Heterologous overexpression of StERF3 triggers cell death in Nicotiana benthamiana.

Programmed cell death plays a crucial role in plant development and disease defense. Here, we report that the expression of StERF3, a potato EAR motif-containing transcription factor, promotes Phytophthora infestans colonization in Nicotiana benthamiana. Transient overexpression of StERF3 induces cell death in N. benthamiana leaves. The substitution of two key amino acids (14th and 19th) in its ERF domain (the DNA binding domain) dramatically altered its cell death-inducing ability. In addition, StERF3△EAR EAR motif-deletion or StERF3AAA mutation abolished the cell death-inducing ability. StERF3 interacted with the co-repressors Topless-related protein 1 (StTPL1) and Topless-related protein 3 (StTPL3) via the EAR motif. Moreover, cell death induced by StERF3 was facilitated by co-expression with StTPL1 or StTPL3. Virus-induced gene silencing (VIGS) of NbTPL1 and NbTPL3 in N. benthamiana compromised the cell death-inducing ability of StERF3. Furthermore, StERF3-induced cell death accompanied with ROS bursts and the upregulation of the respiratory burst oxidase homolog (Rboh) genes NbRbohA and NbRbohC. In addition, several cell death regulator genes, including NbCRTD, NbNCBP, and NbBCPL, and a hypersensitive cell death marker gene Hin1 were upregulated. StERF3 may positively regulate cell death through its EAR motif-mediated transcriptional repressor activity by inhibiting the expression of genes potentially coding the repressor of cell death (CD).

NLR immune receptor RB is differentially targeted by two homologous but functionally distinct effector proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) receptors mediate immune responses by directly or indirectly sensing pathogen-derived effectors. Despite significant advances in the understanding of NLR-mediated immunity, the mechanisms by which pathogens evolve to suppress NLR activation triggered by cognate effectors and gain virulence remain largely unknown. The agronomically important immune receptor RB recognizes the ubiquitous and highly conserved IPI-O RXLR family members (e.g., IPI-O1) from Phytophthora infestans, and this process is suppressed by the rarely present and homologous effector IPI-O4. Here, we report that self-association of RB via the coiled-coil (CC) domain is required for RB activation and is differentially affected by avirulence and virulence effectors. IPI-O1 moderately reduces the self-association of RB CC, potentially leading to changes in the conformation and equilibrium of RB, whereas IPI-O4 dramatically impairs CC self-association to prevent RB activation. We also found that IPI-O1 associates with itself, whereas IPI-O4 does not. Notably, IPI-O4 interacts with IPI-O1 and disrupts its self-association, therefore probably blocking its avirulence function. Furthermore, IPI-O4 enhances the interaction between RB CC and IPI-O1, possibly sequestering RB and IPI-O1 and subsequently blocking their interactions with signaling components. Taken together, these findings considerably extend our understanding of the underlying mechanisms by which emerging virulent pathogens suppress the NLR-mediated recognition of cognate effectors.

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

Mining oomycete proteomes for metalloproteases leads to identification of candidate virulence factors in Phytophthora infestans.

Pathogens deploy a wide range of pathogenicity factors, including a plethora of proteases, to modify host tissue or manipulate host defences. Metalloproteases (MPs) have been implicated in virulence in several animal and plant pathogens. Here we investigated the repertoire of MPs in 46 stramenopile species including 37 oomycetes, 5 diatoms, and 4 brown algae. Screening their complete proteomes using hidden Markov models (HMMs) trained for MP detection resulted in over 4,000 MPs, with most species having between 65 and 100 putative MPs. Classification in clans and families according to the MEROPS database showed a highly diverse MP repertoire in each species. Analyses of domain composition, orthologous groups, distribution, and abundance within the stramenopile lineage revealed a few oomycete-specific MPs and MPs potentially related to lifestyle. In-depth analyses of MPs in the plant pathogen Phytophthora infestans revealed 91 MPs, divided over 21 protein families, including 25 MPs with a predicted signal peptide or signal anchor. Expression profiling showed different patterns of MP gene expression during pre-infection and infection stages. When expressed in leaves of Nicotiana benthamiana, 12 MPs changed the sizes of lesions caused by inoculation with P. infestans; with 9 MPs the lesions were larger, suggesting a positive effect on the virulence of P. infestans, while 3 MPs had a negative effect, resulting in smaller lesions. To the best of our knowledge, this is the first systematic inventory of MPs in oomycetes and the first study pinpointing MPs as potential pathogenicity factors in Phytophthora.

A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms.

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

Cadmium Stress Reprograms ROS/RNS Homeostasis in Phytophthora infestans (Mont.) de Bary.

Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.

Genome-wide identification of Argonautes in Solanaceae with emphasis on potato.

Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.

