RXLR Effector Research Articles

Pathogenesis-related protein 1 suppresses oomycete pathogen by targeting against AMPK kinase complex

IntroductionDuring the arms race between plants and pathogens, pathogenesis-related proteins (PR) in host plants play a crucial role in disease resistance, especially PR1. PR1 constitute a secretory peptide family, and their role in plant defense has been widely demonstrated in both hosts and in vitro. However, the mechanisms by which they control host-pathogen interactions and the nature of their targets within the pathogen remain poorly understood. ObjectivesThe present study was aimed to investigate the anti-oomycete activity of secretory PR1 proteins and elaborate their underlying mechanisms. MethodsThis study was conducted in the potato-Phytophthora infestans pathosystem. After being induced by the pathogen infection, the cross-kingdom translocation of secretory PR1 was demonstrated by histochemical assays and western blot, and their targets in P. infestans were identified by yeast-two-hybrid assays, bimolecular fluorescence complementation assays, and co-immunoprecipitation assay. ResultsThe results showed that the expression of secretory PR1-encoding genes was induced during pathogen infection, and the host could deliver PR1 into P. infestans to inhibit its vegetative growth and pathogenicity. The translocated secretory PR1 targeted the subunits of the AMPK kinase complex in P. infestans, thus affecting the AMPK-driven phosphorylation of downstream target proteins, preventing ROS homeostasis, and down-regulating the expression of RxLR effectors. ConclusionThe results provide novel insights into the molecular function of PR1 in protecting plants against pathogen infection, and uncover a potential target for preventing pre- and post-harvest late blight.

Pathogenicity factors of Phytophthora melonis revealed by comparative proteomics

ABSTRACT Deciphering the transcriptional activities of pathogenicity genes is critical to understand pathogenesis mechanisms of Phytophthora melonis, a devastating pathogen of cucurbits. In this study, we have performed comparative proteomics among P. melonis mutants to evaluate the interacting pathogenicity proteins. Structural characterization of all detected proteins discovered the physicochemical factors behind their pathogenic behavior. Results revealed that gamma radiations have an extreme potential to mutate P. melonis spores, and can be a safe alternative of chemical mutagens. P. melonis is accomplished with seventy-three differentially expressed proteins among which, seven proteins were ranked as strong pathogenicity factors, i.e. Q9AT01 (Elicitin), D0MX11 (Oligopeptidase A, putative), D0NB60 (Secreted RXLR effector peptide protein, putative), D0NIK4 (IpiB3-like protein), D0NIZ1 (Carboxypeptidase), H3GZF4 and H3GZF6. The Ramachandran plot determined the positions of secondary structures (α helix and β sheets) in the 3D structures of pathogenicity proteins. Furthermore, the glycine residues analysis highlighted that six out of seven proteins were functionally and structurally more stable than D0NIK4 protein. The study concluded that the pathogenic proteins must have negative ϕ angles greater than 63°, and ψ angles ≥80° for their proper functioning. This study establishes functional connections between pathogenicity genes and the process of disease establishment.

Blue-light receptor phototropin 1 suppresses immunity to promote Phytophthora infestans infection.

Summary Blue‐light (BL) phototropin receptors (phot1 and phot2) regulate plant growth by activating NPH3/RPT2‐like (NRL) family members. Little is known about roles for BL and phots in regulating plant immunity. We showed previously that Phytophthora infestans RXLR effector Pi02860 targets potato (St)NRL1, promoting its ability to enhance susceptibility by facilitating proteasome‐mediated degradation of the immune regulator StSWAP70. This raises the question: do BL and phots negatively regulate immunity?We employed coimmunoprecipitation, virus‐induced gene silencing, transient overexpression and targeted mutation to investigate contributions of phots to regulating immunity.Whereas transient overexpression of Stphot1 and Stphot2 enhances P. infestans colonization of Nicotiana benthamiana, silencing endogenous Nbphot1 or Nbphot2 reduces infection. Stphot1, but not Stphot2, suppressed the INF1‐triggered cell death (ICD) immune response in a BL‐ and NRL1‐dependent manner. Stphot1, when coexpressed with StNRL1, promotes degradation of StSWAP70, whereas Stphot2 does not. Kinase‐dead Stphot1 fails to suppress ICD, enhance P. infestans colonization or promote StSWAP70 degradation. Critically, BL enhances P. infestans infection, which probably involves phots but not other BL receptors such as cryptochromes and F‐box proteins ZTL1 and FKF1.We demonstrate that Stphot1 and Stphot2 play different roles in promoting susceptibility, and Stphot1 kinase activity is required for BL‐ and StNRL1‐mediated immune suppression.

