes place within minutes and anchors the zoospore to the plant surface via adhesins that are discharged onto the naked zoospore surface [7]. The cyst germinates by developing a germ tube that may penetrate the host either directly, or via an appressorium or appressorium-like structure [44]. Using nutrients acquired from the susceptible host, the intracellular aseptate hyphae ramify through the plant tissue developing a mesh of absorptive mycelium, from which sporulation occurs on the dying seedling and the disease cycle is repeated. The rapidity of this cycle enables the asexual stages to be repeated many times during the course of the growth of a plant. The sexual cycle generates thick walled oospores that are adapted for over-wintering and survival under harsh environmental conditions. Oosporogenesis involves the production of a female oogonium and a male antheridium that grows towards and fuses with the oogonium. The antheridia of heterothallic species of Phytophthora can be either amphigynous where the oogonial stalk is surrounded by the antheridium, or paragynous in which the antheridium makes contact with the oogonium away from the oogonial stalk [29]. Fertilisation occurs through the emptying of some of the contents of the antheridium into the oogonium, leading to the development of an oospore, which has a thick inner wall [128]. This resting spore can exhibit extended dormancy and can over-winter in the soil and then germinate under suitable conditions to produce single or multiple germ tubes. These germ tubes can then form sporangia thereby recapitulating the asexual cycle of the pathogen.

2. Taxonomy position of root pathogenic oomycetes Oomycetes are a group of fungus-like mycelial organisms that belong to the kingdom Straminopila [5, 77], but represent a unique evolutionary line distant from true fungi. In addition to being dispersed via zoospores and generating thick walled sexual oospores, they possess features such as cellulose (b-1,4-glucan) in their cell walls [8], vegetative diploidy [107,108], heterokont agellae (one tinsel and one whiplash) [6], tubular mitochondrial cristae [15], and in the case of Phytophthora species, lack of epoxidation of squalene to sterols [41], that all distinguish them from true fungi. Indeed, phylogenetically they are most closely related to the heterokont algae such as the chrysophytes and to diatoms [112]. Oomycetes are divided into three subclasses: Saprolegniomycetidae, Rhipidiomycetidae, and Peronosporomycetidae. The majority of plant pathogenic oomycetes belong to two orders within the Peronosporomycetidaethe Peronosporales and Pythiales. Most sh pathogenic oomycetes belong to the Saprolegniomycetidae. However, within the Saprolegniomycetidae are species of the genus Aphanomyces, which include root pathogens. The downy mildew pathogens, belonging to genera such as Bremia, Plasmopara and Peronospora, are classied within the order Peronosporales and are all obligate plant pathogens. The majority of the root pathogenic oomycetes are classied within the family Phytiaceae that comprises the genera Pythium and Phytophthora.

4. Cell biology of oomycete root interactions The molecular processes that underlie these developmental stages of oomycetes are now being studied.

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Fig. 1. Life cycle of a typical root infecting Pythium species.

Molecular investigations have focussed on the pre-infection stages and the infection process. 4.1. Sporangial release and zoospore swimming Zoospores are free-swimming propagules that are released from sporangia during wet conditions (see below). This process is commonly referred to as sporangial release and can occur within few minutes (Fig. 2(a) (e)). Sporangia are multinucleate and remain so until cytokinesis is initiated by a cold shock that ultimately compartmentalises single nuclei within each zoospore. Divalent cations, and in particular Ca2 play an important role during sporangial release. Two distinct changes in cytoplasmic free Ca2 are associated with sporangium differentiation of P. cinnamomi [53]. Within the rst minute of cold shock, Ca2 concentrations rise rapidly, but by ten minutes fall back to almost the initial resting concentration. A second gradual and more longlasting increase in Ca2 concentration occurs during the process of cytoplasmic cleavage and is thought to be associated with the regulation of cytokinesis. Osmoregulation and turgor pressure within the sporangium during zoospore release in P. nicotianae is probably regulated by cytoplasmic proline [2]. High concentrations of proline accumulate in the cytoplasm of developing zoospores in the differentiating sporangium. The high proline concentrations may counterbalance

a hyper-osmotic extracellular sporangial lumen. Subsequently, proline is expelled rapidly from the zoospores after their release from the sporangium, thereby preventing osmotic rupture of the zoospore membrane. Once released, the zoospores are unable to build up turgor pressure as part of their osmoregulation and instead, as in many other protists, osmoregulation is maintained by the contractile water expulsion vacuole which pumps water from the cytoplasm. This water expulsion vacuole consists of a reticulum of tubular membranes, the spongiome, surrounding a central bladder [17,94]. The ATPases on the spongiome membranes are presumed to energise the contraction of the water expulsion vacuole [82]. Treatment of zoospores with 2 mM KNO3, an inhibitor of vacuolar H-ATPases, slowed the pulsing of the water expulsion vacuole cycle to nearly half the rate in untreated cells and lead to premature encystment [82]. Zoospores contain several characteristic types of vesicles including large peripheral vesicles, which are thought to function as nutrient stores [78]. Recently, Marshall et al. [78] discovered that these large peripheral vesicles contain at least three high molecular weight proteins, designated LPVs. The Lpv genes contain a variable number of highly conserved 534 bp repeats, which are anked by unique sequences. Expression studies revealed that the LPV transcripts are specically produced after induction of sporangial formation, hinting towards a role in zoospore biology.

