THE SPORES OF PHYTOPHTHORA: WEAPONS OF THE PLANT DESTROYER

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1 THE SPORES OF PHYTOPHTHORA: WEAPONS OF THE PLANT DESTROYER Howard S. Judelson and Flavio A. Blanco Abstract Members of the genus Phytophthora are among the most serious threats to agriculture and food production, causing devastating diseases in hundreds of plant hosts. These fungus-like eukaryotes, which are taxonomically classified as oomycetes, generate asexual and sexual spores with characteristics that greatly contribute to their pathogenic success. The spores include survival and dispersal structures, and potent infectious propagules capable of actively locating hosts. Genetic tools and genomic resources developed over the past decade are now allowing detailed analysis of these important stages in the Phytophthora life cycle. Department of Plant Pathology and Center for Plant Cell Biology, University of California, Riverside, California 92521, USA. Correspondence to H.S.J. doi: /nrmicro1064 Developmental stages commonly known as spores have crucial functions in diverse taxa including ferns, worts, algae, true fungi, bacteria, protozoa and the subject of this review, oomycetes such as Phytophthora. The nomenclature, morphology and physiology of spores vary between these groups; however, all can be classified as having either dispersal or resting functions. Dispersal spores enable geographical spread and escape from deteriorating environments. Resting spores also allow survival in unfavourable conditions, but usually do so while remaining in their place of origin. The physiological and genetic mechanisms that are involved in sporulation and spore germination have been well studied in many prokaryotic and eukaryotic microorganisms, including model species, important pathogens of animals and/or plants, and genera in which sporulation is linked to the production of important metabolites 1 4. One of the earliest cases where the role of spores in spreading disease was recognized involved Phytophthora infestans.this eukaryotic microorganism is notorious for attacking the European potato crop in the mid- 1840s, when it caused the Irish famine 5. P. infestans still limits potato production worldwide, and is also a problem for tomato crops. The genus was named after the Greek words for plant destroyer, and the potato disease was named blight. Blight is estimated to cost growers US$5 billion per year, and necessitates the use of vast quantities of crop-protection chemicals, some of which might pose environmental hazards 6.Most of the other >60 members of the Phytophthora genus also threaten important crops 7.New Phytophthora species continue to be identified for example, Phytophthora ramorum,which was recently found in Europe and North America where it causes sudden oak death 8. The related Pythium and downy mildew groups also include economically significant pathogens. In addition to its economic importance, P. infestans also had a notable role in scientific history. P. infestans was the first microorganism proven to be responsible for disease, at a time when spontaneous generation was more accepted than Pasteur s germ theory 5.Work on P. infestans after the Irish famine initiated the science of plant pathology and was the catalyst for important advances in studies of human and animal diseases. However, the surge of classical genetic and later molecular studies in mycological systems that began in the mid-1900s focused on true fungi, not oomycetes such as Phytophthora.Several ascomycete and basidiomycete fungi became models for studying spore biology 9,10,but the knowledge that was obtained had limited relevance for Phytophthora because a few decades ago it was shown that oomycetes do not belong to the Kingdom Fungi. Instead, although many researchers still consider oomycetes fungi owing to their typical filamentous growth 11, oomycetes belong to the Kingdom Straminipilia, together with groups such as brown algae and diatoms 12,13. One of the many features that distinguish oomycetes from true fungi is that oomycetes NATURE REVIEWS MICROBIOLOGY VOLUME 3 JANUARY

2 Table 1 Major differences between oomycete fungi and true fungi Feature Oomycete True fungi Neighbouring taxonomic Diatoms and golden-brown algae Animals groups Hyphal architecture Aseptate and coenocytic tubular Either single cell or septated hyphae, with one hyphae or more nuclei per compartment Ploidy of vegetative hyphae Diploid, except for transient Typically haploid or dikaryotic; often with a stable haploid nuclei in gametangia or semi-stable diploid stage following mating Typical size of genome Mb Mb Major glucans in cell walls Cellulose (β-1,4-linked glucose), Usually chitin (β-1,4-linked N-acetylglucosamine) and β-1,3, and β-1,6-linked and/or chitosan (β-1,4-linked glucosamine), glucose polymers often with other β-1,3, and β-1,6 glucans Pigmentation Usually unpigmented Very common in hyphae or spores, or secreted (for example, melanin, carotenoids and others) Toxic secondary metabolites None described Common (typically aromatic, heterocyclic compounds) Mating hormones Non-peptide, probably lipid-like Usually small peptides or lipopeptides Predominant asexual spore Undesiccated, unicellular Desiccated single or multicellular conidia sporangia (multinucleate cells) (one nucleus per cell) Motile asexual spores Nearly universal, biflagellated Uncommon, only in chytrids, which are zoospore monoflagellate Sexual spores Oospores, formed on the Various types, often formed in large numbers termini of specialized hyphae, within complex enclosures ( for example, each containing one viable perithecia, mushroom caps and others) zygotic nucleus Major energy reserves used Mycolaminarin and lipid, possibly Glycogen and trehalose, also sugar alcohols by spores polyphosphate and lipid SAPROPHYTE An organism that feeds on dead organic matter. SPORANGIUM A sac-like structure that is capable of converting its cytoplasm into multiple spores. ZOOSPORES Motile, wall-less spores, specialized for dispersal. OOSPORES Non-motile, sexual spores. CHLAMYDOSPORES Thick-walled asexual reproductive structures that are found in many Phytophthora species, but not Phytophthora infestans. are diploid and lack a free haploid life stage (TABLE 1). The resulting masking of recessive mutations, and an absence of colour markers, explains why early mycogeneticists failed to select oomycetes as their models and also why oomycetes like Phytophthora obtained a reputation as being difficult experimental systems, once being described as a nightmare for researchers 14. Fortunately, in the 1990s, effective genetic tools began to be developed