Functional analysis of a Phytophthora host-translocated effector using the yeast model system.

Background Phytophthora plant pathogens secrete effector proteins that are translocated into host plant cells during infection and collectively contribute to pathogenicity. A subset of these host-translocated effectors can be identified by the amino acid motif RXLR (arginine, any amino acid, leucine, arginine). Bioinformatics analysis has identified hundreds of putative RXLR effector genes in Phytophthora genomes, but the specific molecular function of most remains unknown.MethodsHere we describe initial studies to investigate the use of Saccharomyces cerevisiae as a eukaryotic model to explore the function of Phytophthora RXLR effector proteins.Results and ConclusionsExpression of individual RXLR effectors in yeast inhibited growth, consistent with perturbation of a highly conserved cellular process. Transcriptome analysis of yeast cells expressing the poorly characterized P. sojae RXLR effector Avh110 identified nearly a dozen yeast genes whose expression levels were altered greater than two-fold compared to control cells. All five of the most down-regulated yeast genes are normally induced under low phosphate conditions via the PHO4 transcription factor, indicating that PsAvh110 perturbs the yeast regulatory network essential for phosphate homeostasis and suggesting likely PsAvh110 targets during P. sojae infection of its soybean host.

Phytophthora infestans RXLR effector Pi04089 perturbs diverse defense-related genes to suppress host immunity

BackgroundThe oomycete pathogen secretes many effectors into host cells to manipulate host defenses. For the majority of effectors, the mechanisms related to how they alter the expression of host genes and reprogram defenses are not well understood. In order to investigate the molecular mechanisms governing the influence that the Phytophthora infestans RXLR effector Pi04089 has on host immunity, a comparative transcriptome analysis was conducted on Pi04089 stable transgenic and wild-type potato plants.ResultsPotato plants stably expressing Pi04089 were more susceptible to P. infestans. RNA-seq analysis revealed that 658 upregulated genes and 722 downregulated genes were characterized in Pi04089 transgenic lines. A large number of genes involved in the biological process, including many defense-related genes and certain genes that respond to salicylic acid, were suppressed. Moreover, the comparative transcriptome analysis revealed that Pi04089 significantly inhibited the expression of many flg22 (a microbe-associated molecular pattern, PAMP)-inducible genes, including various Avr9/Cf-9 rapidly elicited (ACRE) genes. Four selected differentially expressed genes (StWAT1, StCEVI57, StKTI1, and StP450) were confirmed to be involved in host resistance against P. infestans when they were transiently expressed in Nicotiana benthamiana.ConclusionThe P. infestans effector Pi04089 was shown to suppress the expression of many resistance-related genes in potato plants. Moreover, Pi04089 was found to significantly suppress flg22-triggered defense signaling in potato plants. This research provides new insights into how an oomycete effector perturbs host immune responses at the transcriptome level.

Genome Sequence Resource of Phytophthora colocasiae from China Using Nanopore Sequencing Technology.

