Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance.

Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.

Characterisation and mapping of a Globodera pallida resistance derived from the wild potato species Solanum spegazzinii.

KEY MESSAGE: A new resistance locus acting against the potato cyst nematode Globodera pallida was mapped to chromosome VI in the diploid wild potato species Solanum spegazzinii CPC 7195. The potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis are economically important potato pests in almost all regions where potato is grown. One important management strategy involves deployment through introgression breeding into modern cultivars of new sources of naturally occurring resistance from wild potato species. We describe a new source of resistance to G. pallida from wild potato germplasm. The diploid species Solanum spegazzinii Bitter accession CPC 7195 shows resistance to G. pallida pathotypes Pa1 and Pa2/3. A cross and first backcross of S. spegazzinii with Solanum tuberosum Group Phureja cultivar Mayan Gold were performed, and the level of resistance to G. pallida Pa2/3 was determined in progeny clones. Bulk-segregant analysis (BSA) using generic mapping enrichment sequencing (GenSeq) and genotyping-by-sequencing were performed to identify single-nucleotide polymorphisms (SNPs) that are genetically linked to the resistance, using S. tuberosum Group Phureja clone DM1-3 516 R44 as a reference genome. These SNPs were converted into allele-specific PCR assays, and the resistance was mapped to an interval of roughly 118 kb on chromosome VI. This newly identified resistance, which we call Gpa VIlspg, can be used in future efforts to produce modern cultivars with enhanced and broad-spectrum resistances to the major pests and pathogens of potato.

RLP/K enrichment sequencing; a novel method to identify receptor-like protein (RLP) and receptor-like kinase (RLK) genes.

The identification of immune receptors in crop plants is time-consuming but important for disease control. Previously, resistance gene enrichment sequencing (RenSeq) was developed to accelerate mapping of nucleotide-binding domain and leucine-rich repeat containing (NLR) genes. However, resistances mediated by pattern recognition receptors (PRRs) remain less utilized. Here, our pipeline shows accelerated mapping of PRRs. Effectoromics leads to precise identification of plants with target PRRs, and subsequent RLP/K enrichment sequencing (RLP/KSeq) leads to detection of informative single nucleotide polymorphisms that are linked to the trait. Using Phytophthora infestans as a model, we identified Solanum microdontum plants that recognize the apoplastic effectors INF1 or SCR74. RLP/KSeq in a segregating Solanum population confirmed the localization of the INF1 receptor on chromosome 12, and led to the rapid mapping of the response to SCR74 to chromosome 9. By using markers obtained from RLP/KSeq in conjunction with additional markers, we fine-mapped the SCR74 receptor to a 43-kbp G-LecRK locus. Our findings show that RLP/KSeq enables rapid mapping of PRRs and is especially beneficial for crop plants with large and complex genomes. This work will enable the elucidation and characterization of the nonNLR plant immune receptors and ultimately facilitate informed resistance breeding.

Phytophthora infestans RXLR Effectors Target Parallel Steps in an Immune Signal Transduction Pathway.

The potato (Solanum tuberosum) blight pathogen Phytophthora infestans delivers Arg-X-Leu-Arg (RXLR) effector proteins into host cells to subvert plant immune responses and promote colonization. We show that transient expression and stable transgenic expression of the RXLR effector Pi22926 in Nicotiana benthamiana promotes leaf colonization by P. infestans. Pi22926 suppresses cell death triggered by coexpression of the Cladosporium fulvum avirulence protein Avr4 and the tomato (Solanum lycopersicum) resistance protein Cf4. Pi22926 interacts with a potato mitogen-activated protein kinase kinase kinase, StMAP3Kß2, in the nucleoplasm. Virus-induced gene silencing (VIGS) of the ortholog NbMAP3Kß2 in N. benthamiana enhances P. infestans colonization and attenuates Cf4/Avr4-induced cell death, indicating that this host protein is a positive regulator of immunity. Cell death induced by Cf4/Avr4 is dependent on NbMAP3Kε and NbMAP3Kß2, indicating that these MAP3Ks function in the same signaling pathway. VIGS of NbMAP3Kß2 does not compromise cell death triggered by overexpression of MAP3Kε. Similarly, VIGS of NbMAP3Kε does not attenuate cell death triggered by MAP3Kß2, demonstrating that these MAP3K proteins function in parallel. In agreement, Pi22926 or another RXLR effector, PexRD2, only suppresses cell death triggered by expression of StMAP3Kß2 or StMAP3Kε, respectively. Our data reveal that two P. infestans effectors, PexRD2 and Pi22926, promote P. infestans colonization by targeting MAP3K proteins that act in parallel in the same signal transduction pathway.

