The secreted PAMP-induced peptide StPIP1_1 activates immune responses in potato.

Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

The potato StMKK5-StSIPK module enhances resistance to Phytophthora pathogens through activating the salicylic acid and ethylene signalling pathways.

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant responses to both biotic and abiotic stress. A screen of a Nicotiana benthamiana cDNA virus-induced gene silencing (VIGS) library for altered plant responses to inoculation with Phytophthora infestans previously identified an NbMKK gene, encoding a clade D MAPKK that we renamed as NbMKK5, which is involved in immunity to P. infestans. To study the role of the potato orthologous gene, referred to as StMKK5, in the response to P. infestans, we transiently overexpressed StMKK5 in N. benthamiana and observed that cell death occurred at 2 days postinfiltration. Silencing of the highly conserved eukaryotic protein SGT1 delayed the StMKK5-induced cell death, whereas silencing of the MAPK-encoding gene NbSIPK completely abolished the cell death response. Further investigations showed that StMKK5 interacts with, and directly phosphorylates, StSIPK. Furthermore, both StMKK5 and StSIPK trigger salicylic acid (SA)- and ethylene (Eth)-related gene expression, and co-expression of the salicylate hydroxylase NahG with the negative regulator of Eth signalling CTR1 hampers StSIPK-triggered cell death. This observation indicates that the cell death triggered by StMKK5-StSIPK is dependent on the combination of SA- and Eth-signalling. By introducing point mutations, we showed that the kinase activity of both StMKK5 and StSIPK is required for triggering cell death. Genetic analysis showed that StMKK5 depends on StSIPK to trigger plant resistance. Thus, our results define a potato StMKK5-SIPK module that positively regulates immunity to P. infestans via activation of both the SA and Eth signalling pathways.

Phytophthora infestans RxLR Effector PITG06478 Hijacks 14-3-3 to Suppress PMA Activity Leading to Necrotrophic Cell Death.

Pathogens often induce cell death for their successful proliferation in the host plant. Plasma membrane H+-ATPases (PMAs) are targeted by either pathogens or plant immune receptors in immune response regulation. Although PMAs play pivotal roles in host cell death, the molecular mechanism of effector-mediated regulation of PMA activity has not been described. Here, we report that the Phytophthora infestans RxLR effector PITG06478 can induce cell death in Nicotiana benthamiana but the induced cell death is inhibited by fusicoccin (FC), an irreversible PMA activator. PITG06478, which is localized at the plasma membrane, is not directly associated with the PMA but is associated with Nb14-3-3s, a PMA activator. Immunoblot analyses revealed that the interaction between PITG06478 and Nb14-3-3s was disrupted by FC. PMA activity in PITG06478-expressing plants was eventually inhibited, and cell death likely occurred because the 14-3-3 protein was hijacked. Our results further confirm the significance of PMA activity in host cell death and provide new insight into how pathogens utilize essential host components to sustain their life cycle. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Dynamic localization of a helper NLR at the plant-pathogen interface underpins pathogen recognition.

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet, the subcellular localization of NLRs pre- and postactivation during pathogen infection remains poorly understood. Here, we show that NRC4, from the "NRC" solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extrahaustorial membrane (EHM), presumably to mediate response to perihaustorial effectors that are recognized by NRC4-dependent sensor NLRs. However, not all NLRs accumulate at the EHM, as the closely related helper NRC2 and the distantly related ZAR1 did not accumulate at the EHM. NRC4 required an intact N-terminal coiled-coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that postactivation, NRC4 may undergo a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection, however, NRC4 forms puncta mainly at the EHM and, to a lesser extent, at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

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.

Potato StMPK7 is a downstream component of StMKK1 and promotes resistance to the oomycete pathogen Phytophthora infestans.

