Infestans Genome Research Articles

Transcription factor binding specificities of the oomycete Phytophthora infestans reflect conserved and divergent evolutionary patterns and predict function

BackgroundIdentifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group.ResultsMining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo.ConclusionsWe established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

Identification of miRNAs Involving Potato-Phytophthora infestans Interaction.

sRNAs (small RNAs) play an important role in regulation of plant immunity against a variety of pathogens. In this study, sRNA sequencing analysis was performed to identify miRNAs (microRNAs) during the interaction of potato and Phytophthora infestans. Totally, 171 potato miRNAs were identified, 43 of which were annotated in the miRNA database and 128 were assigned as novel miRNAs in this study. Those potato miRNAs may target 878 potato genes and half of them encode resistance proteins. Fifty-three potato miRNAs may target 194 P. infestans genes. Three potato miRNAs (novel 72, 133, and 140) were predicted to have targets only in the P. infestans genome. miRNAs transient expression and P. infestans inoculation assay showed that miR396, miR166, miR6149-5P, novel133, or novel140 promoted P. infestans colonization, while miR394 inhibited colonization on Nicotiana benthamiana leaves. An artificial miRNA target (amiRNA) degradation experiment demonstrated that miR394 could target both potato gene (PGSC0003DMG400034305) and P. infestans genes. miR396 targets the multicystatin gene (PGSC0003DMG400026899) and miR6149-5p could shear the galactose oxidase F-box protein gene CPR30 (PGSC0003DMG400021641). This study provides new information on the aspect of cross-kingdom immune regulation in potato-P. infestans interaction at the sRNAs regulation level.

Dual RNA Sequencing Reveals the Genome-Wide Expression Profiles During the Compatible and Incompatible Interactions Between Solanum tuberosum and Phytophthora infestans.

Late blight, caused by Phytophthora infestans (P. infestans), is a devastating plant disease. P. infestans genome encodes hundreds of effectors, complicating the interaction between the pathogen and its host and making it difficult to understand the interaction mechanisms. In this study, the late blight-resistant potato cultivar Ziyun No.1 and the susceptible potato cultivar Favorita were infected with P. infestans isolate SCPZ16-3-1 to investigate the global expression profiles during the compatible and incompatible interactions using dual RNA sequencing (RNA-seq). Most of the expressed Arg-X-Leu-Arg (RXLR) effector genes were suppressed during the first 24 h of infection, but upregulated after 24 h. Moreover, P. infestans induced more specifically expressed genes (SEGs), including RXLR effectors and cell wall-degrading enzymes (CWDEs)-encoding genes, in the compatible interaction. The resistant potato activated a set of biotic stimulus responses and phenylpropanoid biosynthesis SEGs, including kirola-like protein, nucleotide-binding site-leucine-rich repeat (NBS-LRR), disease resistance, and kinase genes. Conversely, the susceptible potato cultivar upregulated more kinase, pathogenesis-related genes than the resistant cultivar. This study is the first study to characterize the compatible and incompatible interactions between P. infestans and different potato cultivars and provides the genome-wide expression profiles for RXLR effector, CWDEs, NBS-LRR protein, and kinase-encoding genes.

Satellitome Analysis of Rhodnius prolixus, One of the Main Chagas Disease Vector Species.

The triatomine Rhodnius prolixus is the main vector of Chagas disease in countries such as Colombia and Venezuela, and the first kissing bug whose genome has been sequenced and assembled. In the repetitive genome fraction (repeatome) of this species, the transposable elements represented 19% of R. prolixus genome, being mostly DNA transposon (Class II elements). However, scarce information has been published regarding another important repeated DNA fraction, the satellite DNA (satDNA), or satellitome. Here, we offer, for the first time, extended data about satellite DNA families in the R. prolixus genome using bioinformatics pipeline based on low-coverage sequencing data. The satellitome of R. prolixus represents 8% of the total genome and it is composed by 39 satDNA families, including four satDNA families that are shared with Triatoma infestans, as well as telomeric (TTAGG)n and (GATA)n repeats, also present in the T. infestans genome. Only three of them exceed 1% of the genome. Chromosomal hybridization with these satDNA probes showed dispersed signals over the euchromatin of all chromosomes, both in autosomes and sex chromosomes. Moreover, clustering analysis revealed that most abundant satDNA families configured several superclusters, indicating that R. prolixus satellitome is complex and that the four most abundant satDNA families are composed by different subfamilies. Additionally, transcription of satDNA families was analyzed in different tissues, showing that 33 out of 39 satDNA families are transcribed in four different patterns of expression across samples.

Improved Genome Sequence and Gene Annotation Resource for the Potato Late Blight Pathogen Phytophthora infestans.