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Fig. 2. (a e) Sporangial release of zoospores from Phytophthora palmivora. Pictures were taken from 21 to 26 min after a 4 min cold-shock. Sporangia (sp), papilla (p), discharge vesicle (dv), and zoospores (z) are indicated. The scale bar represents 10 mm. Pictures were taken by Samantha J. Shepherd, Department of Molecular and Cell Biology, University of Aberdeen, Scotland, UK. (f h) Histochemical localization of b-glucuronidase (GUS) activity in infection structures of Pythium aphanidermatum expressing the GUS-reporter gene. Appressorium-like structures (a), cysts (c), an intracellular hypha (ih) penetrating a root hair (rh), an infection site (is), hyphae (h), and an haustorium (ha) are indicated. The scale bars represent 10 mm. Pictures were kindly provided by John J. Weiland, Northern Crop Science Laboratory, SU Station, Fargo, North Dakota, USA. (i) Encysted zoospores in a sporangium. (j) An appressorium from a Phytophthora palmivora strain expressing Green Fluorescent Protein (GFP). An appressorium (a), cysts (c), germinated cyst (gc), nucleus (n), sporangium (sp), and a vacuole (v) are indicated. Scale bars represent 10 mm. (k) Spatial localisation of encysted zoospores of Phytophthora palmivora and Pythium aphanidermatum on a ryegrass root. The ryegrass root was placed in a suspension of GFP-marked P. palmivora zoospores, and then wounded with a scalpel at the point indicated (w). Zoospores of P. aphanidermatum were then added and the root was photographed after 10 min. The uorescence image is overlaid with a bright-eld image and shows the accumulation of non-GFP P. aphanidermatum zoospores at the site of the wound as an aggregate (ag). Root tip (rt) and root hairs (rh) are indicated. Scale bar represents 100 mm.

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4.2. Encystment and docking of zoospores Encystment of zoospores is a rapid process involving detachment of the two agella, and secretion of glycoproteins and other molecules to form the primary cell wall [44]. During docking, zoospores orient with the ventral groove (bearing the agella) facing towards the root surface as they encyst [22,45]. Cysts become rmly attached to the root surface due to the release of these adhesive molecules that are pre-formed in small peripheral vesicles, which are situated near the groove of the ventral surface [45,110]. The molecular composition of the adhesion substance is still unknown, but antibodies that react specically with this material from P. cinnamomi recognise a protein of just over 200 kDa [45]. It has been suggested that the part of the infection cycle involving zoospore release, encystment to cyst germination, and host penetration is dependent on a complex series of calcium signalling events [19,127]. Calcium is required for normal swimming patterns of zoospores at concentrations of at least 0.2 mM [26,42]. Depletion of external calcium with the chelator EGTA caused zoospores of P. aphanidermatum to swim in tight circles. Death and cell lysis followed [26]. External calcium concentrations of up to 1 mM resulted in suppression of zoospore turning movements [103]. Several studies have shown that encystment of zoospores and subsequent germination is stimulated by high concentrations of external Ca2 [22,25,26,51,103,125,134]. Experiments using the calcium-dependent uorophore Fura-2 showed that encystment of zoospores of P. parasitica (syn. P. nicotianae was correlated with an initial Ca2 inux, followed by a larger more progressive efux of Ca2 over a period of 20 30 min [127]. The later efux of intracellular Ca2 correlated with germination of the cysts. This phase was inhibited by TMB-8, which inhibits the release of Ca2 from internal calcium stores, and this inhibition was alleviated by addition of high external Ca2. Some oomycetes, such as P. sojae, are able to release a secondary zoospore from a cyst-a process, which is also regulated by Ca2 [125,134]. In medium with low concentrations of Ca2 a second zoospore was formed from most cysts. However, zoospore formation was suppressed and cyst germination was promoted by higher concentrations of exogenous Ca2 [125]. Other conditions and stimuli also induce zoospore encystment. Agitation is often used in the laboratory to obtain simultaneous encystment of a zoospore suspension. High concentrations of nutrients and peptides also induce encystment [12,25,52,122,125,126]. The soybean isoavone, daidzein, was found to attract P. sojae zoospores and also induced encystment [19]. Detailed experiments indicated that recognition of daizein triggers Ca2 inux into the zoospores. Recently, Latijnhouwers et al. [73] found compelling evidence that zoospore encystment in P. infestans is triggered by mastoparan, phosphatidic acid, and n- and sec-butanol. Mastoparan, which is a G-protein

