Pieter van West

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world’s population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at

A translocation signal for delivery of oomycete effector proteins into host plant cells

6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at ∼240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for ∼74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK—representing a change that conserves physicochemical properties of the protein—P. infestans fails to deliver Avr3a or an Avr3a–GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

Internuclear gene silencing in Phytophthora infestans.

BackgroundPythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.ResultsThe P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans.ConclusionsAccess to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

A Gα subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans

Transformation of the diploid oomycete plant pathogen Phytophthora infestans with antisense, sense, and promoter-less constructs of the coding sequence of the elicitin gene inf1 resulted in transcriptional silencing of both the transgenes and the endogenous gene. Since heterokaryons obtained by somatic fusion of an inf1-silenced transgenic strain and a wild-type strain displayed stable gene silencing, inf1 silencing is dominant and acts in trans. Inf1 remained silenced in nontransgenic homokaryotic progeny from the silenced heterokaryons, thereby demonstrating that the presence of transgenes is not essential for maintaining the silenced status of the endogenous inf1 gene. These findings support a model reminiscent of paramutation and involving a trans-acting factor that is capable of transferring a silencing signal between nuclei.

Advances in research on oomycete root pathogens

The heterotrimeric G‐protein pathway is a ubiquitous eukaryotic signalling module that is known to regulate growth and differentiation in many plant pathogens. We previously identified Pigpa1, a gene encoding a G‐protein α subunit from the potato late blight pathogen Phytophthora infestans. P. infestans belongs to the class oomycetes, a group of organisms in which signal transduction processes have not yet been studied at the molecular level. To elucidate the function of Pigpa1, PiGPA1‐deficient mutants were obtained by homology‐dependent gene silencing. The Pigpa1‐silenced mutants produced zoospores that turned six to eight times more frequently, causing them to swim only short distances compared with wild type. Attraction to the surface, a phenomenon known as negative geotaxis, was impaired in the mutant zoospores, as well as autoaggregation and chemotaxis towards glutamic and aspartic acid. Zoospore production was reduced by 20–45% in different Pigpa1‐silenced mutants. Transformants expressing constitutively active forms of PiGPA1, containing amino acid substitutions (R177H and Q203L), showed no obvious phenotypic differences from the wild‐type strain. Infection efficiencies on potato leaves ranged from 3% to 14% in the Pigpa1‐silenced mutants, compared with 77% in wild type, showing that virulence is severely impaired. The results prove that PiGPA1 is crucial for zoospore motility and for pathogenicity in an important oomycete plant pathogen.

Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato

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 specific 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 significant 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 scientifically neglected group of organisms.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans.

A method for double-stranded RNA-mediated transient gene silencing in Phytophthora infestans.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinklers, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Plasmodium falciparum and Hyaloperonospora parasitica effector translocation motifs are functional in Phytophthora infestans

SUMMARY Gene silencing, triggered by double-stranded RNA (dsRNA), has proved to be a valuable tool for determining and confirming the function of genes in many organisms. For Phytophthora infestans, the cause of late blight on potato and tomato, gene silencing strategies have relied on stable transformation followed by spontaneous silencing of both the endogenous gene and the transgene. Here we describe the first application of transient gene silencing in P. infestans, by delivering in vitro synthesized dsRNA into protoplasts to trigger silencing. A marker gene, gfp, and two P. infestans genes, inf1 and cdc14, both of which have been silenced previously, were selected to test this strategy. Green fluorescent protein (GFP) fluorescence was reduced in regenerating protoplasts up to 4 days after exposure to gfp dsRNA. A secondary reduction in expression of all genes tested was not fully activated until 12-17 days after introduction of the respective homologous dsRNAs. At this time after exposure to dsRNA, reduced GFP fluorescence in gfp dsRNA-treated lines, and reduced INF1 production in inf1 dsRNA-treated lines, was observed. Introduction of dsRNA for the stage-specific gene, cdc14, yielded the expected phenotype of reduced numbers of sporangia when cdc14 expression was significantly reduced compared with control lines. Silencing was shown to be sequence-specific, because analysis of inf1 expression in gfp-silenced lines yielded wild-type levels of gene expression. This report shows that transient gene silencing can be used to generate detectable phenotypes in P. infestans and should provide a high-throughput tool for P. infestans functional genomics.