A cascade formed by phosphorylation events of mitogen-activated protein kinases (MAPKs) takes part in plant stress responses. However, the roles of these MAPKs in resistance of potato (Solanum tuberosum) against Phytophthora pathogens is not well studied. Our previous work showed that a Phytophthora infestans RXLR effector targets and stabilizes the negative regulator of MAPK kinase 1 of potato (StMKK1). Because in Arabidopsis thaliana the AtMPK4 is the downstream phosphorylation target of AtMKK1, we performed a phylogenetic analysis and found that potato StMPK4/6/7 are closely related and are orthologs of AtMPK4/5/11/12. Overexpression of StMPK4/7 enhances plant resistance to P. infestans and P. parasitica. Yeast two-hybrid analysis revealed that StMPK7 interacts with StMKK1, and StMPK7 is phosphorylated on flg22 treatment and by expressing constitutively active StMKK1 (CA-StMKK1), indicating that StMPK7 is a direct downstream signalling partner of StMKK1. Overexpression of StMPK7 in potato enhances potato resistance to P. infestans. Constitutively active StMPK7 (CA-StMPK7; StMPK7D198G, E202A ) was found to promote immunity to Phytophthora pathogens and to trigger host cell death when overexpressed in Nicotiana benthamiana leaves. Cell death triggered by CA-StMPK7 is SGT1/RAR1-dependent. Furthermore, cell death triggered by CA-StMPK7 is suppressed on coexpression with the salicylate hydroxylase NahG, and StMPK7 activation promotes salicylic acid (SA)-responsive gene expression. We conclude that potato StMPK7 is a downstream signalling component of the phosphorelay cascade involving StMKK1 and StMPK7 plays a role in immunity to Phytophthora pathogens via an SA-dependent signalling pathway.

A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.

Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported. We discovered that it is difficult to express CRISPR/Cas9 but not a catalytically inactive form in transformants, suggesting that the active nuclease is toxic. We were able to achieve editing with CRISPR/Cas12a using vectors in which the nuclease and its guide RNA were expressed from a single transcript. Using the elicitor gene Inf1 as a target, we observed editing of one or both alleles in up to 13% of transformants. Editing was more efficient when guide RNA processing relied on the Cas12a direct repeat instead of ribozyme sequences. INF1 protein was not made when both alleles were edited in the same transformant, but surprisingly also when only one allele was altered. We discovered that the isolate used for editing, 1306, exhibited monoallelic expression of Inf1 due to insertion of a copia-like element in the promoter of one allele. The element exhibits features of active retrotransposons, including a target site duplication, long terminal repeats, and an intact polyprotein reading frame. Editing occurred more often on the transcribed allele, presumably due to differences in chromatin structure. The Cas12a system not only provides a tool for modifying genes in P. infestans, but also for other members of the genus by expanding the number of editable sites. Our work also highlights a natural mechanism that remodels oomycete genomes.

Biocontrol mechanism of Myxococcus fulvus B25-I-3 against Phytophthora infestans and its control efficiency on potato late blight.

Phytophthora infestans is the pathogen of potato late blight, which is one of the most serious diseases of the potato. Myxobacteria, especially Myxococcus, become a valuable biological control resource due to their preponderant abilities to produce various secondary metabolites with novel structure and remarkable biological activity. In a previous study, Myxococcus fulvus B25-I-3 with antagonistic activity against P. infestans was isolated from an environmental sample by rabbit fecal induction method. The biocontrol mechanism of M. fulvus B25-I-3 against P. infestans and its control efficiency on potato late blight were studied. The results showed that the active substances produced by strain B25-I-3 had strong inhibitory effect on the asexual reproduction and sexual reproduction of P. infestans. In addition, the active substances could reduce the content of soluble proteins and the activity of the protective enzymes (polyphenol oxidase, peroxidase, phenylalanine ammonia lyase, superoxide dismutase) in P. infestans and increase the oxidative damage and permeability of cell membrane. And the active substances could inhibit the infection of the detached potato leaves by P. infestans significantly. In conclusion, M. fulvus B25-I-3 can produce active substances against P. infestans and has potential value to develop into biological pesticides for the control of potato late blight. The completion of this work may provide basic data for the isolation and identification of active substances and the development of pesticides against potato late blight.

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 nonspecific lipid transfer protein, StLTP10, mediates resistance to Phytophthora infestans in potato.

