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Summary: Phytophthora parasitica causes diseases on a broad range of host plants. It secretes numerous effectors to suppress plant immunity. However, only a few virulence effectors in P. parasitica have been characterized.Here, we highlight that PpE18, a conserved RXLR effector in P. parasitica, was a virulence factor and suppresses Nicotiana benthamiana immunity. Utilizing luciferase complementation, co‐immunoprecipitation, and GST pull‐down assays, we determined that PpE18 targeted NbAPX3‐1, a peroxisome membrane‐associated ascorbate peroxidase with reactive oxygen species (ROS)‐scavenging activity and positively regulates plant immunity in N. benthamiana. We show that the ROS‐scavenging activity of NbAPX3‐1 was critical for its immune function and was hindered by the binding of PpE18. The interaction between PpE18 and NbAPX3‐1 resulted in an elevation of ROS levels in the peroxisome.Moreover, we discovered that the ankyrin repeat‐containing protein NbANKr2 acted as a positive immune regulator, interacting with both NbAPX3‐1 and PpE18. NbANKr2 was required for NbAPX3‐1‐mediated disease resistance. PpE18 competitively interfered with the interaction between NbAPX3‐1 and NbANKr2, thereby weakening plant resistance.Our results reveal an effective counter‐defense mechanism by which P. parasitica employed effector PpE18 to suppress host cellular defense, by suppressing biochemical activity and disturbing immune function of NbAPX3‐1 during infection. [ABSTRACT FROM AUTHOR]

Downy mildew caused by Sclerospora graminicola is a systemic disease that affects the yield and quality of foxtail millet. This obligate biotrophic oomycete manipulates host physiology and immune processes through numerous effectors. A thorough comprehension of effector biology is crucial to unravel disease mechanisms and understand host plant resistance. In this study, bioinformatic analyses revealed 498 potentially secreted proteins in S. graminicola, of which 62 were identified as RXLR effectors; 46 RXLR‐encoding genes exhibited upregulated expression during the early stages of infection. To elucidate the functions of these secreted proteins, a heterogeneous expression system was developed using Nicotiana benthamiana. Twenty‐one RXLR effectors secreted by S. graminicola were transiently expressed in N. benthamiana, of which four could suppress INF1‐triggered cell death. Various defence responses in N. benthamiana were attenuated, including inhibition of defence gene expression, reduction of reactive oxygen species (ROS) accumulation and diminished callose deposition. The expression of SG_RXLR41 also enhanced the growth of Phytophthora capsici on N. benthamiana leaves. These findings indicate that S. graminicola facilitates infection and expansion through the secretion of multiple RXLR effectors, and SG_RXLR41 is an important virulence‐related effector that is involved in manipulating plant immunity by suppressing cell death. [ABSTRACT FROM AUTHOR]

Summary: Arabidopsis Col‐0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown.To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa‐Cala2 and Hpa‐Noks1. We identified ATR2Cala2 as a non‐canonical RxLR‐type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection.ATR2Cala2 is recognized in accession Col‐0 but not in Ler‐0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility.Two additional Col‐0 TIR‐NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa‐Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2‐recognizing accessions. [ABSTRACT FROM AUTHOR]

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. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The development of pepper cultivars with durable resistance to the oomycete Phytophthora capsici has been challenging due to differential interactions between the species that allow certain pathogen isolates to cause disease on otherwise resistant host genotypes. Currently, little is known about the pathogen genes involved in these interactions. To investigate the genetic basis of P. capsici virulence on individual pepper genotypes, we inoculated sixteen pepper accessions, representing commercial varieties, sources of resistance, and host differentials, with 117 isolates of P. capsici, for a total of 1,864 host-pathogen combinations. Analysis of disease outcomes revealed a significant effect of inter-species genotype-by-genotype interactions, although these interactions were quantitative rather than qualitative in scale. Isolates were classified into five pathogen subpopulations, as determined by their genotypes at over 60,000 single-nucleotide polymorphisms (SNPs). While absolute virulence levels on certain pepper accessions significantly differed between subpopulations, a multivariate phenotype reflecting relative virulence levels on certain pepper genotypes compared with others showed the strongest association with pathogen subpopulation. A genome-wide association study (GWAS) identified four pathogen loci significantly associated with virulence, two of which colocalized with putative RXLR effector genes and another with a polygalacturonase gene cluster. All four loci appeared to represent broad-spectrum virulence genes, as significant SNPs demonstrated consistent effects regardless of the host genotype tested. Host genotype–specific virulence variants in P. capsici may be difficult to map via GWAS with all but excessively large sample sizes, perhaps controlled by genes of small effect or by multiple allelic variants that have arisen independently. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