Phytophthora colocasiae is a destructive oomycete pathogen of taro (Colocasia esculenta), which causes taro leaf blight. To date, only one highly fragmented Illumina short-read-based genome assembly is available for this species. To address this problem, we sequenced strain Lyd2019 from China using Oxford Nanopore Technologies long-read sequencing and Illumina short-read sequencing. We generated a 92.51-Mb genome assembly consisting of 105 contigs with an N50 of 1.70 Mb and a maximum length of 4.17 Mb. In the genome assembly, we identified 52.78% repeats and 18,322 protein-coding genes, of which 12,782 genes were annotated. We also identified 191 candidate RXLR effectors and 1 candidate crinkling and necrosis effector. The updated near-chromosome genome assembly and annotation resources will provide a better understanding of the infection mechanisms of P. colocasiae.

An atypical Phytophthora sojae RxLR effector manipulates host vesicle trafficking to promote infection.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.

Interaction of Phytophthora sojae Effector Avr1b With E3 Ubiquitin Ligase GmPUB1 Is Required for Recognition by Soybeans Carrying Phytophthora Resistance Rps1-b and Rps1-k Genes.

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

Insights into the Host Specificity of a New Oomycete Root Pathogen, Pythium brassicum P1: Whole Genome Sequencing and Comparative Analysis Reveals Contracted Regulation of Metabolism, Protein Families, and Distinct Pathogenicity Repertoire.

Pythium brassicum P1 Stanghellini, Mohammadi, Förster, and Adaskaveg is an oomycete root pathogen that has recently been characterized. It only attacks plant species belonging to Brassicaceae family, causing root necrosis, stunting, and yield loss. Since P. brassicum P1 is limited in its host range, this prompted us to sequence its whole genome and compare it to those of broad host range Pythium spp. such as P. aphanidermatum and P. ultimum var. ultimum. A genomic DNA library was constructed with a total of 374 million reads. The sequencing data were assembled using SOAPdenovo2, yielding a total genome size of 50.3 Mb contained in 5434 scaffolds, N50 of 30.2 Kb, 61.2% G+C content, and 13,232 putative protein-coding genes. Pythium brassicum P1 had 175 species-specific gene families, which is slightly below the normal average. Like P. ultimum, P. brassicum P1 genome did not encode any classical RxLR effectors or cutinases, suggesting a significant difference in virulence mechanisms compared to other oomycetes. Pythium brassicum P1 had a much smaller proportions of the YxSL sequence motif in both secreted and non-secreted proteins, relative to other Pythium species. Similarly, P. brassicum P1 had the fewest Crinkler (CRN) effectors of all the Pythium species. There were 633 proteins predicted to be secreted in the P. brassicum P1 genome, which is, again, slightly below average among Pythium genomes. Pythium brassicum P1 had only one cadherin gene with calcium ion-binding LDRE and DxND motifs, compared to Pythium ultimum having four copies. Pythium brassicum P1 had a reduced number of proteins falling under carbohydrate binding module and hydrolytic enzymes. Pythium brassicum P1 had a reduced complement of cellulase and pectinase genes in contrast to P. ultimum and was deficient in xylan degrading enzymes. The contraction in ABC transporter families in P. brassicum P1 is suggested to be the result of a lack of diversity in nutrient uptake and therefore host range.

Draft genome assemblies for tree pathogens Phytophthora pseudosyringae and Phytophthora boehmeriae.

Species of Phytophthora, plant pathogenic eukaryotic microbes, can cause disease on many tree species. Genome sequencing of species from this genus has helped to determine components of their pathogenicity arsenal. Here, we sequenced genomes for two widely distributed species, Phytophthora pseudosyringae and Phytophthora boehmeriae, yielding genome assemblies of 49 and 40 Mb, respectively. We identified more than 270 candidate disease promoting RXLR effector coding genes for each species, and hundreds of genes encoding candidate plant cell wall degrading carbohydrate active enzymes (CAZymes). These data boost genome sequence representation across the Phytophthora genus, and form resources for further study of Phytophthora pathogenesis.

A Phytophthora capsici RXLR effector targets and inhibits the central immune kinases to suppress plant immunity.