Nonspecific lipidtransfer proteins (nsLTPs), which are small, cysteine-rich proteins, belong to the pathogenesis-related protein family, and several of them act as positive regulators during plant disease resistance. However, the underlying molecular mechanisms of these proteins in plant immune responses are unclear. In this study, a typical nsLTP gene, StLTP10, was identified and functionally analysed in potato. StLTP10 expression was significantly induced by Phytophthora infestans, which causes late blight in potato, and defence-related phytohormones, including abscisic acid (ABA), salicylic acid, and jasmonic acid. Characterization of StLTP10-overexpressing and knockdown lines indicated that StLTP10 positively regulates plant resistance to P. infestans. This resistance was coupled with enhanced expression of reactive oxygen species scavenging- and defence-related genes. Furthermore, we identified that StLTP10 physically interacts with ABA receptor PYL4 and affects its subcellular localization. These two proteins work together to regulate stomatal closure during pathogen infection. Interestingly, we also found that wound-induced protein kinase interacts with StLTP10 and positively regulates its protein abundance. Taken together, our results provide insight into the role of StLTP10 in resistance to P. infestans and suggest candidates to enhance broad-spectrum resistance to pathogens in potato.

Transcriptome analysis reveals that exogenous ethylene activates immune and defense responses in a high late blight resistant potato genotype.

Ethylene (ET) is one of the many important signaling hormones that functions in regulating defense responses in plants. Gene expression profiling was conducted under exogenous ET application in the high late blight resistant potato genotype SD20 and the specific transcriptional responses to exogenous ET in SD20 were revealed. Analysis of differentially expressed genes (DEGs) generated a total of 1226 ET-specific DEGs, among which transcription factors, kinases, defense enzymes and disease resistance-related genes were significantly differentially expressed. GO enrichment and KEGG metabolic pathway analysis also revealed that numerous defense regulation-related genes and defense pathways were significantly enriched. These results were consistent with the interaction of SD20 and Phytophthora infestans in our previous study, indicating that exogenous ET stimulated the defense response and initiated a similar defense pathway compared to pathogen infection in SD20. Moreover, multiple signaling pathways including ET, salicylic acid, jasmonic acid, abscisic acid, auxin, cytokinin and gibberellin were involved in the response to exogenous ET, which indicates that many plant hormones work together to form a complex network to resist external stimuli in SD20. ET-induced gene expression profiling provides insights into the ET signaling transduction pathway and its potential mechanisms in disease defense systems in potato.

StRac1 plays an important role in potato resistance against Phytophthora infestans via regulating H2O2 production.

ROP GTPases (Rho-related GTPases from plant), a unique subgroup of the Rho family in plants, is a group of key regulators of different signaling pathways controlling plant growth and development, cell polarity and differentiation, and plant response against biotic and abiotic stresses. The present study determined the potential regulatory mechanism of potato ROP GTPase (StRac1) against Phytophthora infestans (P. infestans) infection. Protein secondary structure analysis indicated that StRAC1 is a Rho GTPase. The expression level of StRac1 was variable in different tissues of potato, with the highest expression in young leaves of both Shepody and Hutou potato varieties. After challenging with P. infestans, the expression level of StRac1was higher in resistance varieties Zihuabai and Longshu 7 than in susceptible varieties Shepody and Desiree. StRAC1 fusion with GFP subcellularly localized at the plasma membrane (PM) in tobacco epidermal cells. The potato with transient or stable over-expression of CA-StRac1 (constitutively active form of StRac1)exhibited a dramatic enhancement of its resistance against P. infestans infections. The increased resistance level in transgenic potato was accompanied with elevated H2O2 levels. Importantly, silencing StRac1 via virus-induced gene silencing (VIGS) in potato resulted in higher susceptibility to P. infestans infection than in control plants. In summary, our data reveal that StRac1 regulates potato resistance against P. infestans via positively modulating the accumulation of H2O2.

Transcriptional induction of capsidiol synthesis genes by wounding can promote pathogen signal-induced capsidiol synthesis.

