Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes.

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

Fragmentation of tRNA in Phytophthora infestans asexual life cycle stages and during host plant infection.

BACKGROUND: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. RESULTS: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. CONCLUSIONS: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans.

Phenotypic diversification by gene silencing in Phytophthora plant pathogens.

Advances in genome sequencing technologies have enabled generation of unprecedented information on genome content and organization. Eukaryote genomes in particular may contain large populations of transposable elements (TEs) and other repeated sequences. Active TEs can result in insertional mutations, altered transcription levels and ectopic recombination of DNA. The genome of the oomycete plant pathogen, Phytophthora infestans, contains vast numbers of TE sequences. There are also hundreds of predicted disease-promoting effector proteins, predominantly located in TE-rich genomic regions. Expansion of effector gene families is also a genomic signature of related oomycetes such as P. sojae. Deep sequencing of small RNAs (sRNAs) from P. infestans has identified sRNAs derived from all families of transposons, highlighting the importance of RNA silencing for maintaining these genomic invaders in an inactive form. Small RNAs were also identified from specific effector encoding genes, possibly leading to RNA silencing of these genes and variation in pathogenicity and virulence toward plant resistance genes. Similar findings have also recently been made for the distantly related species, P. sojae. Small RNA "hotspots" originating from arrays of amplified gene sequences, or from genes displaying overlapping antisense transcription, were also identified in P. infestans. These findings suggest a major role for RNA silencing processes in the adaptability and diversification of these economically important plant pathogens. Here we review the latest progress and understanding of gene silencing in oomycetes with emphasis on transposable elements and sRNA-associated events.

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.

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.

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.

Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in Phytophthora infestans determine virulence on R2 plants.

• A detailed molecular understanding of how oomycete plant pathogens evade disease resistance is essential to inform the deployment of durable resistance (R) genes. • Map-based cloning, transient expression in planta, pathogen transformation and DNA sequence variation across diverse isolates were used to identify and characterize PiAVR2 from potato late blight pathogen Phytophthora infestans. • PiAVR2 is an RXLR-EER effector that is up-regulated during infection, accumulates at the site of haustoria formation, and is recognized inside host cells by potato protein R2. Expression of PiAVR2 in a virulent P. infestans isolate conveys a gain-of-avirulence phenotype, indicating that this is a dominant gene triggering R2-dependent disease resistance. PiAVR2 presence/absence polymorphisms and differential transcription explain virulence on R2 plants. Isolates infecting R2 plants express PiAVR2-like, which evades recognition by R2. PiAVR2 and PiAVR2-like differ in 13 amino acids, eight of which are in the C-terminal effector domain; one or more of these determines recognition by R2. Nevertheless, few polymorphisms were observed within each gene in pathogen isolates, suggesting limited selection pressure for change within PiAVR2 and PiAVR2-like. • Our results direct a search for R genes recognizing PiAVR2-like, which, deployed with R2, may exert strong selection pressure against the P. infestans population.

Identification of appressorial and mycelial cell wall proteins and a survey of the membrane proteome of Phytophthora infestans.

Proteins embedded in the cell wall and plasma membrane of filamentous oomycetes and fungi provide a means by which these organisms can interact with their local environment. However, cell wall and membrane proteins have often proved difficult to isolate using conventional proteomic techniques. Here we have used liquid chromatography tandem mass spectrometry (LC-MS/MS) to facilitate rapid and sensitive quantification of the cell wall proteome. We report the use of LC-MS/MS to identify differentially regulated proteins from the cell walls of three different lifecycle stages of the oomycete plant pathogen Phytophthora infestans: non-sporulating vegetative mycelium, sporulating mycelium, and germinating cysts with appressoria. We have also used quantitative real-time RT-PCR to confirm that the transcripts corresponding to some of these proteins, namely those identified in cell walls of germinating cysts with appressoria, accumulate differentially throughout the lifecycle. These proteins may, therefore, be important for pre-infective development and early pathogenicity. Up to 31 covalently and non-covalently bound cell wall-associated proteins were identified. All of the proteins identified in germinating cysts with appressoria, and several of those from mycelial fractions, were classified as putative effector or pathogen-associated molecular pattern (PAMP) molecules, including members of the CBEL family, the elicitin family, the crinkler (CRN) family and two transglutaminases. Thus, the cell wall of P. infestans may represent an important reservoir for surface-presented, apoplastic effectors or defence activation molecules. Proteins predicted to be cell surface proteins included IPI-B like proteins, mucins, cell wall-associated enzymes and annexin family members. Additionally we identified up to 27 membrane-associated proteins from Triton X-114 phase partitioned mycelial membrane preparations, producing the first inventory of oomycete membrane-associated proteins. Four of these proteins are small Rab-type G-proteins and several are associated with secretion.

Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans.

Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans. Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans. Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H(2)O. Uptake studies with (14)C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, (14)C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.

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

The oomycete potato late blight pathogen, Phytophthora infestans, and the apicomplexan malaria parasite Plasmodium falciparum translocate effector proteins inside host cells, presumably to the benefit of the pathogen or parasite. Many oomycete candidate secreted effector proteins possess a peptide domain with the core conserved motif, RxLR, located near the N-terminal secretion signal peptide. In the Ph. infestans effector Avr3a, RxLR and an additional EER motif are essential for translocation into host cells during infection. Avr3a is recognized in the host cytoplasm by the R3a resistance protein. We have exploited this cytoplasmic recognition to report on replacement of the RxLR-EER of Avr3a with the equivalent sequences from the intracellular effectors ATR1NdWsB and ATR13 from the related oomycete pathogen, Hyaloperonospora parasitica, and the host targeting signal from the Pl. falciparum virulence protein PfHRPII. Introduction of these chimeric transgenes into Ph. infestans and subsequent virulence testing on potato plants expressing R3a demonstrated the alternative motifs to be functional in translocating Avr3a inside plant cells. These results suggest common mechanisms for protein translocation in both malaria and oomycete pathosystems.