for Phytophthora, which proved instrumental in allowing aspects of its biology, including spore development, to be studied. Such methods included approaches for DNA-mediated transformation, which were applied most successfully to P. infestans, making it a model for the genus Homology-based gene-silencing strategies were identified to allow gene function to be analysed 18, although a method for gene replacement has proven elusive due to infrequent homologous recombination. Classical genetic methods were also improved, enabling the development of marker-based maps and chromosome-walking projects in bacterial artificial chromosome (BAC) libraries 19,20.This year, a large expressed sequence tag (EST) dataset became available for P. infestans;when combined with earlier EST data and one-fold coverage of its genome, these data have allowed ~18,000 genes to be predicted 21,22 and are in addition to the EST data that had been reported for Phytophthora sojae and Phytophthora nicotiana Genome-sequencing projects are underway for several species of Phytophthora, and DNA-microarray data are becoming available 26 (see the Online links box for details). In this review, we discuss recent findings concerning the formation and germination of Phytophthora spores, particularly those findings that have been enabled by genetic and genomic strategies. The focus will be on P. infestans as it has been the subject of most molecular studies, although relevant findings from other species will also be discussed. For more details on the cell biology of asexual spores in Phytophthora and other oomycetes, readers are referred to an excellent review 27. Diversity of spore types Several types of spores have important roles in the life and disease cycles of Phytophthora. All spores form after a period of vegetative growth, which involves the extension of aseptate tubular hyphae. Although Phytophthora can be cultured continually as hyphae, in nature new spores must be produced continually. Compared with true fungi, the survival of Phytophthora as a SAPROPHYTE is limited. Therefore, as a colonized plant declines in health, spores are required to allow movement to a new host (FIG. 1). In potato blight, for example, several cycles of sporulation and infection normally occur each season 28. The spores of P. infestans including the predominant asexual spore (the multinucleate SPORANGIUM), motile uninucleate ZOOSPORES released from sporangia and the sexual spore or OOSPORE are illustrated in FIG. 2.These spores resemble those that are produced by other members of the genus; however, thick-walled resting CHLAMYDOSPORES are produced by several species but not P. infestans. 48 JANUARY 2005 VOLUME 3

3 SPORANGIOPHORE A specialized hypha that has a sporangium. CADUCOUS In caducous species the sporangia can detach from the hypha for dispersal. STOMATE An epidermal pore on a leaf or stem that allows the passage of gases and water vapour. LENTICEL An opening in the corky skin of plants that enables gas and vapour to move to and from interior tissues. Sporangia Growing edge of lesion (biotrophic) Overview of asexual sporulation and germination The majority of spores produced by most Phytophthora species are asexual and develop at the termini of specialized hyphae called SPORANGIOPHORES 29 (FIG. 2).These are often branched and bear several multinucleate spores, which are called sporangia (or zoosporangia) as they can release six or more zoospores. As the sporangium matures, an apical papilla forms in most species and a basal septum develops; the latter is notable as hyphae are aseptate. In CADUCOUS species such as P. infestans, wind or water can cause sporangia to detach from the sporangiophore and travel several kilometres 30.This shedding of sporangia is particularly convenient for researchers, as the propagules can be easily purified for analysis. By contrast, sporangia of non-caducous species such as P. sojae remain attached to the hyphae 7. The asexual sporangia have a remarkable ability to germinate by two different pathways. At higher temperatures (>14 C for P. infestans), direct germination occurs, in which hyphae emerge through the sporangial wall. Plant colonization can then occur through openings in a host such as a STOMATE, LENTICEL or wound. Indirect germination also known as zoosporogenesis is predominant at cooler temperatures. Both germination pathways require the sporangia to be immersed in liquid, and are inhibited at high spore concentrations. As the sporangia are not desiccated this might indicate the need to remove an inhibitor of germination. Little effort has been made to characterize this inhibitor, even though it could have applications in crop protection. Stomate Older part of lesion (necrotic) Intercellular hyphae Appressorium Zoospore cyst Haustoria Figure 1 Course of infection by Phytophthora infestans. Typically, a wind-blown sporangium releases zoospores on the plant surface, which encyst and germinate to form appressoria. These enable the host epidermis to be breached, after which hyphae spread throughout the plant. New sporangia usually appear near the boundary between living and necrotic plant cells. Germination by the zoospore pathway is believed to be most important in disease 28.This pathway involves cleavage of the sporangial cytoplasm by nucleus-enveloping membrane networks and the assembly of two flagella per zoospore 27.Dissolution of the sporangial papilla provides an escape route for the wall-less zoospores, which are expelled from the sporangium by turgor pressure 31.The turgor pressure is believed to result in part from high concentrations of proline that accumulate in the cytoplasm of cleaving sporangia 32.The zoospores swim after being released, using an anterior flagellum to pull the cell and a posterior flagellum for steering. This use of flagella for pulling instead of pushing is relatively unconventional, and is enabled by tinsel-like decorations, which accomplish thrust reversal. Zoospores locate host tissue by several mechanisms. Most work in this area has involved root pathogens such as P. sojae and Phytophthora palmivora, and readers are referred to several thorough reviews for more details 33,34. Plants exude nonspecific chemoattractants such as amino acids, which attract zoospores from Phytophthora species (and other oomycetes such as Pythium) regardless of whether they are successful pathogens of that plant. Plants also emit specific attractants including the isoflavones daidzen and genistein, which are released by soybean roots and attract zoospores of the soybean pathogen P. sojae but not of other species 35.Such attractants form concentration gradients