BACKGROUND: Plants are exposed to various forms of environmental stress. Penetration by pathogens is one of the most serious environmental insults. Wounding caused by tissue damage or herbivory also affects the growth and reproduction of plants. Moreover, wounding disrupts physical barriers present at the plant surface and increases the risk of pathogen invasion. Plants cope with environmental stress by inducing a variety of responses. These stress responses must be tightly controlled, because their unnecessary induction is detrimental to plant growth. In tobacco, WIPK and SIPK, two wound-responsive mitogen-activated protein kinases, have been shown to play important roles in regulating wound responses. However, their contribution to downstream wound responses such as gene expression is not well understood. RESULTS: To identify genes regulated by WIPK and SIPK, the transcriptome of wounded WIPK/SIPK-suppressed plants was analyzed. Among the genes down-regulated in WIPK/SIPK-suppressed plants, the largest group consisted of those involved in the production of antimicrobial phytoalexins. Almost all genes involved in the biosynthesis of capsidiol, a major phytoalexin in tobacco, were transcriptionally induced by wounding in WIPK/SIPK-dependent and -independent manners. 5-epi-aristolochene synthase (EAS) is the committing enzyme for capsidiol synthesis, and the promoter of EAS4, a member of the EAS family, was analyzed. Reporter gene analysis revealed that at least two regions each 40-50 bp length were involved in activation of the EAS4 promoter by wounding, as well as by artificial activation of WIPK and SIPK. Unlike transcripts of the capsidiol synthesis genes, accumulation of EAS protein and capsidiol itself were not induced by wounding; however, wounding significantly enhanced their subsequent induction by a pathogen-derived elicitor. CONCLUSIONS: Our results suggest a so-called priming phenomenon since the induction of EAS by wounding is only visible at the transcript level. By inducing transcripts, not the proteins, of EAS and possibly other capsidiol synthesis genes at wound sites, plants can produce large quantities of capsidiol quickly if pathogens invade the wound site, whereas plants can minimize energy loss and avoid the cytotoxic effects of capsidiol where pathogens do not gain entry during wound healing.

Transgenic RXLR Effector PITG_15718.2 Suppresses Immunity and Reduces Vegetative Growth in Potato.

Phytophthora infestans causes the severe late blight disease of potato. During its infection process, P. infestans delivers hundreds of RXLR (Arg-x-Leu-Arg, x behalf of any one amino acid) effectors to manipulate processes in its hosts, creating a suitable environment for invasion and proliferation. Several effectors interact with host proteins to suppress host immunity and inhibit plant growth. However, little is known about how P. infestans regulates the host transcriptome. Here, we identified an RXLR effector, PITG_15718.2, which is upregulated and maintains a high expression level throughout the infection. Stable transgenic potato (Solanum tuberosum) lines expressing PITG_15718.2 show enhanced leaf colonization by P. infestans and reduced vegetative growth. We further investigated the transcriptional changes between three PITG_15718.2 transgenic lines and the wild type Désirée by using RNA sequencing (RNA-Seq). Compared with Désirée, 190 differentially expressed genes (DEGs) were identified, including 158 upregulated genes and 32 downregulated genes in PITG_15718.2 transgenic lines. Eight upregulated and nine downregulated DEGs were validated by real-time RT-PCR, which showed a high correlation with the expression level identified by RNA-Seq. These DEGs will help to explore the mechanism of PITG_15718.2-mediated immunity and growth inhibition in the future.

A potato STRUBBELIG-RECEPTOR FAMILY member, StLRPK1, associates with StSERK3A/BAK1 and activates immunity.

Plant STRUBBELIG (SUB)-RECEPTOR FAMILY (SRF) genes encode putative leucine-rich repeat transmembrane receptor-like kinases. SRFs have been reported to play essential roles in tissue morphogenesis in many plant organs. Here, we show that a potato SRF family gene, StLRPK1, is involved in plant immunity. StLRPK1 is located at the cell plasma membrane and is strongly induced by culture filtrate from in vitro growth of the late blight pathogen Phytophthora infestans. Overexpression of StLRPK1 in stable transgenic potato or ectopic expression in Nicotiana benthamiana plants enhances P. infestans disease resistance, whereas RNA interference (RNAi) of StLRPK1 in potato decreases disease resistance. We found that StLRPK1 constitutively interacts with a pivotal co-receptor, SERK3A/BAK1, which plays a central role in plant immunity. Virus-induced gene silencing of SERK3A/BAK1 in N. benthamiana lines expressing StLRPK1 attenuated P. infestans resistance, indicating that SERK3A/BAK1 is required for StLRPK1-mediated immunity. Finally, we show that StLRPK1-triggered late blight resistance depends on the mitogen-activated protein kinase kinase MEK2 and mitogen-activated protein kinase WIPK. We propose a model in which StLRPK1 associates with SERK3A/BAK1 to positively regulate plant immunity to P. infestans through a MAPK cascade. These data provide new insights into our understanding of SRF function in plant immunity.