Phytophthora infestans is a devastating pathogen causing potato late blight (Solanum tuberosum). Here we report the sequencing, assembly and genome annotation for two Phytophthora infestans isolates sampled in Republic of Korea. Genome sequencing was carried out using long read (Oxford Nanopore) and short read (Illumina Nextseq) sequencing technologies that significantly improved the contiguity and quality of P. infestans genome assembly. Our resources would help researchers better understand the molecular mechanisms by which P. infestans causes late blight disease in the future.

SNP-Based Differentiation of Phytophthora infestans Clonal Lineages Using Locked Nucleic Acid Probes and High-Resolution Melt Analysis

Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic differences can exist between lineages. Whole P. infestans genomes were aligned and single nucleotide polymorphisms (SNPs) identified as targets for the development of clonal-lineage-specific molecular diagnostic tools. Informative SNPs were used to develop high-resolution melt (HRM) assays and locked nucleic acid (LNA) probes to differentiate lineage US-23, the predominant lineage in the Eastern United States for the past several years, from three other U.S. lineages. Three different primer pairs targeting one to three SNPs were capable of separating lineage US-23 from lineages US-8, US-11, and US-24 using HRM analysis. A fourth HRM primer pair targeted a highly variable genomic region containing nine polymorphisms within 63 bp. These primers separated US-23, US-11, and US-8 plus US-24 into three separate groups following HRM analysis but did not separate US-8 from US-24. Additionally, two LNA probes were designed to target a portion of the P. infestans genome containing two SNPs diagnostic for US-23. A single multiplex quantitative polymerase chain reaction assay containing both differentially labeled LNA probes differentiated individuals belonging to lineage US-23 from those belonging to US-8, US-11, and US-24.

Identification of the Dominant Genotypes of Phytophthora infestans in Canada Using Real-Time PCR with ASO-PCR Assays.

Phytophthora infestans, a pathogenic oomycete that is the causal agent of potato and tomato late blight, has devastating effects worldwide. The genetic composition of P. infestans populations in Canada has changed considerably over the last few years, with the appearance of several new genotypes showing different mating types and sensitivity to the fungicide metalaxyl. Genetic markers allowing for a rapid assessment of genotypes from small amounts of biological material would be beneficial for the early detection and control of this pathogen throughout Canada. Mining of the P. infestans genome revealed several regions containing single-nucleotide polymorphisms (SNP) within both nuclear genes and flanking sequences of microsatellite loci. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) assays were developed from 14 of the 50 SNP found by sequencing. Nine optimized ASO-PCR assays were validated using a blind test comprising P. infestans and other Phytophthora spp. The assays revealed diagnostic profiles unique to each of the five dominant genotypes present in Canada. The markers developed in this study can be used with environmental samples such as infected leaves, and will contribute to the genomic toolbox available to assess the genetic diversity of P. infestans at the intraspecific level. For late blight management, early warning about P. infestans genotypes present in potato and tomato fields will help growers select the most appropriate fungicides and application strategies.

Recognition of Phytophthora infestans in potato (Solanum tuberosum L.): Scr74 gene as an example

Abstract Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato worldwide. The P. infestans genome encodes potentially polymorphic genes that evolve continually to evade the recognition of plant R genes, though it has hundreds of predicted and conserved effector proteins recognised by the plant. The gene Scr74 encodes a predicted 74-amino acid secreted cysteine-rich protein belonging to a highly polymorphic gene family within P. infestans. This study screened the recognition of Scr74 genes in wild potato genotypes from August 2013 to January 2014 in the Plant Breeding Laboratory of Wageningen University, the Netherlands. To identify the recognition of the Scr74 gene, we grew potato genotypes in the green house for PVX assays, detached leaf assays and molecular work. Twenty-seven good-quality sequences of the Scr74 gene variant with a length of 74 amino acids were found and more frequent amino acid variation was detected on the mature protein. Seventeen Scr74 constructs were identified as diversified and two effectors were strongly recognised by wild S. verrucosum genotypes via effectoromics from the PVX assay. A strong plant cell death hypersensitive response (HR) was recorded on wild S. verrucosum and S. tuberosum genotypes from the detached leaf assay. This recognition seems to be a useful indicator for the presence of a resistance gene (s) to the polymorphic effectors of P. infestans (as it has seen on Scr74 gene) in the wild potato genotypes.

DINE-1, the highest copy number repeats in Drosophila melanogaster are non-autonomous endonuclease-encoding rolling-circle transposable elements (Helentrons).