from both root tips and wound sites, which presumably represent optimal sites for infection. Many Phytophthora species also exhibit autoattraction (or autoaggregation), a phenomenon in which zoospores move towards each other and which might increase the frequency of successful infections. Although it has been proposed that autoattraction occurs in response to calcium that is released by encysting zoospores, one study has suggested that the autoattractant is species-specific 33,36.Electrotaxis augments chemoattraction and functions as a mechanism for directing zoospores to roots 37.As a consequence of normal ion flux across the surface membranes of plant roots, electrical fields become established within the rhizosphere. Interestingly, the polarity of these fields varies between plant species, which is thought to contribute to the specificity of electrotaxis. For example, zoospores of P. palmivora are attracted to the negatively charged surface of its host, cacao, whereas those of an oomycete that is non-pathogenic for cacao, such as Pythium aphanidermatum,are repelled 38. Eventually, physical and/or chemical stimuli cause the zoospores to lose their flagella and form walled cysts 27.Such stimuli might include the same compounds that are involved in chemotaxis 33,34. Encystment involves discharging the contents of several types of vesicles that reside near the zoospore surface, including one that seems to release a glue that affixes the zoospore to its potential host. Unlike the cysts that are formed by many non-oomycete taxa, which function as survival structures, or the asexual sporangia of oomycetes, Phytophthora cysts immediately start to germinate NATURE REVIEWS MICROBIOLOGY VOLUME 3 JANUARY

4 a d and do not require any particular stimulation. A germ tube extends from the cyst, which in many species swells into an appressorium that adheres tightly to the host plant. Compared with appressoria of most true fungi, these are smaller and are not melanized or pigmented. Both chemotropism and thigmotropism influence the orientation of germ tubes and the formation of appressoria. As with zoospore swimming, chemotropism and thigmotropism presumably help Phytophthora find an optimal infection site, especially the anticlinal walls of epidermal cells 39.At such sites, a combination of mechanical pressure and cell-walldegrading enzymes allows the plant epidermis to be breached 40. As occurs after direct germination, hyphae then ramify through living (not dead) plant tissue, forming feeding relationships that involve haustoria 41. Zoosporogenesis, encystment and appressorium formation can occur within a few hours on plant or artificial surfaces. b e c Advantages of the dual germination strategies The evolution of two different germination pathways is extraordinary and increases the ability of Phytophthora sporangia to colonize plants in diverse environments. In particular, the motile zoospores extend the range of Phytophthora beyond the landing sites of airborne sporangia. Zoospores can remain motile for several hours or even days, and can move >6 cm through water films and farther in surface water 42,43.Therefore, a sporangium that lands some distance from a plant can still infect a host by travelling to stems or leaves that are in contact with soil, or to roots or tubers. Such characteristics make asexual sporangia potent agents for spreading disease, but these features come at a price. To sense and respond rapidly to the environment the sporangia must remain metabolically active. This depletes nutrient reserves and makes sporangia vulnerable to death through desiccation or the effects of solar radiation, with viability under field conditions rarely exceeding a few days 44,45.By contrast, most true fungi produce desiccated spores, which can remain dormant for long periods of time but which consequently miss opportunities for host infection. In addition to the oomycetes, several other eukaryotic taxa make use of flagellated cell types (zoospores). These include relatives such as diatoms and distant groups like chlorophyte algae, foraminiferans, trypanosomes, diplomonads and a few true fungi 46,47.The conservation of the flagella and associated kinetosomes between such species suggests an ancient origin, soon after the nuclear membrane evolved. f h g Figure 2 Stages of the spore cycles of Phytophthora infestans. The bar in each panel represents 5 µm. a Vegetative, non-sporulating hyphae. b The swollen tip of an asexual sporangiophore, typical of a sporangia initial. c A sporangiophore containing four maturing asexual sporangia on lateral branches and a terminal sporangiophore initial. d An ungerminated sporangium. e A mixture of sporangia and zoospores. f The apical tip of a sporangium, showing the opening through which zoospores are released (operculum), which is now filled with an apical plug. g A zoospore with its two flagella attached to a central groove; decorations or mastigonemes can be seen on the upper flagellum. h Sporangia after releasing zoospores, displaying open opercula. i An oospore formed from A1 and A2 hyphae. i Genes induced during spore formation Numerous cytological and physiological studies have examined the asexual spore pathways of Phytophthora,with zoosporogenesis being the most studied 27,39,48.However, the genes that are involved in zoosporogenesis were only recently identified. One general approach has involved isolating differentially expressed genes by subtraction cloning, DNA array or proteomic methods 24,25,49 51.A second strategy examined proteins that had previously been shown to affect spores of true fungi, such as G-proteins, which were cloned using heterologous probes or mined from genomic databases 52,53.A third strategy used antibodies to spore-specific proteins to identify and clone the corresponding genes 54. Analysing differentially expressed genes has been the most successful in identifying genes that are likely to be involved in the spore cycle. In P. infestans, cdna macroarrays identified 60 and 71 genes that were induced >5-fold during sporulation and zoosporogenesis, respectively, one-third of which were only expressed during those stages 49,50.These arrays represented ~4,000 genes, or ~20% of the predicted transcriptome 21,so many important genes probably remain to be identified. EST studies using cdna libraries from sporangia and zoospores of P. nicotianae also identified genes that were expressed during these stages, and RNA blot and macroarray studies indicated that the transcription of many of these genes was also upregulated 24, JANUARY 2005 VOLUME 3