Receptor-like cytoplasmic kinase subfamily VII (RLCK-VII) proteins are the central immune kinases in plant pattern-recognition receptor (PRR) complexes, and they orchestrate a complex array of defense responses against bacterial and fungal pathogens. However, the role of RLCK-VII in plant-oomycete pathogen interactions has not been established. Phytophthora capsici is a notorious oomycete pathogen that infects many agriculturally important vegetables. Here, we report the identification of RXLR25, an RXLR effector that is required for the virulence of P. capsici. In planta expression of RXLR25 significantly enhanced plants' susceptibility to Phytophthora pathogens. Microbial pattern-induced immune activation in Arabidopsis was severely impaired by RXLR25. We further showed that RXLR25 interacts with RLCK-VII proteins. Using nine rlck-vii high-order mutants, we observed that RLCK-VII-6 and RLCK-VII-8 members are required for resistance to P. capsici. The RLCK-VII-6 members are specifically required for Phytophthora culture filtrate (CF)-induced immune responses. RXLR25 directly targets RLCK-VII proteins such as BIK1, PBL8, and PBL17 and inhibits pattern-induced phosphorylation of RLCK-VIIs to suppress downstream immune responses. This study identified a key virulence factor for P. capsici, and the results revealed the importance of RLCK-VII proteins in plant-oomycete interactions.

Expression of several Phytophthora cinnamomi putative RxLRs provides evidence for virulence roles in avocado.

Phytophthora cinnamomi is a plant pathogenic oomycete that causes Phytophthora root rot of avocado (PRR). Currently, there is a limited understanding of the molecular interactions underlying this disease. Other Phytophthora species employ an arsenal of effector proteins to manipulate host physiology, of which the RxLR effectors contribute to virulence by interfering with host immune responses. The aim of this study was to identify candidate RxLR effectors in P. cinnamomi that play a role in establishing PRR, and to infer possible functions for these effectors. We identified 61 candidate RxLR genes which were expressed during infection of a susceptible avocado rootstock. Several of these genes were present in multiple copies in the P. cinnamomi genome, suggesting that they may contribute to pathogen fitness. Phylogenetic analysis of the manually predicted RxLR protein sequences revealed 12 P. cinnamomi RxLRs that were related to characterised effectors in other Phytophthora spp., providing clues to their functions in planta. Expression profiles of nine more RxLRs point to possible virulence roles in avocado–highlighting a way forward for studies of this interaction. This study represents the first investigation of the expression of P. cinnamomi RxLR genes during the course of avocado infection, and puts forward a pipeline to pinpoint effector genes with potential as virulence determinants, providing a foundation for the future functional characterization of RxLRs that contribute to P. cinnamomi virulence in avocado.

SnRK1.1-mediated resistance of Arabidopsis thaliana to clubroot disease is inhibited by the novel Plasmodiophora brassicae effector PBZF1.

Plants have evolved a series of strategies to combat pathogen infection. Plant SnRK1 is probably involved in shifting carbon and energy use from growth‐associated processes to survival and defence upon pathogen attack, enhancing the resistance to many plant pathogens. The present study demonstrated that SnRK1.1 enhanced the resistance of Arabidopsis thaliana to clubroot disease caused by the plant‐pathogenic protozoan Plasmodiophora brassicae. Through a yeast two‐hybrid assay, glutathione S‐transferase pull‐down assay, and bimolecular fluorescence complementation assay, a P. brassicae RxLR effector, PBZF1, was shown to interact with SnRK1.1. Further expression level analysis of SnRK1.1‐regulated genes showed that PBZF1 inhibited the biological function of SnRK1.1 as indicated by the disequilibration of the expression level of SnRK1.1‐regulated genes in heterogeneous PBZF1‐expressing A. thaliana. Moreover, heterogeneous expression of PBZF1 in A. thaliana promoted plant susceptibility to clubroot disease. In addition, PBZF1 was found to be P. brassicae‐specific and conserved. This gene was significantly highly expressed in resting spores. Taken together, our results provide new insights into how the plant‐pathogenic protist P. brassicae employs an effector to overcome plant resistance, and they offer new insights into the genetic improvement of plant resistance against clubroot disease.