As a destructive plant pathogen, Phytophthora infestans secretes diverse host‐entering RxLR effectors to facilitate infection. One critical RxLR effector, PiAvr3b, not only induces effector‐triggered immunity (ETI), which is associated with the potato resistance protein StR3b, but also suppresses pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). To date, the molecular basis underlying such dual activities remains unknown. Based on phylogenetic analysis of global P. infestans isolates, we found two PiAvr3b isoforms that differ by three amino acids. Despite this sequence variation, the two isoforms retain the same properties in activating the StR3b‐mediated hypersensitive response (HR) and inhibiting necrosis induced by three PAMPs (PiNpp, PiINF1, and PsXeg1) and an RxLR effector (Pi10232). Using a combined mutagenesis approach, we found that the dual activities of PiAvr3b were tightly linked and determined by 88 amino acids at the C‐terminus. We further determined that either the W60 or the E134 residue of PiAvr3b was essential for triggering StR3b‐associated HR and inhibiting PiNpp‐ and Pi10232‐associated necrosis, while the S99 residue partially contributed to PTI suppression. Additionally, nuclear localization of PiAvr3b was required to stimulate HR and suppress PTI, but not to inhibit Pi10232‐associated cell death. Our study revealed that PiAvr3b suppresses the plant immune response at different subcellular locations and provides an example in which a single amino acid of an RxLR effector links ETI induction and cell death suppression. [ABSTRACT FROM AUTHOR]

Phytophthora infestans, the etiologic agent of late blight, is a threat to potato production in areas with high humidity during the growing season. The oomycete pathogen is hemi-biotrophic, it establishes infection on living plant cells and then spreads, kills, and feeds off the necrotized plant tissue material. The interaction between host and pathogen is complex with dynamic pathogen RXLR effectors and potato NB-LRR resistance proteins actively competing for dominance and survival. Late blight protection was brought to several cultivars of potato through insertion of the wild potato (Solanum venturii) NB-LRR resistance gene Rpi-vnt1.1. We have established that the late blight protection trait, mediated by Rpi-vnt1.1, is effective despite low expression of RNA. The RNA expression dynamics of Rpi-vnt1.1 and the cognate pathogen RXLR effector, Avr-vnt1, were evaluated following spray inoculation with up to five different contemporary late blight isolates from North America and South America. Following inoculations, RXLR effector transcript profiles provided insight into interaction compatibility in relation to markers of the late blight hemi-biotrophic lifecycle. [ABSTRACT FROM AUTHOR]

Grapevine downy mildew, caused by the oomycete Plasmopara viticola , is one of the most significant production challenges for the grape and wine industry. P. viticola injects a plethora of effectors into its host cells to disrupt immune processes, but the mechanisms by which these effectors act at the molecular level have not been well characterized. Herein, we show that a candidate P. viticola avirulence homolog (Avh) RxLR effector gene, designated PvAvh77 , was strongly up-regulated during the initial stages of P. viticola infection in Vitis vinifera. Further experiments demonstrated that PvAvh77 could trigger non-specific cell death when expressed in the wild grapevine Vitis riparia and in tobacco (Nicotiana benthamiana and Nicotiana tabacum). In addition, a truncated form of PvAvh77, designated PvAvh77-M2, was more active in inducing cell death in N. benthamiana and V. riparia than full-length PvAvh77. Ectopic expression of PvAvh77 in V. vinifera 'Thompson Seedless' leaves neutralized host immunity and enhanced colonization by P. viticola , and the immune-inhibiting activity of PvAvh77 on susceptible Eurasian grapevine depended on its nuclear localization. Using a yeast signal sequence trap approach, we showed that the signal peptide of PvAvh77 is functional in yeast. Moreover, PvAvh77 with a signal peptide stimulated plant immune responses in the apoplast. Notably, application of exogenous purified PvAvh77-M2 effectively initiated defence responses in grapevine extracellularly, as evidenced by increased accumulation of salicylic acid and H2O2, and reduced infection of inoculated P. viticola. In summary, we identified a novel effector, PvAvh77, from P. viticola , which has the potential to serve as an inducer of plant immunity. [ABSTRACT FROM AUTHOR]

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici , whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant. [ABSTRACT FROM AUTHOR]

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The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors that enter the plant cells and suppress host immunity. Only a few of these genes are conserved across different strains and species. Such core effectors might target hub genes and immune pathways in hosts. Here, we describe the functional characterization of the core P. capsici RXLR effector RXLR242. The expression of RXLR242 was up‐regulated during infection, and its ectopic expression in Nicotiana benthamiana, an experimental plant host, further promoted Phytophthora infection. RXLR242 physically interacted with a group of RAB proteins that belong to the small GTPase family and play a role in regulating transport pathways in the intracellular membrane trafficking system. In addition, RXLR242 impeded the secretion of PATHOGENESIS‐RELATED 1 (PR1) protein to the apoplast. This phenomenon resulted from the competitive binding of RXLR242 to RABE1‐7. We also found that RXLR242 interfered with the association between RABA4‐3 and its binding protein, thereby disrupting the trafficking of the membrane receptor FLAGELLIN‐SENSING 2. Thus, RXLR242 manipulates plant immunity by targeting RAB proteins and disrupting protein trafficking in the host plants. [ABSTRACT FROM AUTHOR]