5 a b Cdc14 PK3 c M90 e f g h i j d NIFS NIFC Figure 3 Promoter activation during asexual sporulation and germination. a Schematic representation of the stages of asexual spore development, showing where the promoters of representative genes (Cdc14, PK3, M90, NIFS and NIFC) are induced based on promoter GUS fusion studies. Illustrated from left to right are an aseptate hypha; sporangiophore initials emerging from hyphae, into which nuclei migrate; a sporangiophore in mid-development, containing multiple nuclei; a sporangium forming at the sporangiophore terminus, into which nuclei and cytoplasm migrate; a mature sporangium delimited from a well-vacuolated sporangiophore by a septum; and zoospores being released. b g Progression of Cdc14::GUS activity during sporulation, ranging from its first detection within narrow zones adjacent to sporangiophore initials (b), to mature sporangia and zoospores (g). h The stage at which PK3 activity is first detected, usually before significant sporangiophore initials are evident. i An intermediate stage of sporangiophore development, where M90 activity is first detected. j The constitutive pattern of expression in vegetative hyphae that results when a transcription factor binding site is mutated in the spore-specific M90 promoter. Based on the predicted amino acid sequences, these stage-specific genes seem to encode proteins with diverse roles, which range from metabolic to regulatory to structural functions. Those of the latter class include mucin-like proteins (which might protect against desiccation), dynein components of the flagella and cell-wall-strengthening transglutaminases 49,55,56.Many predicted protein sequences for which no matches were detected in GenBank contain repeated motifs of threonine, serine and proline; these might form rigid, linear molecules with high potential for glycosylation, which are often associated with structural or adhesive functions 57.Several such genes belonged to families, with different forms being expressed in hyphae, sporangia, zoospores or oospores 55. Several of the transcriptionally regulated, stagespecific genes encode proteins with homologues that in other species primarily exhibit post-translational regulation. For example, the sequences of two of four stage-specific protein kinases identified from P. infestans are most similar to calcium-regulated kinases from other kingdoms, with the exception that the P. infestans kinases lack the calcium- or calmodulin-binding domains that are normally used to confer post-translational regulation 49,50,58.Similarly, the sporulationspecific Picdc14 gene from P. infestans seems to be a homologue of genes that are constitutively transcribed in all other taxa 59.Does this indicate a disproportionate reliance on transcriptional versus post-translational regulation in oomycetes compared with other eukaryotes? One challenge in predicting the roles of many of the genes that are induced during sporulation and zoosporogenesis has been a dearth of close matches in the available databases to genes of known function. This is not unexpected, as close relatives of Phytophthora and other oomycetes have been little studied compared with other systems. Although 35 85% of the spore-induced genes from the various studies matched proteins in GenBank at E<10 5,many hits were weak and usually did not represent authentic homologues. Despite several notable cases where gene silencing was used to assign roles to spore-relevant genes in P. infestans (as described below), functional studies will be a limiting step as silencing is still a low-throughput technology. The published studies on spore-induced sequences were limited by the lack of a complete database of Phytophthora genes, but more comprehensive data will become available as genome projects advance. For example, draft sequences of the P. ramorum and P. sojae genomes are now being completed by the US Department of Energy Joint Genome Institute (see the Online links box), and are certain to be exploited for expression-profiling studies. Also, all stages of the P. infestans life cycle were recently profiled using Affymetrix GeneChips, and 18,000 genes were predicted using 79,000 ESTs from 20 conditions of growth and development and one-fold genome data 21.In these chip studies, major changes in mrna abundance were observed through the stages of the spore pathway, with mrna levels of >15% of the genes varying >10-fold at the various stages (H.S.J. and F.A.B., unpublished observations). The fraction of genes that were shown to be stagespecific in these GeneChip experiments and the earlier cdna macroarray studies 24,25,49,50 is larger than that predicted from two-dimensional protein gel studies. In P. palmivora, for example, according to protein gel studies only 1% of proteins appeared specific for mycelia, sporangia, zoospores, cysts or germinated cysts 51.Technical factors might be responsible for this difference because only about 800 protein spots were resolved per gel and most weakly expressed proteins would not have been scored. Another explanation is that alterations in mrna abundance might not reflect corresponding changes in their cognate proteins. Although a recent study in mammalian cells indicated NATURE REVIEWS MICROBIOLOGY VOLUME 3 JANUARY

6 that the overall pattern of protein expression is similar to that of mrna 60,many differences were observed in Saccharomyces 61.Future work will be required to determine if mrna expression profiles accurately represent protein levels in Phytophthora. When do the induced genes act? Identifying developmentally regulated genes is trivial compared with identifying the stage of the life cycle at which their encoded proteins function. Although sporulation requires de novo protein and RNA biosynthesis, sporangia seem to be largely pre-programmed for zoospore release and encystment as these processes are not blocked by actinomycin D and cycloheximide 62,63 (although the ability of inhibitors to enter sporangia was recently questioned 58 ). Cell-biology studies also indicate that sporangia contain vesicles with components that are required by the zoospore, such as flagella and a water expulsion apparatus 27. Therefore, sporangia-specific transcripts might encode proteins that are involved in spore maturation or later stages such as germination or encystment. mrnas that are required early in sporulation might also persist in mature sporangia, because cytoplasm from sporangiophores is propelled into sporangia during differentiation; this differs from the situation in most true fungi, where conidia develop from specialized cells such as phialides 9,10. Knowing when a gene is induced can suggest its role, but further experimental methods such