activator, induced the accumulation of phosphatidic acid. Furthermore, it was shown that the mastoparan-induced increase by phosphatidic acid was due to stimulation of phospholipase-D. Therefore P. infestans (and presumably also other oomycetes) have a mastoparan- and butanolinducible phospholipase-D pathway involved in the regulation of encystment [73]. 4.3. Germination and formation of appressoria-like structures Formation of the germ tube occurs on the ventral surface that is orientated towards the root surface [45]. Germ tubes may penetrate the root directly via the anticlinal walls between the epidermal cells. Alternatively an appressorium may be formed that enables direct cell penetration. Appressorium production can also be induced in the absence of host tissue on various articial membranes [69]. If the surface displays topographic micro-discontinuities, as occurs on polypropylene foil or Paralm, appressoria are produced more readily [69]. Sometimes under these in vitro conditions a germinated cyst may produce multiple appressoria [88]. This indicates that the germinated cyst contains enough energy and biomass to produce secondary or tertiary appressoria. During the formation of the appressorium, cytoplasmic migration takes place from the cyst into the germ tube and the appressorial vesicle. A septum is formed that separates the now empty cyst from the cytoplasm-lled appressorium [69]. The mechanism by which turgor pressure is generated in the appressoria remains to be dened, although preliminary data suggests that accumulation of proline might play a role in this (van West and Gow, unpublished observations). The molecular processes involved in cyst germination and appressoria formation in oomycete plant pathogens are unknown. G-protein-mediated signalling may be hypothesised to play a role during formation and germination of cysts. This is supported by expression analysis of the G protein a and b subunit genes of P. infestans, which are upregulated during these developmental stages [74]. In P. infestans, a gene family encoding Car (cyst-germinationspecic acidic repeat) proteins, was identied. Car gene expression was found specically during cyst germination and appressorium stages [34]. Car proteins are homologous to mammalian mucins and are localized on the surface of the pre-infection structures. These adhesive proteins may therefore be important components of the mucous layer that protects the germinated cyst from desiccation, physical damage, or adverse effects of the plant defence response [34]. Attempts to identify proteins that are produced specically in germinated cysts and appressoria are in progress. For example a proteomics based approach, employing P. infestans and P. palmivora is being followed. Several newly synthesised proteins were found on 2D gels containing proteins from germinated cysts and also appressoria of

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P. infestans. These are now being analysed (Grenville, Taylor, Gow, and van West, unpublished data). 4.4. Pathogen plant interactions Considerable interest has focussed on identifying factors from oomycetes that induce defence responses in host and non-host plants (reviewed in Refs. [59,61,117]). Pathogenicity comprises all the cellular and molecular processes that are necessary for the pathogen to establish and maintain a successful infection. This denition implies that genes which are generally considered as house-keeping genes do not encode pathogenicity factors. However, proteins and other macromolecules involved in overcoming physical and chemical barriers (such as cutinases or plant cell wall degrading enzymes), detoxication of plant substances (such as phytoalexins), production of toxins, and formation of infection structures, are considered to be potential pathogenicity determinants [93,119]. Only a few potential pathogenicity factors have been isolated and studied in oomycete plant pathogens. These consist mainly of wall degrading enzymes such as endocellulases, 1,3- b-glucanases, b-glucosidases, cutinases, pectin-esterases, galactanases and endopolygalacturonases [119]. An 85-kDa N-mannosylated betaglucosidase/xylosidase (BGX1) was recently puried from culture ltrates of P. infestans [16]. BGX1 did not hydrolyse carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting it has a high substrate specicity for native polysaccharides and/or specic cofactor requirements for enzymatic activity. Interestingly, the bgx1 gene was found in various Phytophthora species but not in Pythium spp. Detailed proteomic analysis of secreted proteins in P. infestans culture ltrates by 2D gel electrophoresis and peptide mass mapping identied Bgx1 amongst 100 major secreted proteins (Li, Kamoun, Gow and van West, unpublished data). Furthermore, an endopolygalacturonidase gene from P. infestans has been identied [115]. Polygalacturonases (PGs) are pectin-degrading enzymes that have been implicated in the invasion of plant tissue by plant pathogenic oomycetes. They are secreted during saprophytic and parasitic growth. At least 17 different PG genes were found in P. cinnamomi [35]. Therefore oomycetes have a wide range of plant cell wall degrading enzymes that are presumed to function in host penetration and pathogenesis. Plants can also secrete enzymes that are able to degrade the cell wall of the invading pathogen. These enzymes are often classied as pathogenesis-related (PR) proteins because expression is normally induced upon infection (reviewed in Ref. [114]). Furthermore, in response to attack by cell wall degrading enzymes, plants also produce proteins that inhibit these enzymes, including inhibitors of polygalacturonidase and xylanase [23,32,79]. It has also been shown that oomycete plant pathogens secrete proteins

that can inhibit PR proteins such as endo-1,3-b-glucanases secreted by plants [43,105]. Analysis of a group of glucanase inhibitor proteins (GIPs) isolated from P. sojae, revealed sequence homology with the trypsin class of Ser proteases [105]. However the catalytic sites of the GIPs are mutated, suggesting that they are not functionally active proteases. GIP1 is able to form a complex with an endoglucanase of soybean (EgaseA) during pathogenesis, and interestingly it suppresses the release of oligoglucoside elicitors. The latter is a remarkable nding, since previous research demonstrated that EgaseA is able to release elicitors from P. sojae. Recognition of the elicitors by the soybean plant reduced the infection rate signicantly [92]. The fact that GIP1 is able to reduce the release of potential elicitors and thus acts as a defence suppressor clearly suggests a potentially important role in pathogenicity.