Divergent Evolution of PcF/SCR74 Effectors in Oomycetes Is Associated with Distinct Recognition Patterns in Solanaceous Plants.

Plants deploy cell surface receptors known as pattern-recognition receptors (PRRs) that recognize non-self molecules from pathogens and microbes to defend against invaders. PRRs typically recognize microbe-associated molecular patterns (MAMPs) that are usually widely conserved, some even across kingdoms. Here, we report an oomycete-specific family of small secreted cysteine-rich (SCR) proteins that displays divergent patterns of sequence variation in the Irish potato famine pathogen Phytophthora infestans A subclass that includes the conserved effector PcF from Phytophthora cactorum activates immunity in a wide range of plant species. In contrast, the more diverse SCR74 subclass is specific to P. infestans and tends to trigger immune responses only in a limited number of wild potato genotypes. The SCR74 response was recently mapped to a G-type lectin receptor kinase (G-LecRK) locus in the wild potato Solanum microdontum subsp. gigantophyllum. The G-LecRK locus displays a high diversity in Solanum host species compared to other solanaceous plants. We propose that the diversification of the SCR74 proteins in P. infestans is driven by a fast coevolutionary arms race with cell surface immune receptors in wild potato, which contrasts the presumed slower dynamics between conserved apoplastic effectors and PRRs. Understanding the molecular determinants of plant immune responses to these divergent molecular patterns in oomycetes is expected to contribute to deploying multiple layers of disease resistance in crop plants.IMPORTANCE Immune receptors at the plant cell surface can recognize invading microbes. The perceived microbial molecules are typically widely conserved and therefore the matching surface receptors can detect a broad spectrum of pathogens. Here we describe a family of Phytophthora small extracellular proteins that consists of conserved subfamilies that are widely recognized by solanaceous plants. Remarkably, one subclass of SCR74 proteins is highly diverse, restricted to the late blight pathogen Phytophthora infestans and is specifically detected in wild potato plants. The diversification of this subfamily exhibits signatures of a coevolutionary arms race with surface receptors in potato. Insights into the molecular interaction between these potato-specific receptors and the recognized Phytophthora proteins are expected to contribute to disease resistance breeding in potato.

Phytophthora Effectors Modulate Genome-wide Alternative Splicing of Host mRNAs to Reprogram Plant Immunity.

Alternative splicing (AS) of pre-mRNAs increases transcriptome and proteome diversity, regulates gene expression through multiple mechanisms, and plays important roles in plant development and stress responses. However, the prevalence of genome-wide plant AS changes during infection and the mechanisms by which pathogens modulate AS remain poorly understood. Here, we examined the global AS changes in tomato leaves infected with Phytophthora infestans, the infamous Irish famine pathogen. We show that more than 2000 genes exhibiting significant changes in AS are not differentially expressed, indicating that AS is a distinct layer of transcriptome reprogramming during plant-pathogen interactions. Furthermore, our results show that P. infestans subverts host immunity by repressing the AS of positive regulators of plant immunity and promoting the AS of susceptibility factors. To study the underlying mechanism, we established a luminescence-based AS reporter system in Nicotiana benthamiana to screen pathogen effectors modulating plant AS. We identified nine splicing regulatory effectors (SREs) from 87 P. infestans effectors. Further studies revealed that SRE3 physically binds U1-70K to manipulate the plant AS machinery and subsequently modulates AS-mediated plant immunity. Our study not only unveils genome-wide plant AS reprogramming during infection but also establishes a novel AS screening tool to identify SREs from a wide range of plant pathogens, providing opportunities to understand the splicing regulatory mechanisms through which pathogens subvert plant immunity.

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.

AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity.

To be successful plant pathogens, microbes use "effector proteins" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant-pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virus-induced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.

Comparative transcriptome analysis shows the defense response networks regulated by miR482b.

KEY MESSAGE: The transcriptomic profile in the leaves of miR482b-overexpressing tomato plants revealed that miR482b may suppress alpha-linolenic acid metabolism, cysteine and methionine metabolism, plant-pathogen interaction, and the MAPK pathway to reduce resistance to Phytophthora infestans. Our previous study showed that tomato miR482b acted as a negative regulator during tomato resistance to Phytophthora infestans by silencing NBS-LRR genes. To investigate pathways related to miR482b, the transcriptomic profile of tomato plants that overexpressed miR482b was constructed. A total of 47,124,670 raw sequence reads from the leaves of miR482b-overexpressing tomato plants were generated by Illumina sequencing. A total of 746 genes in miR482b-overexpressing tomato plants were found to show significantly differential expression relative to those in wild-type tomato plants, including 132 up-regulated genes and 614 down-regulated genes. GO and KEGG enrichment analyses showed that plant-pathogen interaction, the MAPK pathway, and the pathways related to JA and ET biosynthesis were affected by miR482b in tomato. qRT-PCR results showed that all the enriched genes in these pathways were down-regulated in tomato plants that overexpressed miR482b and up-regulated in tomato plants that overexpressed an NBS-LRR gene (Soly02g036270.2, the target gene of miR482b). After P. infestans infection, the expression of the enriched genes showed a time-dependent response, and the genes played different roles between resistant tomato (Solanum pimpinellifolium L3708) and tomato susceptible to P. infestans (S. lycopersicum Zaofen No. 2). Our results have, therefore, demonstrated that miR482b is an important component of defense response network. This will also help to identify candidate genes involved in plant-pathogen interaction.