A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato.

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans.

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.

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

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.

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

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

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.

A novel non-protein-coding infection-specific gene family is clustered throughout the genome of Phytophthora infestans.

Phytophthora infestans is the cause of late blight, a devastating and re-emerging disease of potato. Significant advances have been made in understanding the biology of P. infestans, and in the development of molecular tools to study this oomycete. Nevertheless, little is known about the molecular bases of the establishment or development of disease in this hemibiotrophic pathogen. Suppression subtractive hybridization (SSH) was used to generate cDNA enriched for sequences upregulated during potato infection. To identify pathogen-derived cDNAs, and eliminate host sequences from further study, SSH cDNA was hybridized to a P. infestans bacterial artificial chromosome library. A new gene family was identified called Pinci1, comprising more than 400 members arranged in clusters of up to nine copies throughout the P. infestans draft genome sequence. Real-time RT-PCR was used to quantify the expression of five classes of transcript within the family, relative to the constitutively expressed PiactA gene, and it revealed them to be significantly upregulated from 12 to 33 h post-inoculation, a period defining the biotrophic phase of infection. Computational analysis of sequences suggested that transcripts were non-protein coding, and this was confirmed by transient expression of FLAG-tagged ORFs in P. infestans.

An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm.

The oomycete Phytophthora infestans causes late blight, the potato disease that precipitated the Irish famines in 1846 and 1847. It represents a reemerging threat to potato production and is one of >70 species that are arguably the most devastating pathogens of dicotyledonous plants. Nevertheless, little is known about the molecular bases of pathogenicity in these algae-like organisms or of avirulence molecules that are perceived by host defenses. Disease resistance alleles, products of which recognize corresponding avirulence molecules in the pathogen, have been introgressed into the cultivated potato from a wild species, Solanum demissum, and R1 and R3a have been identified. We used association genetics to identify Avr3a and show that it encodes a protein that is recognized in the host cytoplasm, where it triggers R3a-dependent cell death. Avr3a resides in a region of the P. infestans genome that is colinear with the locus containing avirulence gene ATR1(NdWsB) in Hyaloperonospora parasitica, an oomycete pathogen of Arabidopsis. Remarkably, distances between conserved genes in these avirulence loci were often similar, despite intervening genomic variation. We suggest that Avr3a has undergone gene duplication and that an allele evading recognition by R3a arose under positive selection.

Families of short interspersed elements in the genome of the oomycete plant pathogen, Phytophthora infestans.

The first known families of tRNA-related short interspersed elements (SINEs) in the oomycetes were identified by exploiting the genomic DNA sequence resources for the potato late blight pathogen, Phytophthora infestans. Fifteen families of tRNA-related SINEs, as well as predicted tRNAs, and other possible RNA polymerase III-transcribed sequences were identified. The size of individual elements ranges from 101 to 392 bp, representing sequences present from low (1) to highly abundant (over 2000) copy number in the P. infestans genome, based on quantitative PCR analysis. Putative short direct repeat sequences (6-14 bp) flanking the elements were also identified for eight of the SINEs. Predicted SINEs were named in a series prefixed infSINE (for infestans-SINE). Two SINEs were apparently present as multimers of tRNA-related units; four copies of a related unit for infSINEr, and two unrelated units for infSINEz. Two SINEs, infSINEh and infSINEi, were typically located within 400 bp of each other. These were also the only two elements identified as being actively transcribed in the mycelial stage of P. infestans by RT-PCR. It is possible that infSINEh and infSINEi represent active retrotransposons in P. infestans. Based on the quantitative PCR estimates of copy number for all of the elements identified, tRNA-related SINEs were estimated to comprise 0.3% of the 250 Mb P. infestans genome. InfSINE-related sequences were found to occur in species throughout the genus Phytophthora. However, seven elements were shown to be exclusive to P. infestans.

Elevated amino acid biosynthesis in Phytophthora infestans during appressorium formation and potato infection.

Appressorium formation is believed to be an important event in establishing a successful interaction between the late blight pathogen, Phytophthora infestans, and its host plants potato and tomato. An understanding of molecular events occurring in appressorium development could suggest new strategies for controlling late blight. We used parallel studies of the transcriptome and proteome to identify genes and proteins that are up-regulated in germinating cysts developing appressoria. As a result, five distinct genes involved in amino acid biosynthesis were identified that show increased expression in germinating cysts with appressoria. These are a methionine synthase (Pi-met1), a ketol-acid reductoisomerase (Pi-kari1), a tryptophan synthase (Pi-trp1), an acetolactate synthase (Pi-als1), and a threonine synthase (Pi-ts1). Four of these P. infestans genes were also up-regulated, although to lower levels, during the early, biotrophic phase of the interaction in potato and all five were considerably up-regulated during the transition (48 hpi) to the necrotrophic phase of the interaction. Real-time RT-PCR revealed that expression of potato homologues of the amino acid biosynthesis genes increased during biotrophic and necrotrophic infection phases. Furthermore, we investigated levels of free amino acids in the pre-infection stages and found that in most cases there was a decrease in free amino acids in zoospores and germinating cysts, relative to sporangia, followed by a sharp increase in germinating cysts with appressoria. Amino acid biosynthesis would appear to be important for pathogenicity in P. infestans, providing a potential metabolic target for chemical control.

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