as gene silencing will ultimately be required to define protein function. Nevertheless, understanding transcriptional patterns will reveal the networks regulating development. RNA blot or array studies can provide some resolution of processes such as zoosporogenesis or cyst germination, which occur relatively synchronously within no more than 90 minutes. However, these methods provide less detail about sporulation, which occurs with only partial synchrony over several hours in most Phytophthora species 64. Following expression patterns using reporters To complement RNA blot and array studies, the temporal and spatial patterns of transcription can be followed in transformants expressing reporters that are driven by spore-specific promoters. This is not as direct as measuring expression by immunolocalization, which has been used successfully against several spore-specific proteins in Phytophthora cinnamomi and P. nicotiana 27,but might be more suited to the analysis of large numbers of genes. Using fusions with the β-glucuronidase (GUS) gene, discrete stages of transcriptional regulation in P. infestans were identified (FIG. 3). One P. infestans gene that is transcribed early in asexual sporulation (Picdc14) encodes the Cdc14 cellcycle phosphatase 59.The promoter of this gene becomes active near and within small lateral branches protruding from hyphae in sporulation-competent cultures, which are defined as sporangiophore-organizing centres (FIG.3b). The identification of this stage with the reporter is notable, because the incipient sporangiophore cannot be distinguished morphologically from young vegetative branches. Cdc14::GUS activity persists as sporangiophores extend (FIG. 3c) and as sporangia form (FIG. 3d,e), mature (FIG.3f) and germinate (FIG.3g). Each of several other sporulation-induced genes that were examined using the reporter-gene approach were activated at different times, indicating that the transcriptional patterns during sporulation are complex. For example, M90 is induced later than Picdc14 in mid-stage sporangiophores 65 (FIG. 3i). Pisp14 is induced at an even later stage as it is first detected in maturing sporangia 66. PK3 is first transcribed in regions of hyphal tubes that later produce sporangiophores (FIG. 3h).Its expression spans a larger zone than Picdc14 (~100 µm in length), which might represent where the initial inducer of sporulation is perceived. PK3 encodes a protein kinase 50,which might be an important regulator of early events during sporulation. The GUS reporter has also been used with zoosporogenesis-specific promoters. Promoters from members of the NIFC family of transcriptional regulators from P. infestans successfully expressed GUS in cleaving sporangia 49.However, as zoosporogenesis proceeds rapidly, with little time to translate newly made mrnas, histochemical staining seemed to underestimate the activation of the promoter 66. In addition to allowing wild-type expression patterns to be observed, GUS reporter constructs with mutagenized promoters have been used to identify transcriptional regulatory sites. This approach identified a 7-bp binding site for an activating transcription factor on several P. infestans zoosporogenesis-induced promoters (S. Tani and H.S.J., unpublished observations). By contrast, de-repression was suggested to determine the transcription pattern of several sporulationspecific genes (A. Ah Fong, K. S. Kim, C. Cvitanich and H.S.J., unpublished observations). For example, a normally sporulation-specific M90 promoter was expressed constitutively in vegetative hyphae once the regulatory region was mutated (FIG. 3j; the wild type is shown in FIG. 3i). As sporulation comprises only a small portion of the life cycle compared with vegetative growth (typically ~10 hours versus 5 days), the use of the de-repression strategy by Phytophthora might seem uneconomical. However, it might represent an efficient mechanism for initiating development when resources become limiting. What regulates asexual sporulation? Arguably, the features of sporulation that are most important to understand are the physiological and molecular triggers that directly or indirectly activate the promoters described above. However, identifying the initiators of sporulation represents a major challenge. The sensors for such signals are presumably already present in hyphae and would not be expected to appear on lists of spore-induced genes (although they might exhibit autoactivation like some genes that participate in conidiation in ascomycetes 9 ). It is often assumed that nutrient limitation induces sporulation 29,but this is an oversimplification and other environmental factors are also involved. In P. infestans, 52 JANUARY 2005 VOLUME 3

7 COENOCYTIC Non-septate, with nuclei residing in a common cytoplasm. some, but not all, sporulation-induced genes are also activated by nitrogen or carbon limitation 50.Moreover, some Phytophthora species only sporulate when deprived of nutrients, whereas others such as P. infestans sporulate after two days on rich media. This indicates that in many Phytophthora species complex factors could be involved, and it could be necessary to achieve competence as in true fungi like Aspergillus 67.High humidity and high oxygen and low carbon dioxide concentrations are also required for sporulation in Phytophthora 29.Such requirements favour dispersal by encouraging sporulation in conditions that retard desiccation and in aerial environments or non-flooded spaces between soil particles. Plant factors might help integrate these signals; P. infestans sporulates continually and asynchronously in laboratory cultures, but synchronously in field-infected plants 64. Two genes with regulatory functions have been proven to be essential for sporulation in P. infestans;these were the first genes to be shown to have an important role in the developmental cycle of any oomycete. One gene encodes a G-protein β-subunit, which when silenced reduced sporulation by 99% (REF. 52). Its homologue in the true fungus Cryphonectria parasitica is also required for conidiation 68.Due to its potential interaction with sensor proteins, P. infestans Gβ might be an early regulator of sporulation (either alone or as part of the heterotrimeric G-protein complex). However, it is unlikely to be the initial regulator, as Gβ-silenced strains produce excess aerial hyphae of the type that are formed just before sporulation. The other P. infestans gene shown to be essential for sporulation based on gene silencing is Picdc14,which encodes a phosphatase that has been implicated in regulating various aspects of mitosis 59. Picdc14 might act after Gβ because