BackgroundThe Drosophila INterspersed Elements-1 (DINE-1/INE1) transposable elements (TEs) are the most abundant component of the Drosophila melanogaster genome and have been associated with functional gene duplications. DINE-1 TEs do not encode any proteins (non-autonomous) thus are moved by autonomous partners. The identity of the autonomous partners has been a mystery. They have been allied to Helitrons (rolling-circle transposons), MITEs (DNA transposons), and non-LTR retrotransposons by different authors.ResultsWe report multiple lines of bioinformatic evidence that illustrate the relationship of DINE-1 like TEs to endonuclease-encoding rolling-circle TEs (Helentrons). The structural features of Helentrons are described, which resemble the organization of the non-autonomous partners, but differ significantly from canonical Helitrons. In addition to the presence of an endonuclease domain fused to the Rep/Helicase protein, Helentrons have distinct structural features. Evidence is presented that illustrates that Helentrons are widely distributed in invertebrate, fish, and fungal genomes. We describe an intermediate family from the Phytophthora infestans genome that phylogenetically groups with Helentrons but that displays Helitron structure. In addition, evidence is presented that Helentrons can capture gene fragments in a pattern reminiscent of canonical Helitrons.ConclusionsWe illustrate the relationship of DINE-1 and related TE families to autonomous partners, the Helentrons. These findings will allow their proper classification and enable a more accurate understanding of the contribution of rolling-circle transposition to the birth of new genes, gene networks, and genome composition.

Mitochondrial DNA polymorphisms in Phytophthora infestans: New haplotypes are identified and re-defined by PCR

Polymorphisms of mitochondrial DNA (mt-DNA) are particularly useful for monitoring specific pathogen populations like Phytophthora infestans. Basically type I and II of P. infestans mt-DNA were categorized by means of polymorphism lengths caused by an ~2kb insertion, which can be detected via restriction enzyme digestion. In addition genome sequencing of haplotype Ib has been used as a simple Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR–RFLP) method to indirectly identify type I and II alterations through EcoR I restriction enzyme DNA fragment patterns of the genomic P4 area. However, with the common method, wrong mt-DNA typing occurs due to an EcoR I recognition site mutation in the P4 genomic area. Genome sequencing of the four haplotypes (Ia, Ib, IIa, and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and we indentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRi length polymorphism caused by a 2kb insertion/deletion was utilized to identify mt-DNA types I and II, while another length polymorphism in the HVRii region is caused by a variable number of tandem repeats (n=1, 2, or 3) of a 36bp sized DNA stretch and was further used to determine mt-DNA sub-types, which were described as Rn=1, 2, or 3. Finally, the P. infestans mt-DNA haplotypes were re-defined as IR1 or IIR2 according to PCR derived HVRi and HVRii length polymorphisms. Twenty-three isolates were chosen to verify the feasibility of our new approach for identifying mt-DNA haplotypes and a total of five haplotypes (IR1, IR2, IR3, IIR2 and IIR3) were identified. Additionally, we found that six isolates determined as type I by our method were mistakenly identified as type II by the PCR–RFLP technique. In conclusion, we propose a simple and rapid PCR method for identification of mt-DNA haplotypes based on sequence analyses of the mitochondrial P. infestans genome.

Genome-wide Prediction and Functional Validation of Promoter Motifs Regulating Gene Expression in Spore and Infection Stages of Phytophthora infestans

Most eukaryotic pathogens have complex life cycles in which gene expression networks orchestrate the formation of cells specialized for dissemination or host colonization. In the oomycete Phytophthora infestans, the potato late blight pathogen, major shifts in mRNA profiles during developmental transitions were identified using microarrays. We used those data with search algorithms to discover about 100 motifs that are over-represented in promoters of genes up-regulated in hyphae, sporangia, sporangia undergoing zoosporogenesis, swimming zoospores, or germinated cysts forming appressoria (infection structures). Most of the putative stage-specific transcription factor binding sites (TFBSs) thus identified had features typical of TFBSs such as position or orientation bias, palindromy, and conservation in related species. Each of six motifs tested in P. infestans transformants using the GUS reporter gene conferred the expected stage-specific expression pattern, and several were shown to bind nuclear proteins in gel-shift assays. Motifs linked to the appressoria-forming stage, including a functionally validated TFBS, were over-represented in promoters of genes encoding effectors and other pathogenesis-related proteins. To understand how promoter and genome architecture influence expression, we also mapped transcription patterns to the P. infestans genome assembly. Adjacent genes were not typically induced in the same stage, including genes transcribed in opposite directions from small intergenic regions, but co-regulated gene pairs occurred more than expected by random chance. These data help illuminate the processes regulating development and pathogenesis, and will enable future attempts to purify the cognate transcription factors.

Evidence for small RNAs homologous to effector-encoding genes and transposable elements in the oomycete Phytophthora infestans.

Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19–40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.

Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans

Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5Δ(5, 8, 11, 14, 17)) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.

Can silencing of transposons contribute to variation in effector gene expression inPhytophthora infestans?