Genome Sequence Data of Peronophythora litchii, an Oomycete Pathogen Causing Litchi Downy Blight.

Peronophythora litchii is an oomycete pathogen that exclusively infects litchi, with infection stages affecting a broad range of tissues. In this study, we obtained a near chromosome-level genome assembly of P. litchii ZL2018 from China using Oxford Nanopore Technologies long-read sequencing and Illumina short-read sequencing. The genome assembly was 64.15 Mb in size and consisted of 81 contigs with an N50 of 1.43 Mb and a maximum length of 4.74 Mb. Excluding 34.67% of repeat sequences, 14,857 protein-coding genes were identified, among which 14,447 genes were annotated. We also predicted 306 candidate RxLR effectors in the assembly. The high-quality genome assembly and annotation resources reported in this study will provide new insight into the infection mechanisms of P. litchii.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

Allelic variants of the NLR protein Rpi-chc1 differentially recognize members of the Phytophthora infestans PexRD12/31 effector superfamily through the leucine-rich repeat domain.

SUMMARYPhytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that trigger effective defense responses upon the recognition of cognate RXLR avirulence (Avr) effector proteins. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition. In this study, we cloned 16 allelic variants of the Rpi‐chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study effector recognition and co‐evolution. Functional and non‐functional alleles of Rpi‐chc1 encode coiled‐coil nucleotide‐binding leucine‐rich repeat (CNL) proteins, being the first described representatives of the CNL16 family. These alleles have distinct patterns of RXLR effector recognition. While Rpi‐chc1.1 recognized multiple PexRD12 (Avrchc1.1) proteins, Rpi‐chc1.2 recognized multiple PexRD31 (Avrchc1.2) proteins, both belonging to the PexRD12/31 effector superfamily. Domain swaps between Rpi‐chc1.1 and Rpi‐chc1.2 revealed that overlapping subdomains in the leucine‐rich repeat (LRR) domain are responsible for the difference in effector recognition. This study showed that Rpi‐chc1.1 and Rpi‐chc1.2 evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host–pathogen co‐evolution can lead to novel recognition specificities. These insights will guide future strategies to breed durable resistant varieties.

Haustorium formation and a distinct biotrophic transcriptome characterize infection of Nicotiana benthamiana by the tree pathogen Phytophthora kernoviae.

Phytophthora species cause some of the most serious diseases of trees and threaten forests in many parts of the world. Despite the generation of genome sequence assemblies for over 10 tree‐pathogenic Phytophthora species and improved detection methods, there are many gaps in our knowledge of how these pathogens interact with their hosts. To facilitate cell biology studies of the infection cycle we examined whether the tree pathogen Phytophthora kernoviae could infect the model plant Nicotiana benthamiana. We transformed P. kernoviae to express green fluorescent protein (GFP) and demonstrated that it forms haustoria within infected N. benthamiana cells. Haustoria were also formed in infected cells of natural hosts, Rhododendron ponticum and European beech (Fagus sylvatica). We analysed the transcriptome of P. kernoviae in cultured mycelia, spores, and during infection of N. benthamiana, and detected 12,559 transcripts. Of these, 1,052 were predicted to encode secreted proteins, some of which may function as effectors to facilitate disease development. From these, we identified 87 expressed candidate RXLR (Arg‐any amino acid‐Leu‐Arg) effectors. We transiently expressed 12 of these as GFP fusions in N. benthamiana leaves and demonstrated that nine significantly enhanced P. kernoviae disease progression and diversely localized to the cytoplasm, nucleus, nucleolus, and plasma membrane. Our results show that N. benthamiana can be used as a model host plant for studying this tree pathogen, and that the interaction likely involves suppression of host immune responses by RXLR effectors. These results establish a platform to expand the understanding of Phytophthora tree diseases.