it is not expressed in Gβ-silenced strains 52,59.Expression of Gβ but not Picdc14 was induced during starvation, indicating that sporulation and starvation induce overlapping but non-identical transcriptional patterns. As nutrient limitation is used in some species to induce spore formation, researchers need to carefully distinguish sporulation-associated genes from those that are mainly starvation-related. Establishing sporangial dormancy Sporangia do not desiccate, and their cytoplasm remains largely functional despite some quantitative changes such as a reduction in Golgi stacks 27. Nevertheless, normal cellular processes such as mitosis and hyphal extension are arrested. How is this state of quiescence established? Two genes that are induced during sporulation, Picdc14 and pumilio,might provide clues as both may function by repressing mitosis. Pumilio proteins are RNA-binding factors that sequester or deadenylate transcripts to suppress their translation, often acting with other proteins 69. One known target of such proteins is cyclin B mrna 70, which, together with a cyclin B-dependent kinase, regulates the end of mitosis. Transcription of the P. infestans pumilio gene begins during sporulation and ends during cyst germination 65.Although it remains to be proven whether the P. infestans protein binds cyclin B mrna, it was proposed that Pumilio might help establish mitotic dormancy in sporangia and zoospores by preventing cyclin B synthesis. As discussed above, Picdc14 expression is first detected in sporangiophores and is essential for sporulation. It is a member of the Cdc14 family of protein phosphatases, which are well-studied regulators of the cell cycle. Although their functions can vary between species, in yeast and animals they end mitosis by suppressing cyclin B and its kinase, and might also regulate the spindle apparatus and cytokinesis 71,72.Its role in P. infestans was proposed to involve synchronizing nuclear division in sporangiophores, regulating the movement of nuclei into sporangia, and/or arresting mitosis once sporangia form; its ability to arrest mitosis was supported by its complementation of a mitotic arrest defect in a temperature-sensitive cdc14 ts strain of Saccharomyces cerevisiae 59.Expression of Picdc14 continues until a few hours before nuclear division resumes in germinated cysts, which is also consistent with a role in mitotic arrest 59. Do Picdc14 and Pumilio combine to block mitosis, and is this sufficient to establish dormancy of the sporangia? It is probably no coincidence that two conserved regulators of cyclin B are induced during sporulation. However, it is also possible that Picdc14 dephosphorylates and thereby regulates proteins that control processes besides mitosis, and Pumilio might bind more than one mrna target as in animals 69,70. Readers of this review might be confused to hear that Cdc14 exhibits spore-specific expression in Phytophthora, as it is constitutively expressed but regulated post-translationally in all other studied species, such as S. cerevisiae. The absence of Cdc14 during vegetative growth might explain why mitosis is asynchronous in Phytophthora hyphae even though their nuclei reside in a common cytoplasm 73 ;apparently these COENOCYTIC organisms have learned to live without a traditional mitotic cycle! Studies of spore biology in Phytophthora may therefore have solved a long mystery related to hyphal growth. How does cold trigger zoosporogenesis? The mechanism by which a short exposure to low temperatures induces multinucleate sporangia to reform into zoospores is unknown, but the rapidity of this process and its insensitivity to RNA and protein synthesis inhibitors have caused researchers to investigate several signalling molecules, which presumably act on preformed proteins. It has long been known that an increase in cytoplasmic Ca 2+ concentrations is required for zoosporogenesis 27,and recent data suggest this might be triggered by phospholipid signals. Inhibitors of phospholipase C (U-73122) and the inositol trisphosphate (IP 3 )-gated Ca 2+ channel (2-APB) were shown to block zoospore cleavage 49.This suggests a model whereby low temperatures reduce the fluidity of the plasma membrane, which stimulates a membrane-bound sensor such as phospholipase or an associated G-protein. Phospholipid signals (possibly NATURE REVIEWS MICROBIOLOGY VOLUME 3 JANUARY

8 Box 1 Priming the spores for attack The spores of Phytophthora spp. like those of most other phytopathogenic fungi germinate to form hyphae that are specialized to penetrate the plant cuticle and cell wall. These penetration hyphae normally emerge from appressoria (swellings on cyst germ tubes that adhere to the cuticle, as shown in the figure), although some root colonizers can penetrate without appressoria 39. As cyst germination and appressoria formation occur rapidly, factors that are involved in pathogenesis might already be accumulating in zoospores. Expressed sequence tag (EST) and other studies 21,23,81,107,108 found that zoospores already contain transcripts for several secreted plant-cellwall-degrading enzymes and potential suppressors of plant defences, including those listed below: Cutinases These degrade cutin, which is an insoluble polyester of C hydroxy fatty acids and which is the main component of the plant surface. Polygalacturonases These hydrolyse a galacturonan-containing plant polysaccharide, pectin, which is mixed with cutin on the plant surface and also forms the major cement between cells. Pectate lyases Pectin is also broken down by these enzymes, but by β-elimination rather than hydrolysis. Cellulases (β-1,4-glucanases) The core structural unit of the plant cell wall is digested by these enzymes. Hemicellulases (xylanases and others) These degrade various complex polysaccharide polymers that are found within the plant wall and between cells. Glucanase inhibitor proteins (GIPs) These are thought to provide a counter-defence against plant β-1,3-glucanases that would otherwise degrade the Phytophthora cell wall 109. Protease inhibitors Several members of the Kazal group of serine protease inhibitors are secreted by Phytophthora and are proposed to block the activity of enzymes made by the plant for defence against pathogens 110. The figure shows the development of appressoria. The upper panel shows a young appressorium of Phytophthora infestans (ya) developing from a germinated cyst (c) on the surface of a tomato leaflet, whereas the lower panel shows a more mature appressorium (a). IP 3 ) then cause Ca 2+ to enter the cytoplasm, which activates proteins that drive zoosporogenesis. Perhaps relevant to this are several