Natural resistance to Potato virus Y in Solanum tuberosum Group Phureja.

KEY MESSAGE: Novel major gene resistance against Potato virus Y in diploid populations of Solanum tuberosum Groups Phureja and Tuberosum was biologically and genetically characterised. Named Ry(o)phu, it mapped to chromosome 9. A new source of genetic resistance derived from Solanum tuberosum Group Phureja against Potato virus Y (PVY) was identified and genetically characterised in three diploid biparental potato populations. Segregation data for two populations (05H1 and 08H1) suggested the presence of a single dominant gene for resistance to PVY which, following DaRT analysis of the 08H1 cross, was mapped to chromosome 9. More detailed genetic analysis of resistance utilised a well-characterised SNP-linkage map for the 06H1 population, together with newly generated marker data. In these plants, which have both S. tuberosum Group Phureja and S. tuberosum Group Tuberosum in their pedigree, the resistance was shown to map to chromosome 9 at a locus not previously associated with PVY resistance, although there is evidence for at least one other genetic factor controlling PVY infection. The resistance factor location on chromosome 9 (named as Ry(o)phu) suggests a potential role of NB-LRR genes in this resistance. Phenotypic analysis using a GUS-tagged virus revealed that a small amount of PVY replication occurred in occasional groups of epidermal cells in inoculated leaves of resistant plants, without inducing any visible hypersensitive response. However, the virus did not enter the vascular system and systemic spread was completely prevented.

New Findings on the Resistance Mechanism of an Elite Diploid Wild Potato Species JAM1-4 in Response to a Super Race Strain of Phytophthora infestans.

Late blight is a devastating potato disease worldwide, caused by Phytophthora infestans. The P. infestans strain 2013-18-306 from Yunnan is a "supervirulent race" that overcomes all 11 known late blight resistance genes (R1 to R11) from Solanum demissum. In a previous study, we identified a diploid wild-type potato JAM1-4 (S. jamesii) with high resistance to 2013-18-306. dRenSeq analysis indicated the presence of novel R genes in JAM1-4. RNA-Seq was used to analyze the late blight resistance response genes and defense regulatory mechanisms of JAM1-4 against 2013-18-306. Gene ontology enrichment and KEGG pathway analysis showed that many disease-resistant pathways were significantly enriched. Analysis of differentially expressed genes (DEGs) revealed an active disease resistance mechanism of JAM1-4, and the essential role of multiple signal transduction pathways and secondary metabolic pathways comprised of SA-JA-ET in plant immunity. We also found that photosynthesis in JAM1-4 was inhibited to promote the immune response. Our study reveals the pattern of resistance-related gene expression in response to a super race strain of potato late blight and provides a theoretical basis for further exploration of potato disease resistance mechanisms, discovery of new late blight resistance genes, and disease resistance breeding.

Tracking disease resistance deployment in potato breeding by enrichment sequencing.

Following the molecular characterisation of functional disease resistance genes in recent years, methods to track and verify the integrity of multiple genes in varieties are needed for crop improvement through resistance stacking. Diagnostic resistance gene enrichment sequencing (dRenSeq) enables the high-confidence identification and complete sequence validation of known functional resistance genes in crops. As demonstrated for tetraploid potato varieties, the methodology is more robust and cost-effective in monitoring resistances than whole-genome sequencing and can be used to appraise (trans) gene integrity efficiently. All currently known NB-LRRs effective against viruses, nematodes and the late blight pathogen Phytophthora infestans can be tracked with dRenSeq in potato and hitherto unknown polymorphisms have been identified. The methodology provides a means to improve the speed and efficiency of future disease resistance breeding in crops by directing parental and progeny selection towards effective combinations of resistance genes.

Phytophthora infestans effector SFI3 targets potato UBK to suppress early immune transcriptional responses.

