The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of nonflowering plants retain immune-related functions through translineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to nonflowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable with motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the nonflowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent nonflowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.

Candidate effector proteins from the oomycetes Plasmopara viticola and Phytophthora parasitica share similar predicted structures and induce cell death in Nicotiana species.

Effector proteins secreted by plant pathogens are essential for infection. Cytoplasmic RXLR effectors from oomycetes are characterized by the presence of RXLR and EER motifs that are frequently linked to WY- and/or LWY-domains, folds that are exclusive to this effector family. A related family of secreted candidate effector proteins, carrying WY-domains and the EER motif but lacking the canonical RXLR motif, has recently been described in oomycetes and is mainly found in downy mildew pathogens. Plasmopara viticola is an obligate biotrophic oomycete causing grapevine downy mildew. Here we describe a conserved Pl. viticola secreted candidate non-RXLR effector protein with cell death-inducing activity in Nicotiana species. A similar RXLR effector candidate from the broad host range oomycete pathogen Phytophthora parasitica also induces cell death in Nicotiana. Through comparative tertiary structure modelling, we reveal that both proteins are predicted to carry WY- and LWY-domains. Our work supports the presence of LWY-domains in non-RXLR effectors and suggests that effector candidates with similar domain architecture may exert similar activities.

An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface.

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization.

With its long filaments reaching deep inside its prey, the tiny fungi-like organism known as Phytophthora infestans has had a disproportionate impact on human history. Latching onto plants and feeding on their cells, it has caused large-scale starvation events such as the Irish or Highland potato famines. Many specialized proteins allow the parasite to accomplish its feat. For instance, PexRD54 helps P. infestans hijack a cellular process known as autophagy. Healthy cells use this 'self-eating' mechanism to break down invaders or to recycle their components, for example when they require specific nutrients. The process is set in motion by various pathways of molecular events that result in specific sac-like 'vesicles' filled with cargo being transported to specialized compartments for recycling. PexRD54 can take over this mechanism by activating one of the plant autophagy pathways, directing cells to form autophagic vesicles that Phytophthora could then possibly use to feed on or to destroy antimicrobial components. How or why this is the case remains poorly understood. To examine these questions, Pandey, Leary et al. used a combination of genetic and microscopy techniques and tracked how PexRD54 alters autophagy as P. infestans infects a tobacco-related plant. The results show that PexRD54 works by bridging two proteins: one is present on cellular vesicles filled with cargo, and the other on autophagic structures surrounding the parasite. This allows PexRD54 to direct the vesicles to the feeding sites of P. infestans so the parasite can potentially divert nutrients. Pandey, Leary et al. then went on to develop a molecule called the AIM peptide, which could block autophagy by mimicking part of PexRD54. These results help to better grasp how a key disease affects crops, potentially leading to new ways to protect plants without the use of pesticides. They also shed light on autophagy: ultimately, a deeper understanding of this fundamental biological process could allow the development of plants which can adapt to changing environments.

Deep learning-based quantification of arbuscular mycorrhizal fungi in plant roots.

Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the abundance of hyphal structures in mutant roots rely on staining and human scoring involving simple yet repetitive tasks which are prone to variation between experimenters. We developed Automatic Mycorrhiza Finder (AMFinder) which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks. AMFinder delivered high-confidence predictions on image datasets of roots of multiple plant hosts (Nicotiana benthamiana, Medicago truncatula, Lotus japonicus, Oryza sativa) and captured the altered colonisation in ram1-1, str, and smax1 mutants. A streamlined protocol for sample preparation and imaging allowed us to quantify mycobionts from the genera Rhizophagus, Claroideoglomus, Rhizoglomus and Funneliformis via flatbed scanning or digital microscopy, including dynamic increases in colonisation in whole root systems over time. AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed at https://github.com/SchornacklabSLCU/amfinder.

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation.

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process.

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity.