predicted calcium-regulated kinases and ion channels that are induced during sporulation 50.Supporting this model are studies of the effects of dimethyl sulphoxide (DMSO) and benzyl alcohol, which decrease and increase membrane fluidity, respectively 74 ; DMSO induces zoosporogenesis and the transcription of many cleavage-specific genes, whereas benzyl alcohol has the opposite effect (S. Tani and H.S.J., unpublished observations). Some features of this model remain to be proven, and might be oversimplified as, on the basis of the effect of different inhibitors on cleavage-specific genes, additional signal cascades seem to be involved 49.Although U and 2-APB blocked the induction of ~40% of such c c ya a genes, 20% were insensitive to both inhibitors and 30% were sensitive only to U The latter pattern implicates diacylglycerol, another phospholipase C product, as a messenger. Calcium agonists also failed to block the normal induction of several genes. Steps after zoospore release As in zoosporogenesis, Ca 2+ helps regulate each of the swimming, encystment, cyst germination, and appressorium stages 34,58,75 77, and phosphatidic acid might also be involved 78.Studies of transcriptional changes during these stages have been limited; however, many components are probably made during the preceding sporulation and zoosporogenesis steps. This was shown in P. infestans by studies of a sporulationinduced Gα subunit 53 and a cleavage-induced protein kinase 58.Silencing of the Gα protein impaired the directional swimming, chemotaxis, autoattraction and pathogenicity of zoospores. The kinase was found to interact with a bzip transcription factor, which, when silenced, resulted in a perpetual turning phenotype in zoospores and an inability to form appressoria (H.S.J. and F.A.B., unpublished observations). Future studies of the G-protein signalling pathway and genes that are regulated by the transcription factor should reveal whether the aberrant swimming behaviours that are observed in these silenced strains are due to defects in the composition of the flagella or kinetosome, or in signalling pathways that regulate their activities. Many post-encystment events require de novo RNA and protein synthesis 62, including the production of several factors that are believed to be important for pathogenesis. These include cell-wall-degrading enzymes (BOX 1),mucin-like proteins 56 and IPI-O, which may interact with the plant wall 79.In P. nicotianae, two genes of unknown function were shown to be highly induced in germlings versus zoospores 25, and similarly upregulated proteins were identified in protein gel studies 80.It is unknown, however, whether genes that are overexpressed in germlings versus zoospores are specific to the former, or if their induction simply reflects a return to the hyphal condition. For example, several glucanases that are expressed in germlings but not in zoospores are also expressed in hyphae 81. Energy management in asexual spores Stored nutrients must be relied on after sporangia are delimited from hyphae and until a host is colonized 62. Substantial energy reserves are required as sporangia are metabolically active and zoospores are energydemanding; the ATPase activity per volume in zoospores is similar to that in contracting skeletal muscle 82,83. Lipids, a novel β-1,3-glucan called mycolaminarin and polyphosphate are proposed to provide most energy for Phytophthora spores, although there are contradictory data on the contribution of polyphosphate Polyols are a major store of carbon in true fungi, but do not accumulate significantly in oomycetes 88. How does the spore prepare itself to meet its energy needs? Several genes relevant to energy utilization are induced during sporulation 50.Particularly intriguing 54 JANUARY 2005 VOLUME 3

9 a c A1 A2 Figure 4 Sex in Phytophthora. a A1 (left) and A2 (right) isolates of Phytophthora infestans paired within the same culture. Where the isolates meet, oospores form beneath the agar surface, whereas aerial hyphae and asexual sporulation are suppressed. The colony morphology of A1 strains are generally fluffy, whereas many A2 strains are lumpy as in the example shown. b d Representative sexual interactions observed when a GUS-expressing A1 strain meets a GUS A2 strain. Usually, 50% or more oospores result from hybridization, as in (b) where the GUS + strain is the female, having formed the oosphere (o), while the GUS strain has formed the male antheridia (a). However, once both mating hormones are detected, selfing is possible as in (c) and (d) where each strain has produced both male and female gametangia. b d o are spore-specific forms of enzymes that are known to buffer ATP concentrations in other species, namely adenylate kinase and a phosphagen kinase. The involvement of a phosphagen kinase was unexpected as such enzymes are largely restricted to animals 89.It will be interesting to learn whether these enzymes simply maintain ATP concentrations, or shuttle ATP from mitochondria to sites of high utilization such as the flagellar kinetosome (temporal versus spatial energy buffering). Also induced fold during sporulation were several Krebs cycle enzymes and a mitochondrial succinate fumarate carrier, which can mobilize carbon from lipids; three proteases that might catabolize proteins that are not required; and phosphenolpyruvate carboxykinase, which converts Krebs intermediates to phosphoenolpyruvate 50. Another gene in the phosphoenolpyruvate pathway, which might be important for gluconeogenesis, was also reported to be induced during sporulation in P. cinnamomi 90. a Mating strategies and sexual spores Due to their energy needs and fragility, asexual sporangia are ineffective resting structures. Instead the thick-walled and durable sexual spores, called oospores, can act as resting structures. As they remain viable between growing seasons, oospores are an important inoculum for disease, particularly for the Phytophthora species that are homothallic (self-fertile; approximately half of the species are of this class). Oospores can also be significant for heterothallics when both mating types (A1 and A2) share the same geographical space. Until recently, A1 and A2 P. infestans were rarely associated in most parts of the world. However, this is no longer true and soil-borne oospores now make controlling potato light blight difficult on several continents 91,92. In both homo- and heterothallics, male and female gametangia develop, which later fuse and form oospores (FIG. 4).Oospores later germinate to produce either a hyphal tube, which can directly infect a plant, or a germ sporangium, which acts like an asexual