A Plasmopara viticola RXLR effector targets a chloroplast protein PsbP to inhibit ROS production in grapevine.

Pathogens secrete a large number of effectors that manipulate host processes to create an environment conducive to pathogen colonization. However, the underlying mechanisms by which Plasmopara viticola effectors manipulate host plant cells remain largely unclear. In this study, we reported that RXLR31154, a P. viticola RXLR effector, was highly expressed during the early stages of P. viticola infection. In our study, stable expression of RXLR31154 in grapevine (Vitis vinifera) and Nicotiana benthamiana promoted leaf colonization by P. viticola and Phytophthora capsici, respectively. By yeast two-hybrid screening, the 23-kDa oxygen-evolving enhancer 2 (VpOEE2 or VpPsbP), encoded by the PsbP gene, in Vitis piasezkii accession Liuba-8 was identified as a host target of RXLR31154. Overexpression of VpPsbP enhanced susceptibility to P. viticola in grapevine and P. capsici in N. benthamiana, and silencing of NbPsbPs, the homologs of PsbP in N. benthamiana, reduced P. capcisi colonization, indicating that PsbP is a susceptibility factor. RXLR31154 and VpPsbP protein were co-localized in the chloroplast. Moreover, VpPsbP reduced H2 O2 accumulation and activated the 1 O2 signaling pathway in grapevine. RXLR31154 could stabilize PsbP. Together, our data revealed that RXLR31154 reduces H2 O2 accumulation and activates the 1 O2 signaling pathway through stabilizing PsbP, thereby promoting disease.

The Phytophthora effector Avh241 interacts with host NDR1-like proteins to manipulate plant immunity.

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization. Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection, the molecular basis of Avh241 virulence remains poorly understood. Here we identified non-race specific disease resistance 1 (NDR1)-like proteins, the critical components in plant effector-triggered immunity (ETI) responses, as host targets of Avh241. Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses. Silencing of GmNDR1s increases the susceptibility of soybean to P. sojae infection, and overexpression of GmNDR1s reduces infection, which supports its positive role in plant immunity against P. sojae. Furthermore, we demonstrate that GmNDR1 interacts with itself, and Avh241 probably disrupts the self-association of GmNDR1. These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses, likely by disturbing the function of NDR1 during infection.

Phytophthora infestans RXLR effector AVR1 disturbs the growth of Physcomitrium patens without affecting Sec5 localization

Plant pathogens often exploit a whole range of effectors to facilitate infection. The RXLR effector AVR1 produced by the oomycete plant pathogen Phytophthora infestans suppresses host defense by targeting Sec5. Sec5 is a subunit of the exocyst, a protein complex that is important for mediating polarized exocytosis during plant development and defense against pathogens. The mechanism by which AVR1 manipulates Sec5 functioning is unknown. In this study, we analyzed the effect of AVR1 on Sec5 localization and functioning in the moss Physcomitrium patens. P. patens has four Sec5 homologs. Two (PpSec5b and PpSec5d) were found to interact with AVR1 in yeast-two-hybrid assays while none of the four showed a positive interaction with AVR1ΔT, a truncated version of AVR1. In P. patens lines carrying β-estradiol inducible AVR1 or AVR1ΔT transgenes, expression of AVR1 or AVR1ΔT caused defects in the development of caulonemal protonema cells and abnormal morphology of chloronema cells. Similar phenotypes were observed in Sec5- or Sec6-silenced P. patens lines, suggesting that both AVR1 and AVR1ΔT affect exocyst functioning in P. patens. With respect to Sec5 localization we found no differences between β-estradiol-treated and untreated transgenic AVR1 lines. Sec5 localizes at the plasma membrane in growing caulonema cells, also during pathogen attack, and its subcellular localization is the same, with or without AVR1 in the vicinity.

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.