The potato blight agent Phytophthora infestans secretes a range of RXLR effectors to promote disease. Recent evidence indicates that some effectors suppress early pattern-triggered immunity (PTI) following perception of microbe-associated molecular patterns (MAMPs). Phytophthora infestans effector PiSFI3/Pi06087/PexRD16 has been previously shown to suppress MAMP-triggered pFRK1-Luciferase reporter gene activity. How PiSFI3 suppresses immunity is unknown. We employed yeast-two-hybrid (Y2H) assays, co-immunoprecipitation, transcriptional silencing by RNA interference and virus-induced gene silencing (VIGS), and X-ray crystallography for structure-guided mutagenesis, to investigate the function of PiSFI3 in targeting a plant U-box-kinase protein (StUBK) to suppress immunity. We discovered that PiSFI3 is active in the host nucleus and interacts in yeast and in planta with StUBK. UBK is a positive regulator of specific PTI pathways in both potato and Nicotiana benthamiana. Importantly, it contributes to early transcriptional responses that are suppressed by PiSFI3. PiSFI3 forms an unusual trans-homodimer. Mutation to disrupt dimerization prevents nucleolar localisation of PiSFI3 and attenuates both its interaction with StUBK and its ability to enhance P. infestans leaf colonisation. PiSFI3 is a 'WY-domain' RXLR effector that forms a novel trans-homodimer which is required for its ability to suppress PTI via interaction with the U-box-kinase protein StUBK.

Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes.

The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world-wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor. Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up < 1% of the entire genome. PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes. PenSeq represents a cost-effective alternative to whole-genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively neutral markers and consequently have limited utility in the analysis of adaptive evolution. The approach can be adapted to diverse microbes and pathogens.

The Potato MAP3K StVIK Is Required for the Phytophthora infestans RXLR Effector Pi17316 to Promote Disease.

Plant pathogens deliver effectors to manipulate processes in their hosts, creating a suitable environment for invasion and proliferation. Yet, little is known about the host proteins that are targeted by effectors from filamentous pathogens. Here, we show that stable transgenic expression in potato (Solanum tuberosum) and transient expression in Nicotiana benthamiana of the arginine-any amino acid-leucine-arginine effector Pi17316 enhances leaf colonization by the late blight pathogen Phytophthora infestans Expression of Pi17316 also attenuates cell death triggered by the pathogen-associated molecular pattern Infestin1 (INF1), indicating that the effector suppresses pattern-triggered immunity. However, this effector does not attenuate cell death triggered by a range of resistance proteins, showing that it specifically suppresses INF1-triggered cell death (ICD). In yeast two-hybrid assays, Pi17316 interacts directly with the potato ortholog of VASCULAR HIGHWAY1-interacting kinase (StVIK), encoding a predicted MEK kinase (MAP3K). Interaction in planta was confirmed by coimmunoprecipitation and occurs at the plant plasma membrane. Virus-induced gene silencing of VIK in N. benthamiana attenuated P. infestans colonization, whereas transient overexpression of StVIK enhanced colonization, indicating that this host protein acts as a susceptibility factor. Moreover, VIK overexpression specifically attenuated ICD, indicating that it is a negative regulator of immunity. The abilities of Pi17316 to enhance P. infestans colonization or suppress ICD were compromised significantly in NbVIK-silenced plants, demonstrating that the effector activity of Pi17316 is mediated by this MAP3K. Thus, StVIK is exploited by P. infestans as a susceptibility factor to promote late blight disease.

Mapping the H2 resistance effective against Globodera pallida pathotype Pa1 in tetraploid potato.

KEY MESSAGE: The nematode resistance gene H2 was mapped to the distal end of chromosome 5 in tetraploid potato. The H2 resistance gene, introduced into cultivated potatoes from the wild diploid species Solanum multidissectum, confers a high level of resistance to the Pa1 pathotype of the potato cyst nematode Globodera pallida. A cross between tetraploid H2-containing breeding clone P55/7 and susceptible potato variety Picasso yielded an F1 population that segregated approximately 1:1 for the resistance phenotype, which is consistent with a single dominant gene in a simplex configuration. Using genome reduction methodologies RenSeq and GenSeq, the segregating F1 population enabled the genetic characterisation of the resistance through a bulked segregant analysis. A diagnostic RenSeq analysis of the parents confirmed that the resistance in P55/7 cannot be explained by previously characterised resistance genes. Only the variety Picasso contained functionally characterised disease resistance genes Rpi-R1, Rpi-R3a, Rpi-R3b variant, Gpa2 and Rx, which was independently confirmed through effector vacuum infiltration assays. RenSeq and GenSeq independently identified sequence polymorphisms linked to the H2 resistance on the top end of potato chromosome 5. Allele-specific KASP markers further defined the locus containing the H2 gene to a 4.7 Mb interval on the distal short arm of potato chromosome 5 and to positions that correspond to 1.4 MB and 6.1 MB in the potato reference genome.

Potato late blight field resistance from QTL dPI09c is conferred by the NB-LRR gene R8.