5. Strategies for zoospore root targeting Given the brief intervals of opportunity for zoospores to be released and nd their target, it would be surprising from a strategic point of view if the targeting mechanism were to be restricted to specic host plant species. Instead it seems that zoospores have a variety of types of mechanism that maximise the chances of the zoospore locating and encysting on any plant surface, including plant roots. The specicity of interactions between particular pathogens and certain plants is encoded in specic gene-for-gene interactions that occur later, at the time of attempted penetration and invasion of the plant [24]. The zoospore-phase is nonspecic as far as host selection is concerned. There are several mechanisms that account for ability of zoospores to accumulate at the surface of a root or at certain sites of shoots and leaves [38]. It may be convenient to consider these mechanisms individually, but it is certain that not one is sufcient on its own to explain the observation that zoospores often accumulate at certain specic sites in high numbers on the plant surface. The only role of the zoospore is in transmission from plant to plant, not in invasion of the host surface. 5.1. Mechanisms of targeting and accumulation of zoospores It is self-evident that when zoospores locate potential invasion sites on a plant, they must take advantage of natural cues at plant surfaces to guide them to their target. There are two basic types of mechanisms employedzoospore taxis and induced zoospore immobilisation. With tactic mechanisms the zoospores exhibit directional swimming responses with respect to a chemical, nutrient, ionic or electrical gradient. Importantly, the immobilisation of zoospores at a site of the plant surface can give the appearance that taxis resulted in the observed accumulation. However,

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immobilisation can occur in the absence of taxis since this leads to selective entrapment of the pathogen at the plant surface. Distinction between these two non-exclusive mechanisms requires careful measurement of directional swimming using some form of image analysis to track the net movement of individual zoospores. Many video microscopy packages offer such facilities and these have been applied recently to the analysis of zoospore movements [26,27,122]. One commonly cited guidance cue is that of the natural gradient of nutrient exudates that exist at root tips or around wound sites [21,47,49,106]. Zoospores have been reported to be chemotactic to root exudates and to their component solutes [28,38,125]. Zoospores may well exhibit directional swimming in such gradients, however, in many cases bone de taxis has not been demonstrated unequivocally. This is relevant since it is clear that high concentrations of nutrients and certain ions such as Ca2 lead to the immobilisation (encystment) of swimming cells [25,52,125,126,127]. Therefore capillary tube swim-in tests, which have been used extensively to study chemotaxis have to be interpreted with this observation in mind. However, some notable examples of true chemotaxis to plant-derived compounds include that of attraction of P. sojae zoospores to daidzein and genisteintwo isoavones of soybeans [89]. Also relevant to the use of capillary tube assays and other assays of apparent chemotaxis is the phenomenon of autoaggregation [68,101,103]. Some zoospore species swim spontaneously together in groups of hundreds or thousands (Fig. 3(a) (e)). Encystment may follow and further zoospores are then actively attracted to the cyst aggregate. Germ tubes that emanate from peripheral cysts exhibit clear tropic growth towards the aggregate (Fig. 3(f)).

The agents that induce these behaviours are not known, but in P. palmivora the agent is heat stable and pronase-resistant (Campbell and Gow, unpublished observations). Moreover, these autoattractants are species-specic since an aggregate formed by one zoospore species is not attractive to zoospores of other species or genus [103]. Agents that induce encystment in a localised area are likely to also induce autoaggregation [103]. These include physical collisions, high nutrient or calcium concentrations and a range of other stimuli. Therefore groups of encysted zoospores within a capillary tube may establish an autorecruitment signal, distinct from that which induced the primary encystment of the rst zoospores to arrive in the tube. Molecules at the surface of a plant, including cellulose [84], components of root mucilage and border cells, polyuronates [136] and certain fucosyl compounds may also induce zoospore encystment [48,75]. Furthermore, antibodies that recognise the specic agella components can induce encystment [12,30,46]. Therefore chemical interactions at the surface components of plants may initiate the encystment process that may be underpinned and enhanced by subsequent autoaggregation phenomena (reviewed in Ref. [38]). However, coating of roots with a thick layer of calcium alginate did not affect the overall accretion pattern suggesting that plant contact was not obligatorily required for successful aggregation at the root surface [28,54]. 5.2. Electrotaxis as an alternative guidance cue for zoospores Plant roots act as batteries in the soil. They establish endogenous circulating ionic currents and associated electrical elds in the rhizosphere that can be measured with voltage-sensitive vibrating electrodes [9,39,50,80,81, 91,132]. The ionic currents are due to spatial asymmetries in the distributions of electrogenic ion transport protein in the cortical cells of roots. When ion pumps are spatially segregated from ion channels, ionic current ows between them and a current-carrying ionic circulation is generated that has both an intracellular loop and an extracellular one in the rhizosphere. This circulation may serve the plant in maintaining ionic balance, and may also be relevant to the mechanism of polarised root development and gravitropism [37 39]. The main current-carrying ions of plant roots are H, Cl2, K and Ca2 [18,50,80,132]. As a consequence pH and ionic gradients are also established at the root surface and can be detected using ion-selective microelectrodes [39]. The consequences of these ionic gradients to oomycetes-root interactions is important in several aspects. Zoospores exhibit pH-taxis [87], but buffering of the standing pH gradient around roots did not inuence the pattern of accumulation of P. palmivora zoospores, suggesting that this tactic response is not relevant to root targeting.