sporangium. In contrast to asexual sporulation, which occurs on aerial hyphae, oospores form within plant tissue or beneath the surface of media. This would be expected for a resting spore as rapid dissemination is not desirable. This can also be particularly effective in potato blight, where oospores that have established in a tuber in one season can germinate when the tuber sends up new shoots during the next season, resulting in an infected plant that can disseminate copious asexual sporangia. The sexual cycle has been well-described cytologically 93, and genetic studies have examined the basis of mating type and gene expression during oosporogenesis. In P. infestans, a single mating-type locus was identified genetically and flanked by markers as part of chromosome walks 20.The precise gene(s) determining mating type are unidentified, but A1 and A2 mating types behave as heterozygotes and homozygotes, respectively The mating locus is located in an unusual region of the genome that is characterized by interchromosomal polymorphisms (possibly mediated by transposable elements 97 ) and genetic aberrations that include grossly distorted segregation, balanced lethals and translocations 20,98,99.Until the mating-type locus is cloned, the role of such aberrations will remain unclear; however, they might reflect a mechanism to restrict recombination within a complex locus along the lines of a primitive sex chromosome. The mating-type locus is thought to control the synthesis and/or response to the two mating hormones. These are undescribed but might be small, polar and lipid-like 100.The hormones are the basis of heterothallism, as there is no evidence for self-incompatibility as seen in true fungi 10, and most homothallics produce both hormones 101. Once a heterothallic is stimulated by the opposite mating type, both hybrid and selfed oospores can form 102.The ability of most strains to develop male and female gametangia enables selfing, as is evident in matings involving a GUS-expressing strain (FIG. 4).However,some Phytophthora strains have a clear sexual predisposition or can switch preference when NATURE REVIEWS MICROBIOLOGY VOLUME 3 JANUARY

10 influenced by another strain 102, indicating that courtship involves complex interactions, as in Achlya 103. There are also reports that chemical stresses can occasionally induce oosporogenesis in single heterothallic strains 104.This indicates that some regulators of sexual development, which act downstream of the mating locus, might be unstable. Although the contribution of this to the epidemiology of Phytophthora diseases is unclear, such a leaky regulatory system might confer an advantage by providing an additional mechanism to generate resting spores. Only 10 mating-specific genes have been described 105, so much work remains to be done. However, promoter GUS fusion experiments have helped to show where and when such genes are expressed 65,and insights into oosporogenesis have been provided by their predicted products. Four matingspecific genes seem to encode structural features of gametangia or oospores. Interestingly, two are members of protein families that are known to trigger the innate defence responses of plants. Another belongs to a sterol-binding family, which could conceivably be related to the processing of mating hormones. Three interact with RNA (as helicases, nucleases or other binding proteins), which might be relevant for the storage or recycling of RNA in oospores. With one exception, the mating-specific genes were not induced during asexual sporulation, indicating that the two processes are quite distinct. The exception was the pumilio homologue, which in Phytophthora is expressed during both mating and asexual spore development but not during other life stages 65.This protein might participate in one of the few aspects that seem to be conserved between the two pathways: the need to sequester RNAs for use at later stages of development and the need to arrest mitosis 70. Conclusions and practical applications After 150 years of research on Phytophthora,genetics and genomics have joined to enable progress to be made in dissecting molecular aspects of its spores. This is important for understanding the novel biology and evolutionary history of oomycetes, and why they are such successful and persistent pathogens. Numerous spore-specific genes have now been identified, and understanding the functions of their protein products will be a major and necessary activity it cannot be assumed that all have essential roles. In the case of zoosporogenesis, it is fortunate that a good cytological and cell-biological framework exists for such studies 27, as many of the induced genes probably relate to structures that were observed in that earlier work. However, other areas are less well understood, such as the triggers and early cellular events of spore development. One reason for the latter is that neither asexual or sexual spores develop from discrete cells that can be easily identified in their early stages, unlike in true fungi 10. Related to this is the absence of septation in the hyphae of Phytophthora,which is also unlike true fungi and raises an associated question: how can developmental signals be targeted to narrow sites within a large and coenocytic cellular compartment? As shown in FIG. 3 for Cdc14 and PK3, mechanisms for accomplishing this clearly exist. The induced genes should therefore be useful markers for better understanding Phytophthora cell biology, as well as vice versa. Studies of the spore pathways might also lead to environmentally safe and effective strategies for controlling disease. Asexual spores represent stages of the life cycle that are particularly vulnerable to crop-protection chemicals as they are exposed to the environment, can persist only briefly outside a host and have limited nutrient reserves that limit their ability to resist toxins. Several fungicides that were developed for true fungi were found to target germination pathways 10,106.Other mechanisms by which manipulations of spore biology might enable disease control could involve chemicals with antisporulant activity, which would arrest the spread of disease; stimulators of oospore germination, which if applied before planting would reduce oospore infection; and modified mating hormones that might lead to growth arrest or sterility. 1. Roncal, T. & Ugalde, U. Conidiation induction in Penicillium. Res. Microbiol. 154, (2003). 2. Williams, H. P. & Harwood, A. J. Cell polarity and Dictyostelium development. Curr. Opin. Microbiol. 6, (2003). 3. Hilbert, D. W. & Piggot, P. J. 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