Partial resistance in plants generally exerts a low selective pressure on pathogens, and thus ensuring their durability in agrosystems. However, little is known about the effect of partial resistance on the molecular mechanisms of pathogenicity, a knowledge that could advance plant breeding for sustainable plant health. Here we investigate the gene expression of Phytophthora capsici during infection of pepper (Capsicum annuum L.), where only partial genetic resistance is reported, using Illumina RNA-seq. Comparison of transcriptomes of P. capsici infecting susceptible and partially resistant peppers identified a small number of genes that redirected its own resources into lipid biosynthesis to subsist on partially resistant plants. The adapted and non-adapted isolates of P. capsici differed in expression of genes involved in nucleic acid synthesis and transporters. Transient ectopic expression of the RxLR effector genes CUST_2407 and CUST_16519 in pepper lines differing in resistance levels revealed specific host-isolate interactions that either triggered local necrotic lesions (hypersensitive response or HR) or elicited leave abscission (extreme resistance or ER), preventing the spread of the pathogen to healthy tissue. Although these effectors did not unequivocally explain the quantitative host resistance, our findings highlight the importance of plant genes limiting nutrient resources to select pepper cultivars with sustainable resistance to P. capsici. [ABSTRACT FROM AUTHOR]

Plants have developed intricate immune mechanisms to impede Phytophthora colonization. In response, Phytophthora secretes RxLR effector proteins that disrupt plant defense and promote infection. The specific molecular interactions through which Phytophthora RxLR effectors undermine plant immunity, however, remain inadequately defined. In this study, we delineate the role of the nuclear-localized RxLR effector PcAvh87 , which is pivotal for the full virulence of Phytophthora cinnamomi. Gene expression analysis indicates that PcAvh87 expression is significantly upregulated during the initial infection stages, interacting with the immune responses triggered by the elicitin protein INF1 and pro-apoptotic protein BAX. Utilizing PEG/CaCl 2 -mediated protoplast transformation and CRISPR/Cas9-mediated gene editing, we generated PcAvh87 knockout mutants, which demonstrated compromised hyphal growth, sporangium development, and zoospore release, along with a marked reduction in pathogenicity. This underscores PcAvh87's crucial role as a virulence determinant. Notably, PcAvh87, conserved across the Phytophthora genus, was found to modulate the activity of plant immune protein 113, thereby attenuating plant immune responses. This implies that the PcAvh87-mediated regulatory mechanism could be a common strategy in Phytophthora species to manipulate plant immunity. Our findings highlight the multifaceted roles of PcAvh87 in promoting P. cinnamomi infection, including its involvement in sporangia production, mycelial growth, and the targeting of plant immune proteins to enhance pathogen virulence. [ABSTRACT FROM AUTHOR]

Partial resistance in plants generally exerts a low selective pressure on pathogens, and thus ensuring their durability in agrosystems. However, little is known about the effect of partial resistance on the molecular mechanisms of pathogenicity, a knowledge that could advance plant breeding for sustainable plant health. Here we investigate the gene expression of Phytophthora capsici during infection of pepper (Capsicum annuum L.), where only partial genetic resistance is reported, using Illumina RNA-seq. Comparison of transcriptomes of P. capsici infecting susceptible and partially resistant peppers identified a small number of genes that redirected its own resources into lipid biosynthesis to subsist on partially resistant plants. The adapted and non-adapted isolates of P. capsici differed in expression of genes involved in nucleic acid synthesis and transporters. Transient ectopic expression of the RxLR effector genes CUST_2407 and CUST_16519 in pepper lines differing in resistance levels revealed specific host-isolate interactions that either triggered local necrotic lesions (hypersensitive response or HR) or elicited leave abscission (extreme resistance or ER), preventing the spread of the pathogen to healthy tissue. Although these effectors did not unequivocally explain the quantitative host resistance, our findings highlight the importance of plant genes limiting nutrient resources to select pepper cultivars with sustainable resistance to P. capsici.