Fig. 3. Autoaggregation of zoospores from Phytophthora palmivora. (a e) A zoospore suspension of P. palmivora was left at room temperature for 2 min. Images were taken at 30 s intervals. Aggregation of zoospores started after 30 s (b). Aggregation centres are visible as white condensed areas. The zoospores are free-swimming and can be dispensed by gentle shaking. (f) Zoospore aggregate of P. palmivora after 45 min at room temperature. Germ tubes that emanate from peripheral cysts exhibit tropic growth towards the aggregate.

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The electric eld generated by plant roots in the rhizosphere will depend on the resistivity and hence salt content of the water lms that surround the root. The higher the salt content, the lower the electrical eld for the same ionic current density. Also the resistivity of water-saturated soil will be higher due to the dilution of salts and will support a larger endogenous electrical eld. For soils with a resistivity of around 5000 V cm, roots will typically generate a eld of around 05 to 50 V cm21 [38,39,81,85]. Wounds generally generate stronger but more transient elds which may be maximally around 100 500 mV cm21 [50,83]. These electrical elds have a reproducible prole along the rhizoplane and the total inward and outward ionic current remains balanced as a whole. Therefore it is important to view the root as a dynamic and electrically contiguous tissue with a physiology that is dependent on its integrity over many centimetres. Zoospore electrotaxis was described many years ago [49, 65,66,116]. However early studies often applied electrical elds using bare electrodes that can generate electrode products that immobilise zoospores rapidly. More recently, chambers for electrotaxis experiments were made in which zoospores were protected from electrode products via long agarose bridges. These studies conrmed that zoospores were genuinely electrotactic in elds that were smaller than those capable of being generated in the rhizosphere [85,86]. Zoospores of P. palmivora were found to be anodotactic while those of Pythium aphanidermatum were cathodotactic [85]. The mechanism of electrotaxis reected differences in the electrical dipole of the anterior and posterior agella since metabolically-paralysed zoospores rotated in the exogenous electrical eld with the anterior agellum towards the electrical pole to which they swim. In addition, the turning frequency of the zoospores increased 3-4-fold in a physiological electrical eld. The obvious extension of these experiments was to determine whether the electrical eld around the root had any inuence on zoospore behaviour and attraction in the rhizosphere. A recent study showed that there was indeed a close correlation between the in vitro electrotactic behaviour and the sites of zoospore accumulation at natural root anodes and cathodes [122]. In this study the endogenous electrical elds were rst mapped with voltage-sensitive vibrating electrodes and then the pattern of zoospore accumulation and the swimming of individual zoospores was measured by image analysis to distinguish tactic and entrapment-based responses. Anodotactic zoospores of P. palmivora swam towards and encysted preferentially on anodic regions of roots of rye, wheat and cacao plants but were repelled from cathodic wound sites. In contrast, cathodotactic zoospores of P. aphanidermatum were attracted to wounds and to the cathodic regions behind the root apex. These zoospores were actively repelled from the root apex. Indeed mixtures of these two zoospore species became spatially segregated at the root surface when added together (Fig. 2(k)). Further experiments tested

the robustness of this correlation by examining the consequences of natural or induced perturbations of the normal electrical eld pattern around roots. The electrical eld declines progressively after wounding. The attractiveness of the wound for P. aphanidermatum zoospores declined concomitantly. Treatment of roots with fusicoccin lead to electrical eld reversalthis again had predictable effects on the zoospore-swimming pattern adjacent to the roots. Furthermore, focal electrical elds applied using microelectrodes that were held adjacent to the root surface with a micromanipulator could be used to recruit zoospores to sites of roots that were normally repellent. Therefore, electrical signals could override normal endogenous zoospore recruitment signals at the root surface. Focal electrical elds were also used to demonstrate that prolonged exposure to an electrical eld induced zoospore encystmentas found when zoospores are exposed to high nutrient concentrations [122]. Taken together these experiments demonstrate that the endogenous electrical eld prole of a root is the best predictor of zoospore accumulation sites. To invoke a chemotaxis-based explanation for these observations would require that wounds exude both specic zoospore attractants (e.g. for P. aphanidermatum) and zoospore repellents (e.g. for P. palmivora) and that root apices exude attractants for P. palmivora and repellents for P. aphanidermatum. The fairly expansive literature on zoospore chemotaxis provides little supporting evidence for these specic chemical-signalling events. Rather, the available evidence seems to point to electrotaxis being an important and dominant short-range guidance cue that determines zoospore localisation patterns around roots.