Following the often short-lived protection that major nucleotide binding, leucine-rich-repeat (NB-LRR) resistance genes offer against the potato pathogen Phytophthora infestans, field resistance was thought to provide a more durable alternative to prevent late blight disease. We previously identified the QTL dPI09c on potato chromosome 9 as a more durable field resistance source against late blight. Here, the resistance QTL was fine-mapped to a 186 kb region. The interval corresponds to a larger, 389 kb, genomic region in the potato reference genome of Solanum tuberosum Group Phureja doubled monoploid clone DM1-3 (DM) and from which functional NB-LRRs R8, R9a, Rpi-moc1, and Rpi_vnt1 have arisen independently in wild species. dRenSeq analysis of parental clones alongside resistant and susceptible bulks of the segregating population B3C1HP showed full sequence representation of R8. This was independently validated using long-range PCR and screening of a bespoke bacterial artificial chromosome library. The latter enabled a comparative analysis of the sequence variation in this locus in diverse Solanaceae. We reveal for the first time that broad spectrum and durable field resistance against P. infestans is conferred by the NB-LRR gene R8, which is thought to provide narrow spectrum race-specific resistance.

Identification and rapid mapping of a gene conferring broad-spectrum late blight resistance in the diploid potato species Solanum verrucosum through DNA capture technologies.

KEY MESSAGE: A broad-spectrum late blight disease-resistance gene from Solanum verrucosum has been mapped to potato chromosome 9. The gene is distinct from previously identified-resistance genes. We have identified and characterised a broad-spectrum resistance to Phytophthora infestans from the wild Mexican species Solanum verrucosum. Diagnostic resistance gene enrichment (dRenSeq) revealed that the resistance is not conferred by previously identified nucleotide-binding, leucine-rich repeat genes. Utilising the sequenced potato genome as a reference, two complementary enrichment strategies that target resistance genes (RenSeq) and single/low-copy number genes (Generic-mapping enrichment Sequencing; GenSeq), respectively, were deployed for the rapid, SNP-based mapping of the resistance through bulked-segregant analysis. Both approaches independently positioned the resistance, referred to as Rpi-ver1, to the distal end of potato chromosome 9. Stringent post-enrichment read filtering identified a total of 64 informative SNPs that corresponded to the expected ratio for significant polymorphisms in the parents as well as the bulks. Of these, 61 SNPs are located on potato chromosome 9 and reside within 27 individual genes, which in the sequenced potato clone DM locate to positions 45.9 to 60.9 Mb. RenSeq- and GenSeq-derived SNPs within the target region were converted into allele-specific PCR-based KASP markers and further defined the position of the resistance to a 4.3 Mb interval at the bottom end of chromosome 9 between positions 52.62-56.98 Mb.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease.

Phytophthora infestans RXLR effector PexRD2 interacts with host MAPKKK ε to suppress plant immune signaling.

Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity-related signaling.

An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus.

The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

Relocalization of late blight resistance protein R3a to endosomal compartments is associated with effector recognition and required for the immune response.

An important objective of plant-pathogen interactions research is to determine where resistance proteins detect pathogen effectors to mount an immune response. Many nucleotide binding-Leucine-rich repeat (NB-LRR) resistance proteins accumulate in the plant nucleus following effector recognition, where they initiate the hypersensitive response (HR). Here, we show that potato (Solanum tuberosum) resistance protein R3a relocates from the cytoplasm to endosomal compartments only when coexpressed with recognized Phytophthora infestans effector form AVR3a(KI) and not unrecognized form AVR3a(EM). Moreover, AVR3a(KI), but not AVR3a(EM), is also relocalized to endosomes in the presence of R3a. Both R3a and AVR3a(KI) colocalized in close physical proximity at endosomes in planta. Treatment with brefeldin A (BFA) or wortmannin, inhibitors of the endocytic cycle, attenuated both the relocalization of R3a to endosomes and the R3a-mediated HR. No such effect of these inhibitors was observed on HRs triggered by the gene-for-gene pairs Rx1/PVX-CP and Sto1/IpiO1. An R3a(D501V) autoactive MHD mutant, which triggered HR in the absence of AVR3a(KI), failed to localize to endosomes. Moreover, BFA and wortmannin did not alter cell death triggered by this mutant. We conclude that effector recognition and consequent HR signaling by NB-LRR resistance protein R3a require its relocalization to vesicles in the endocytic pathway.

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 $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 approximately 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 approximately 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.

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.

Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1.

Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a(KI), which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a(EM), which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a(KI/Y147del), a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a(KI) or Avr3a(EM) but not the Avr3a(KI/Y147del) mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.