Phytophthora palmivora is a destructive oomycete plant pathogen with a wide host range. So far, little is known about the factors governing its infection structure development and pathogenicity. From the culture filtrate of a P. palmivora strain isolated from papaya, we identified a secreted glycoprotein of 15 kDa, designated as Ppal15kDa, using liquid chromatography tandem mass spectrometry. Two gene variants, Ppal15kDaA and Ppal15kDaB were amplified from a P. palmivora papaya isolate. Transient expression of both variants in Nicotiana benthamiana by agroinfiltration enhanced P. palmivora infection. Six Ppal15kDa mutants with diverse mutations were generated via CRISPR/Cas9-mediated gene editing. All mutants were compromised in infectivity on N. benthamiana and papaya. Two mutants with all Ppal15kDa copies mutated almost completely lost pathogenicity. The pathogenicity of the other four containing at least one wild-type copy of Ppal15kDa was compromised at varying levels. The mutants were also affected in development as they produced smaller sporangia, shorter germ tubes, and fewer appressoria. The affected levels in development corresponded to the levels of reduction in pathogenicity, suggesting that Ppal15kDa plays an important role in normal development of P. palmivora infection structures. Consistent with its role in infection structure development and pathogenicity, Ppal15kDa was found to be highly induced during appressorium formation. In addition, Ppal15kDa homologs are broadly present in Phytophthora spp., but none were characterized. Altogether, this study identified a novel component involved in development and pathogenicity of P. palmivora and possibly other Phytophthora spp. known to contain a Ppal15kDa homolog.

A secreted WY-domain-containing protein present in European isolates of the oomycete Plasmopara viticola induces cell death in grapevine and tobacco species.

Plasmopara viticola is a biotrophic oomycete pathogen causing grapevine downy mildew. We characterized the repertoire of P. viticola effector proteins which may be translocated into plants to support the disease. We found several secreted proteins that contain canonical dEER motifs and conserved WY-domains but lack the characteristic RXLR motif reported previously from oomycete effectors. We cloned four candidates and showed that one of them, Pv33, induces plant cell death in grapevine and Nicotiana species. This activity is dependent on the nuclear localization of the protein. Sequence similar effectors were present in seven European, but in none of the tested American isolates. Together our work contributes a new type of conserved P. viticola effector candidates.

Conserved Biochemical Defenses Underpin Host Responses to Oomycete Infection in an Early-Divergent Land Plant Lineage.

The expansion of plants onto land necessitated the evolution of robust defense strategies to protect against a wide array of microbial invaders. Whereas host responses to microbial colonization are extensively explored in evolutionarily young land plant lineages such as angiosperms, we know relatively little about plant-pathogen interactions in early-diverging land plants thought to better represent the ancestral state. Here, we define the transcriptional and proteomic response of the early-divergent liverwort Marchantia polymorpha to infection with the oomycete pathogen Phytophthora palmivora. We uncover a robust molecular response to oomycete colonization in Marchantia that consists of conserved land plant gene families. Direct macroevolutionary comparisons of host infection responses in Marchantia and the model angiosperm Nicotiana benthamiana further reveal a shared set of orthologous microbe-responsive genes that include members of the phenylpropanoid metabolic pathway. In addition, we identify a role for the Marchantia R2R3-MYB transcription factor MpMyb14 in activating phenylpropanoid (flavonoid) biosynthesis during oomycete infection. Mpmyb14 mutants infected with P. palmivora fail to activate phenylpropanoid biosynthesis gene expression and display enhanced disease susceptibility compared to wild-type plants. Conversely, the ectopic induction of MpMyb14 led to the accumulation of anthocyanin-like pigments and dramatically enhanced liverwort resistance to P. palmivora infection. Collectively, our results demonstrate that the Marchantia response to oomycete infection displays evolutionarily conserved features indicative of an ancestral pathogen deterrence strategy centered on phenylpropanoid-mediated biochemical defenses.

Glycerol-3-phosphate acyltransferase 6 controls filamentous pathogen interactions and cell wall properties of the tomato and Nicotiana benthamiana leaf epidermis.

The leaf outer epidermal cell wall acts as a barrier against pathogen attack and desiccation, and as such is covered by a cuticle, composed of waxes and the polymer cutin. Cutin monomers are formed by the transfer of fatty acids to glycerol by glycerol-3-phosphate acyltransferases, which facilitate their transport to the surface. The extent to which cutin monomers affect leaf cell wall architecture and barrier properties is not known. We report a dual functionality of pathogen-inducible GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 6 (GPAT6) in controlling pathogen entry and cell wall properties affecting dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to infection by the oomycetes Phytophthora infestans and Phytophthora palmivora, whereas overexpression of NbGPAT6a-GFP rendered leaves more resistant. A loss-of-function mutation in tomato SlGPAT6 similarly resulted in increased susceptibility of leaves to Phytophthora infection, concomitant with changes in haustoria morphology. Modulation of GPAT6 expression altered the outer wall diameter of leaf epidermal cells. Moreover, we observed that tomato gpat6-a mutants had an impaired cell wall-cuticle continuum and fewer stomata, but showed increased water loss. This study highlights a hitherto unknown role for GPAT6-generated cutin monomers in influencing epidermal cell properties that are integral to leaf-microbe interactions and in limiting dehydration.

Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors.

BACKGROUND: Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. RESULTS: We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. CONCLUSIONS: These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

The plant defense and pathogen counterdefense mediated by Hevea brasiliensis serine protease HbSPA and Phytophthora palmivora extracellular protease inhibitor PpEPI10.

Rubber tree (Hevea brasiliensis Muell. Arg) is an important economic crop in Thailand. Leaf fall and black stripe diseases caused by the aggressive oomycete pathogen Phytophthora palmivora, cause deleterious damage on rubber tree growth leading to decrease of latex production. To gain insights into the molecular function of H. brasiliensis subtilisin-like serine proteases, the HbSPA, HbSPB, and HbSPC genes were transiently expressed in Nicotiana benthamiana via agroinfiltration. A functional protease encoded by HbSPA was successfully expressed in the apoplast of N. benthamiana leaves. Transient expression of HbSPA in N. benthamiana leaves enhanced resistance to P. palmivora, suggesting that HbSPA plays an important role in plant defense. P. palmivora Kazal-like extracellular protease inhibitor 10 (PpEPI10), an apoplastic effector, has been implicated in pathogenicity through the suppression of H. brasiliensis protease. Semi-quantitative RT-PCR revealed that the PpEPI10 gene was significantly up-regulated during colonization of rubber tree by P. palmivora. Concurrently, the HbSPA gene was highly expressed during infection. To investigate a possible interaction between HbSPA and PpEPI10, the recombinant PpEPI10 protein (rPpEPI10) was expressed in Escherichia coli and purified using affinity chromatography. In-gel zymogram and co-immunoprecipitation (co-IP) assays demonstrated that rPpEPI10 specifically inhibited and interacted with HbSPA. The targeting of HbSPA by PpEPI10 revealed a defense-counterdefense mechanism, which is mediated by plant protease and pathogen protease inhibitor, in H. brasiliensis-P. palmivora interactions.

Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2.

Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways.

Variation in capsidiol sensitivity between Phytophthora infestans and Phytophthora capsici is consistent with their host range.

Plants protect themselves against a variety of invading pathogenic organisms via sophisticated defence mechanisms. These responses include deployment of specialized antimicrobial compounds, such as phytoalexins, that rapidly accumulate at pathogen infection sites. However, the extent to which these compounds contribute to species-level resistance and their spectrum of action remain poorly understood. Capsidiol, a defense related phytoalexin, is produced by several solanaceous plants including pepper and tobacco during microbial attack. Interestingly, capsidiol differentially affects growth and germination of the oomycete pathogens Phytophthora infestans and Phytophthora capsici, although the underlying molecular mechanisms remain unknown. In this study we revisited the differential effect of capsidiol on P. infestans and P. capsici, using highly pure capsidiol preparations obtained from yeast engineered to express the capsidiol biosynthetic pathway. Taking advantage of transgenic Phytophthora strains expressing fluorescent markers, we developed a fluorescence-based method to determine the differential effect of capsidiol on Phytophtora growth. Using these assays, we confirm major differences in capsidiol sensitivity between P. infestans and P. capsici and demonstrate that capsidiol alters the growth behaviour of both Phytophthora species. Finally, we report intraspecific variation within P. infestans isolates towards capsidiol tolerance pointing to an arms race between the plant and the pathogens in deployment of defence related phytoalexins.

Interactions of beneficial and detrimental root-colonizing filamentous microbes with plant hosts.

Understanding commonalities and differences of how symbiotic and parasitic microbes interact with plants will improve advantageous interactions and allow pathogen control strategies in crops. Recently established systems enable studies of root pathogenic and symbiotic interactions in the same plant species.

The Irish potato famine pathogen Phytophthora infestans translocates the CRN8 kinase into host plant cells.

Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8(R469A;D470A) resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface.

In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.

Phosphatidylinositol monophosphate-binding interface in the oomycete RXLR effector AVR3a is required for its stability in host cells to modulate plant immunity.

The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steady-state levels of non-PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1-dependent immunity.

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana.

BACKGROUND: The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. PRINCIPAL FINDINGS: We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. CONCLUSIONS/SIGNIFICANCE: We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.