6. Biological control The use of biological control strategies to suppress root infecting oomycetes using bacterial, oomycete or fungal antagonists, is an area of increasing research interest [133]. Interest in this area is driven in part by concerns over the use of chemicals in the environment and the need to identify alternative disease control strategies. The development of such biocontrol strategies relies on a thorough understanding of the interactions between plant, pathogen and antagonist, the conditions under which biocontrol can be achieved, and the ecological impact of the microbial antagonist in the soil environment. The full potential for biological control of oomycete pathogens has not been explored but several recent studies have described progress in the control of pythiaceous species by mycoparasitism [133]. Oomycete mycoparasites such as Pythium oligandrum, Pythium acanthicum, and Pythium periplocum are capable of suppressing various soilborne oomycete and fungal plant pathogens [1,10,11,76,95,98,104]. The release of diffusible inhibitors (antibiosis), can affect the hyphae of the host

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before contact with the antagonist takes place [10,133]. Growth of P. oligandrum hyphae towards P. parasitica cells correlated with changes in the host cells, including retraction of the plasma membrane and cytoplasmic disorganisation [98]. Deposition of cellulose-enriched material occurs on the inner host cell surface. Interestingly, P. oligandrum is able to penetrate these thickened host cell walls with this cellulose-enriched material, suggesting that it secretes cellulolytic enzymes. The initial stimulus for P. oligandrum to grow towards its hosts is not known, but probably involves chemical stimuli or chemotropic growth towards host metabolites [98]. In addition to its antagonistic effect against a wide variety of fungal and oomycete plant pathogens, P. oligandrum is also able to penetrate the tomato root system without causing extensive cell damage, and thereby antagonise cell invasion of both Fusarium oxysporum and P. parasitica in situ [10,97]. Interestingly, this apparently induced resistance phenomena seemed to be due to de novo formation of structural and biochemical barriers in the root rather than to mycoparasitism. It was concluded that callose depositions inltrated with phenolic compounds restricted growth of Fusarium to the outermost root cortical layers. Picard et al. identied a 10 kDa protein produced by P. oligandrin (oligandrin), that induces a systemic plant defence reaction [97]. Tomato plants treated with oligandrin and subsequently challenged with P. parasitica show signicantly decreased disease. Ultrastructural investigations revealed that Phytophthora cells were damaged and that less pathogen biomass was found in oligandrintreated plants. At present it is unclear whether oligandrin formed in planta has a direct anti-oomycete effect, or whether it induces synthesis of other anti-microbial compounds in the plant. However, treatment of P. parasitica cells with oligandrin did not result in morphological or structural changes in challenged hyphae. These ndings are important because oligandrin is the rst example of a protein of oomycete or fungal origin that is able to exert an effect on pathogen colonisation in a host plant [97]. Oligandrin shares some similarities with several elicitins from various oomycetes and it has been proposed that it is also involved in sterol binding [72]. Elicitins are secreted proteins that can induce a hypersensitive response (HR) in a restricted number of plants, particularly in the genus Nicotiana within the family of Solanaceae [62,64]. Unfortunately, Picard and co-authors did not examine the effect of inltration of oligandrin into tobacco. Therefore at present it is not known whether oligandrin induces a HR in tobacco. The P. infestans elicitin, INF1, other elicitins [62, 64], and oligandrin [97], all failed to provoke a HR in tomato, indicating that tomato plants are not sensitive to these potential elicitors. It would be interesting to nd out whether tomato plants inltrated with elicitins from various oomycetes exhibit similar defence responses towards P. parasitica as oligandrin.