Phytophthora infestans RXLR effectors act in concert at diverse subcellular locations to enhance host colonization.

Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP-PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP-RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.

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.

Phytophthora infestans small phospholipase D-like proteins elicit plant cell death and promote virulence.

The successful invasion of host tissue by (hemi-)biotrophic plant pathogens is dependent on modifications of the host plasma membrane to facilitate the two-way transfer of proteins and other compounds. Haustorium formation and the establishment of extrahaustorial membranes are probably dependent on a variety of enzymes that modify membranes in a coordinated fashion. Phospholipases, enzymes that hydrolyse phospholipids, have been implicated as virulence factors in several pathogens. The oomycete Phytophthora infestans is a hemibiotrophic pathogen that causes potato late blight. It possesses different classes of phospholipase D (PLD) proteins, including small PLD-like proteins with and without signal peptide (sPLD-likes and PLD-likes, respectively). Here, we studied the role of sPLD-like-1, sPLD-like-12 and PLD-like-1 in the infection process. They are expressed in expanding lesions on potato leaves and during in vitro growth, with the highest transcript levels in germinating cysts. When expressed in planta in the presence of the silencing suppressor P19, all three elicited a local cell death response that was visible at the microscopic level as autofluorescence and strongly boosted in the presence of calcium. Moreover, inoculation of leaves expressing the small PLD-like genes resulted in increased lesion growth and greater numbers of sporangia, but this was abolished when mutated PLD-like genes were expressed with non-functional PLD catalytic motifs. These results show that the three small PLD-likes are catalytically active and suggest that their enzymatic activity is required for the promotion of virulence, possibly by executing membrane modifications to support the growth of P. infestans in the host.

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants.

RXLR effectors encoded by Phytophthora species play a central role in pathogen-plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.

Oxathiapiprolin-based fungicides provide enhanced control of tomato late blight induced by mefenoxam-insensitive Phytophthora infestans.

Oxathiapiprolin is a new fungicide with extremely high efficacy against oomycete plant pathogens. Solo components oxathiapiprolin (OXPT), chlorothalonil (CHT), azoxystrobin (AZ), mandipropamid (MPD), and mefenoxam (MFX) were compared with each other and with four oxathiapiprolin pre-packed fungicidal mixtures, OXPT+CHT 1+66.7, OXPT+AZ 1+10.3, OXPT+MPD 1+8.3, and OXPT+MFX 1+3 (weight active ingredient ratio), for control efficacy of late blight induced by MFX-insensitive Phytophthora infestans strains in tomato in growth chambers and the field. Mixtures performed better than all partner fungicides alone, except OXPT. Of the four mixtures, OXPT+MFX outperformed, with the highest preventive, curative, translaminar, and systemic efficacies. In the field, OXPT+MFX was superior to other fungicides in controlling late blight epidemics induced by MFX-insensitive isolates. Its deployment in the field will combat the dominating MFX-insensitive isolates, reduce the selection pressure imposed on P. infestans and delay the buildup of subpopulations resistant to oxathiapiprolin.

Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen.

During plant cell invasion, the oomycete Phytophthora infestans remains enveloped by host-derived membranes whose functional properties are poorly understood. P. infestans secretes a myriad of effector proteins through these interfaces for plant colonization. Recently we showed that the effector protein PexRD54 reprograms host-selective autophagy by antagonising antimicrobial-autophagy receptor Joka2/NBR1 for ATG8CL binding (Dagdas et al., 2016). Here, we show that during infection, ATG8CL/Joka2 labelled defense-related autophagosomes are diverted toward the perimicrobial host membrane to restrict pathogen growth. PexRD54 also localizes to autophagosomes across the perimicrobial membrane, consistent with the view that the pathogen remodels host-microbe interface by co-opting the host autophagy machinery. Furthermore, we show that the host-pathogen interface is a hotspot for autophagosome biogenesis. Notably, overexpression of the early autophagosome biogenesis protein ATG9 enhances plant immunity. Our results implicate selective autophagy in polarized immune responses of plants and point to more complex functions for autophagy than the widely known degradative roles.

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.