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.

PAMP-responsive ATL gene StRFP1 and its orthologue NbATL60 positively regulate Phytophthora infestans resistance in potato and Nicotiana benthamiana.

Ubiquitination is a post-translational modification that plays a crucial role during the regulation of plant immune signalling. The plant ATL family consists of a large number of putative RING type ubiquitin ligases. We show that potato ATL family gene StRFP1 and its orthologue NbATL60 from N. benthamiana both respond to Phytophthora infestans culture filtrate (CF) and flg22 induction. StRFP1 positively regulates immunity against P. infestans in potato. Ectopic transient expression of StRFP1 or expression of NbATL60 in N. benthamiana also enhances late blight resistance. By contrast, silencing NbATL60 in N. benthamiana reduces late blight resistance and leads to plant growth inhibition. Both StRFP1 and NbATL60 localize to the plasma membrane and intracellular puncta and possess E3 Ligase activity in vitro. Furthermore we demonstrate that the RING finger domain mutants of StRFP1 and NbATL60 lost E3 ligase activity and fail to suppress P. infestans colonization in N. benthamiana, indicating that E3 ligase activity is critical for StRFP1 and NbATL60 to regulate immunity. Overexpression or RNA interference of StRFP1 in transgenic potato led to increased or decreased expression of PTI maker genes (WRKY7, WRKY8, ACRE31 and Pti5) respectively. Similarly silencing of NbATL60 in N. benthamiana decreases expression of these PTI marker genes. Moreover, VIGS of NbATL60 in N. benthamiana did not compromise P. infestans PAMP INF1 or R2/Avr2, R3a/AVR3a, Rx/Cp and Pto/AvrPto triggered cell death. These results indicate that ATL genes StRFP1 and NbATL60 contribute to basal immunity (PTI) in Solanaceous plants.

Solanaceous exocyst subunits are involved in immunity to diverse plant pathogens.

The exocyst, a multiprotein complex consisting of eight subunits, plays an essential role in many biological processes by mediating secretion of post-Golgi-derived vesicles towards the plasma membrane. In recent years, roles for plant exocyst subunits in pathogen defence have been uncovered, largely based on studies in the model plant Arabidopsis. Only a few studies have been undertaken to assign the role of exocyst subunits in plant defence in other plants species, including crops. In this study, predicted protein sequences from exocyst subunits were retrieved by mining databases from the Solanaceous plants Nicotiana benthamiana, tomato, and potato. Subsequently, their evolutionary relationship with Arabidopsis exocyst subunits was analysed. Gene silencing in N. benthamiana showed that several exocyst subunits are required for proper plant defence against the (hemi-)biotrophic plant pathogens Phytophthora infestans and Pseudomonas syringae. In contrast, some exocyst subunits seem to act as susceptibility factors for the necrotrophic pathogen Botrytis cinerea. Furthermore, the majority of the exocyst subunits were found to be involved in callose deposition, suggesting that they play a role in basal plant defence. This study provides insight into the evolution of exocyst subunits in Solanaceous plants and is the first to show their role in immunity against multiple unrelated pathogens.

HSP70s Enhance a Phytophthora infestans Effector-Induced Cell Death via an MAPK Cascade in Nicotiana benthamiana.

A destructive pathogen, Phytophthora infestans, secretes hundreds of effectors for successful survival in its host plants. The effectors modulate the plant defense system at diverse cellular compartments to take an advantage of pathogen survivals. A few research studies have shown the mode of action of each effector and their interacting proteins in plant cells. Here, we investigated the mode of action of a P. infestans effector, Pi23226, which induces cell death in Nicotiana benthamiana. To identify its host factors, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry, and selected members of heat shock protein 70 (HSP70s) as candidates. These HSP70s, known to function as chaperones, were associated with Pi23226 in planta and accelerated Pi23226-induced cell death. Additionally, they were found to be involved in plant basal defense by suppressing the growth of P. infestans. We also found that specific components of a mitogen-activated protein kinase cascade were involved in Pi23226-induced cell death. Our findings show that HSP70s functions in defense systems by regulating effector-triggered cell death and by suppressing the growth of the pathogen. This suggests that host plants manipulate the ubiquitous proteins to detect pathogen effectors for functioning in the defense system.