7. Molecular tools for oomycete research 7.1. Transformation methods The principal impediment to progress in the molecular analysis of oomycetes has been the lack of efcient genetic and molecular biological tools, and in particular the absence of an efcient transformation system. Judelson et al. [58] rst developed a CaCl2- and polyethylene-glycol-based (PEG) transformation protocol for the late blight pathogen P. infestans. At that time, transformation and even directed mutagenesis was well established in several plant pathogenic fungi [70,113] but oomycete molecular biology had barely taken its rst steps. Subsequently stable transformation procedures based on the Ca2-PEG transformation have been developed for several other oomycete species ca [90], P. including P. sojae [57], Saprolegnia mono palmivora [123], P. parasitica [13], and Pythium aphanidermatum [130] (Fig. 2(f) (h)). Experiments are also in progress to transform Aphanomyces cochlioides and Aphanomyces euteiches, and regeneration of protoplasts of these root pathogens has already been achieved successful [131]. Unfortunately, a key enzyme preparation to generate protoplasts of oomycetes, Novozym 234 (Novo Nordisk, Denmark) was withdrawn from production in 2000 and therefore alternative methods are being developed to transform various oomycete species without the need for protoplasts. An Agrobacterium tumefaciens-based transformation system [124] and a biolistic particle bombardment method [20] have been developed recently for P. infestans. In addition, an electroporation-based transformation system was developed for P. palmivora,P. infestans and P. sojae (B. Tyler, personal communication). The latter method has been used successfully to generate P. infestans strains with altered expression proles of the G-alpha and G-beta genes (Latijnhouwers and Govers, personal communication). All currently available transformation methods have transformation efciencies yielding only up to 50 transformants per experiment. More efcient transformation protocols would signicantly enhance progress and would open the door for new molecular approaches. 7.2. Mutagenesis studies Many traditional mutagenesis approaches such as gene disruption are not feasible in oomycetes due to their diploid nature, and alternative strategies have therefore been investigated for analysis of gene function. Gene silencing has been used successfully in P. infestans to suppress expression of endogenous genes [121]. This seems to be the best option for the development of functional analysis systems in other oomycetes root pathogenic species. Transformation with antisense and sense constructs and with a cDNA clone of the inf1 gene all led to silencing of the endogenous genes. Inf1 encodes for the most abundant

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secreted elicitor protein in culture ltrates [62] and neither inf1 mRNA nor INF1 protein could be detected in up to 20% of the transformants. The silenced state of the inf1 gene was shown to be mitotically stable under various conditions in vitro and in planta [63]. This indicated that gene silencing would be appropriate for detailed analyses of genes in Phytophthora. Several groups have since reported successful silencing of particular genes in oomycete plant pathogens. Latijnhouwers et al. (personal communication) have successfully silenced two G-proteins from P. infestans. Furthermore, CBEL, a glycoprotein with a cellulose-binding motif that elicits defence responses in plants, has been silenced in P. nicotianae with both antisense and sense constructs [33]. It is anticipated that gene silencing will have broad application in oomycete research and the methods developed in P. infestans may be utilised in the analysis of other species. 7.3. Mechanism of gene silencing Experiments to investigate the mechanism of gene silencing in P. infestans revealed that inf1 gene silencing is regulated at the transcriptional level [121]. DNA methylation, a feature often associated with transcriptionally regulated gene silencing was not detected in the inf1 gene sequences. Heterokaryons obtained by somatic fusion of an inf1-silenced transgenic strain and a wild type strain were also stably silent, demonstrating that inf1 silencing is dominant and acts in trans. The inf1 gene remained silenced in non-transgenic homokaryotic single zoospore isolates released from the silenced heterokaryons. Apparently, the presence of transgenes is not essential for maintaining the silenced state of the endogenous inf1 gene. Karyogamy was not demonstrated and hence it is unlikely that the silenced state of the inf1 gene is transmitted from nucleus to nucleus by specic DNA-DNA interactions. Consequently, it was proposed that a novel-silencing phenomenon exists in which internuclear gene silencing is maintained by a diffusible silencing factor. The nature of this factor is not known, but it may consist of a diffusible protein, aberrant RNA molecule or a complex consisting of RNA and protein. Such a diffusible molecule could function by inducing changes in chromatin structure of the target gene or regions surrounding the target gene thereby inuencing transcription. Recently, we found evidence that alterations in heterochromatin formation of the inf1-silenced gene do occur in silenced strains (van West, Shepherd, Govers and Gow, unpublished data). Internuclear gene silencing is therefore a trans-inactivation phenomenon that is reminiscent of paramutation [121]. Further research is in progress to identify the internuclear silencing signal. 7.4. Reporter gene studies An additional transformation-based technology that is now assisting in the characterisation of oomycete genes