Transposable elements are ubiquitous residents in eukaryotic genomes. Often considered to be genomic parasites, they can lead to dramatic changes in genome organization, gene expression, and gene evolution. The oomycete plant pathogen Phytophthora infestans has evolved a genome organization where core biology genes are predominantly located in genome regions that have relatively few resident transposons. In contrast, disease effector-encoding genes are most frequently located in rapidly evolving genomic regions that are rich in transposons. P. infestans, as a eukaryote, likely uses RNA silencing to minimize the activity of transposons. We have shown that fusion of a short interspersed element (SINE) to an effector gene in P. infestans leads to the silencing of both the introduced fusion and endogenous homologous sequences. This is also likely to occur naturally in the genome of P. infestans, as transcriptional inactivation of effectors is known to occur, and over half of the translocated “RXLR class” of effectors are located within 2 kb of transposon sequences in the P. infestans genome. In this commentary, we review the diverse transposon inventory of P. infestans, its control by RNA silencing, and consequences for expression modulation of nearby effector genes in this economically important plant pathogen.

Exploiting Knowledge of Pathogen Effectors to Enhance Late Blight Resistance in Potato

Late blight disease of potato, caused by Phytophthora infestans, is the most serious disease of this crop on a global scale and is thus a threat to food security. Use of resistant potato cultivars to prevent late blight does not have a very successful history, as P. infestans genotypes have overcome the deployed resistances. Thus, there is a need to identify more durable resistances, or identify and develop novel forms of resistance that exploit vulnerabilities in the biology of P. infestans. Application of molecular biology tools in P. infestans research has recently culminated in the identification of several avirulence effectors matching specific resistances in potato, the sequencing of the P. infestans genome and identification of hundreds of candidate translocated pathogen ‘RXLR’ effector proteins that may promote disease progression. Strategies for prioritising these effectors for further research are revealing those that are highly expressed during infection, difficult for the pathogen to alter rapidly, essential for P. infestans pathogenesis and recognized by resistant accessions of Solanum spp. These effector characteristics are being used to identify and characterise resistances from Solanum germplasm that may prove more durable. In addition to RXLR effectors, P. infestans also produces a broad spectrum of additional secreted proteins. These are exposed to plant cells and may potentially act to trigger resistance, either as broad spectrum pathogen-associated molecular patterns or as specific effectors of resistance. Alternatively, conserved secreted proteins may be attractive targets for novel agrichemical development. We have silenced a diverse selection of these candidate secreted proteins in P. infestans and demonstrated their effects on late blight disease development. Results from these studies are aiding a deeper understanding of P. infestans disease development and identifying potential pathogen weaknesses for exploitation in future control measures.

Silencing of the PiAvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element

Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5′- infSINEm:: PiAvr3a-3′ or 5′- PiAvr3a:: SINEm-3′ chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements.

Genome-wide analysis of the secreted proteins of phytophthora infestans

Based on the Phytophthora infestans genome sequence, we used bioinformatics and computer-based prediction algorithms to predict the secreted proteins of P infestans in detail, which would help us to elucidate the molecular mechanism underlying the interaction between the host plants and the P infestans. In this study, the signal peptide prediction algorithms SignalP v3.0 and PSORT, transmembrane helix prediction algorithms TMHMM-2.0 and THUMBUP, GPI-anchoring site prediction algorithm big-PI Predictor, and subcellular protein location distribution algorithm TargetP v1.01 were used to analyze the 22658 protein sequences of P infestans published. Our results suggested that there might be 671 secreted pro- teins, accounting for 3.0% of the total proteins. Among them, the functions of the 45 secreted proteins had been described previously. Their functions involved cellular metabolism and signal transduction etc. In addition, some of the secreted proteins were functionally similar to elicitin, which were likely to be associated with the virulence of P. infestans.

Evidence for involvement of Dicer‐like, Argonaute and histone deacetylase proteins in gene silencing in Phytophthora infestans

Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD helicases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.

Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans

BackgroundPhytophthora infestans is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The P. infestans genome experienced a repeat-driven expansion relative to the genomes of Phytophthora sojae and Phytophthora ramorum and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN) families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation.ResultsWe used in silico approaches to predict and describe the repertoire of P. infestans secreted proteins (the secretome). We defined the "plastic secretome" as a subset of the genome that (i) encodes predicted secreted proteins, (ii) is excluded from genome segments orthologous to the P. sojae and P. ramorum genomes and (iii) is encoded by genes residing in gene sparse regions of P. infestans genome. Although including only ~3% of P. infestans genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced in planta. We highlight 19 plastic secretome genes induced in planta but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors.ConclusionsThis work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes.

Discovery of Phytophthora infestans genes expressed in planta through mining of cDNA libraries.

Background Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta.Methodology/Principal FindingsWe describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family.Conclusions/SignificanceWe annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.