involves introduction of reporter genes such as b-glucuronidase (GUS), b-galactosidase, luciferase (LUC), and green-uorescent protein (GFP) [13,55,56,120,122,123, 130]. Transgenic root pathogenic P. palmivora strains that constitutively produce GUS or GFP, were obtained after stable DNA integration using the PEG and Ca2-based transformation protocol [123]. GUS and GFP production was found in all pre-infection stages of the life cycle of P. palmivora and facilitated identication of root infection sites in mixed zoospore infection assays [122] (Fig. 2(i) (k)). Similar approaches have been taken with P. nicotianae where the GFP-tagged strains were also used to study root infection processes in seedlings [13]. Weiland [130] performed the rst successful transformation of a Pythium spp. with a reporter gene that resulted in constitutive b-glucuronidase expression (Fig. 2 (f) (h)). 7.5. Sequencing projects Several sequencing projects are now in progress for P. sojae and P. infestans genomes. The Phytophthora Genome Consortium initiated the rst pilot-sequencing project [60,102,129]. Several thousand expressed sequence tags (ESTs) of each species are currently available in the database at the website of the National Centre for Genome Resources (NCGR) (http://www.ncgr.org/pgc). A follow-up project by the Phytophthora Genome Consortium to develop a further 41 000 P. sojae ESTs and 14 000 P. infestans ESTs was funded by the USDA-IFAFS ([96], Tyler personal communications). Several ESTs from P. infestans and P. sojae are also included in the Phytopathogenic Fungi database from the Consortium for the Functional Genomics of Microbial Eukaryotes (COGEME) which is funded by the British Biotechnology and Biological Sciences Research Council (BBRSC) [111]. In addition, a large number of ESTs from P. infestans that have been sequenced by an international consortium funded by Syngenta, are expected to become publicly available in the near future. A small pilot project to sequence one thousand ESTs from Pythium aphanidermatum mycelium and zoospores is also underway (van West, Appiah and Gow, unpublished data). Sequencing of the genome of P. sojae to a 8X coverage has also been funded by the USDA and NSF (B. Tyler, personal communication). These resources provide signicant new opportunities for gene discovery and comparative genome projects for root pathogenic oomycetes. 7.6. Proteomic approaches Proteomic approaches are now being employed to identify stage-specic and extra cellular proteins from several oomycete species, including P. nicotianae , P. palmivora, P. infestans, and Pythium aphanidermatum ([83,137], van West, Li, and Gow, unpublished data and Fig. 4). Differences in protein proles of plasma membrane and

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spectrometry techniques will aid in the identication of proteins from the 2-D gels. This will provide the basis for the construction of databases of protein proles from the selected oomycetes. It is expected that proteomic approaches in combination with genomic approaches will accelerate the discovery of novel extra cellular and stage-specic proteins and their corresponding genes in oomycete plant pathogens. In addition, a comparative analysis of protein diversity among oomycete plant pathogens will be possible. We envisage that proteomic experiments will serve as one of the principle tools underpinning future research projects aimed at functional analysis of proteins of relevance to fundamental molecular processes, host specicity, pathogenicity and resistance in oomycetes. 7.7. Phage display approaches Phage display libraries offer the exiting possibility of identifying oomycete proteins that are involved in various developmental and infection processes. This method can be used to generate a phage clone that expresses a specic antibody on the end of its tubular capsid. This method therefore allows specic antibody-like reagents to be generated to single or multiple epitopes by a process called bio-panning. Phage display protocols to identify antibodies and peptides that bind to surface molecules of zoospores from P. nicotianae and P. capsici and also germinated cysts of P. infestans have been developed [12,36]. For P. capsici, about half of the selected phages that were tested induced encystment, and one phage was found that bind to zoospores but not to cysts [12]. Phage reagents will therefore be a useful alternative to conventional antibodies in cytological and molecular analyses in the future.

Fig. 4. Two-dimensional gel electrophoresis of proteins isolated from sporangia (a) and zoospores (b) of Phytophthora palmivora. Gels were silver-stained. Some proteins that are present in one stage and absent in the other are indicated with arrows. The direction of increasing iso-electric point and decreasing molecular weight are indicated. The two-dimensional gels were analysed by samantha shepherd, Department of Molecular and cell biology, University of Aberdeen, Scotland, UK.

endomembranes of zoospore and cysts from P. nicotianae microsomal fractions have been investigated on 1-D and 2D gels [83]. At least 53 proteins were found to be specic for one or the other spore type. Future identication of these proteins will establish their functions. Earlier work by mer et al. [69] described a number of stage-specic Kra expressed proteins. The most marked changes in the proteome occurred between the cyst stage and the time of germ tube development. In particular several new and abundant proteins were synthesized during appressorium formation [69]. We have conrmed this observation based on analyses of proteins isolated from P. infestans and P. palmivora zoospores, germinated cysts and appressorium stages. Furthermore, it was found that approximately 1% of proteins isolated from P. palmivora appeared to be specic for each of the mycelium, sporangia, zoospores, cysts and germinated cysts stages of the life cycle [137]. From comparisons of proteins produced by equivalent developmental stages of P. palmivora and P. infestans, approximately 30% of proteins co-migrate on 2-D gels. Thus proteomics can be used across species boundaries to examine some of the changes occurring during differentiation of Phytophthora plant pathogens [137]. Matrix-assisted laser/desorption ionisation-time of ight mass spectrometry (MALDI-TOF MS) and other mass

8. Summary Root pathogens, which include Phytophthora and Pythium species, remain relatively poorly studied organisms despite their undoubted commercial importance. Progress and the impetus for the investment of academic focus on root pathogenic oomycetes have in the past been hampered by the paucity of molecular tools, the lack of DNA sequence information, and in particular a route for functional analysis of genes. However, work in the last few years has signicantly altered these aspects of research and it is now possible, although not yet easy, to examine the relationship between host and pathogen at the molecular level. Gene silencing is beginning to be established as a robust and generally applicable technology engendering optimism that the consequences of gene knock-down can be used to explore gene-for-gene interactions as well as key aspects of the remarkable oomycete life cycle. In particular the asexual stages from zoosporogenesis through zoospore liberation, host targeting, encystment, germination and appressorium

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