Phytophthora sojae is a destructive soybean pathogen that orchestrates various secreted proteins (effectors) to modulate plant immunity and facilitate infection. Although a number of effectors have been identified and functionally studied in P. sojae, the way these molecules are regulated is marginally known. In this study, we performed a weighted gene correlation network analysis (WGCNA) based on digital RNA-seq, which enabled the identification of a transcription factor (PsCZF3) in P. sojae. This transcription factor is a C2H2-type zinc finger protein that regulates the transcription of 35 RxLR effectors during the early infection stage. Phylogenetic analysis revealed that PsCZF3 is a highly conserved protein across oomycetes, suggesting that this regulation mechanism may broadly exist in oomycete species. In addition, by building a subnetwork of PsCZF3 and correlated genes, we also found that PsCZF3 contributed to the transcriptional regulation of carbohydrate-active enzymes. Our findings suggest that the activation of PsCZF3 facilitates P. sojae infection by up-regulating RxLR effectors and carbohydrate-active enzymes. [ABSTRACT FROM AUTHOR]

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3 , plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica , showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (Q RGG YD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25–26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25–26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3 , strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25–26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica , thereby contributing to infection and pathogenicity of the pathogen. [ABSTRACT FROM AUTHOR]

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3, plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica, showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (QRGGYD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25–26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25–26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3, strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25–26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica, thereby contributing to infection and pathogenicity of the pathogen.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2 Cala2 effector was unknown. To reveal ATR2 Cala2 , an F 2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2 Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2 Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2 Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2 Emoy2 and ATR2 Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2 Cala2 . Stable and transient expression of ATR2 Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2 Cala2 -recognizing accessions., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

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. [ABSTRACT FROM AUTHOR]

A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate-glutamate-alanine-histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

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.

Phytophthora sojae is a destructive soybean pathogen that orchestrates various secreted proteins (effectors) to modulate plant immunity and facilitate infection. Although a number of effectors have been identified and functionally studied in P. sojae, the way these molecules are regulated is marginally known. In this study, we performed a weighted gene correlation network analysis (WGCNA) based on digital RNA-seq, which enabled the identification of a transcription factor (PsCZF3) in P. sojae. This transcription factor is a C2H2-type zinc finger protein that regulates the transcription of 35 RxLR effectors during the early infection stage. Phylogenetic analysis revealed that PsCZF3 is a highly conserved protein across oomycetes, suggesting that this regulation mechanism may broadly exist in oomycete species. In addition, by building a subnetwork of PsCZF3 and correlated genes, we also found that PsCZF3 contributed to the transcriptional regulation of carbohydrate-active enzymes. Our findings suggest that the activation of PsCZF3 facilitates P. sojae infection by up-regulating RxLR effectors and carbohydrate-active enzymes.

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. [ABSTRACT FROM AUTHOR]

SUMMARY: Phytophthora 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. Significance statement: In this manuscript the cloning of novel late blight resistance genes and their cognate effectors is described. The mechanism of recognition, host pathogen co‐evolution, and implications for deployment of the knowledge in agriculture are discussed. [ABSTRACT FROM AUTHOR]

SUMMARY: 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 Vitispiasezkii 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 H2O2 accumulation and activated the 1O2 signaling pathway in grapevine. RXLR31154 could stabilize PsbP. Together, our data revealed that RXLR31154 reduces H2O2 accumulation and activates the 1O2 signaling pathway through stabilizing PsbP, thereby promoting disease. [ABSTRACT FROM AUTHOR]

Summary: The oomycete pathogen Hyaloperonospora arabidopsidis delivers diverse effector proteins into host plant cells to suppress the plant's innate immunity. In this study, we investigate the mechanism of action of a conserved RxLR effector, HaRxLL470, in suppressing plant immunity.Genomic, molecular and biochemical analyses were performed to investigate the function of HaRxLL470 and the mechanism of the interaction between HaRxLL470 and the target host protein during H. arabidopsidis infection.We report that HaRxLL470 enhances plant susceptibility to H. arabidopsidis isolate Noco2 by interacting with the host photomorphogenesis regulator protein HY5. Our results demonstrate that HY5 is not only an important component in the regulation of light signalling, but also positively regulates host plant immunity against H. arabidopsidis by transcriptional activation of defense‐related genes. We show that the interaction between HaRxLL470 and HY5 compromises the function of HY5 as a transcription factor by attenuating its DNA‐binding activity.The present study demonstrates that HY5 positively regulates host plant defense against H. arabidopsidis whereas HaRxLL470, a conserved RxLR effector across oomycete pathogens, enhances pathogenicity by interacting with HY5 and suppressing transcriptional activation of defense‐related genes. [ABSTRACT FROM AUTHOR]

Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen Phytophthora sojae can overcome resistance conferred by the host resistance gene Rps1b through natural silencing of its corresponding effector gene, Avr1b-1. With the Phytophthora CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the Avr1b-1 gene was deleted from the P. sojae genome using a homology-directed recombination strategy that replaced Avr1b-1 with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on Rps1b plants, while infection of plants lacking Rps1b was not compromised. When a sgRNA-resistant version of Avr1b-1 was reintroduced into the Avr1b-1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed Avr1b-1 gene were unable to infect Rps1b-containing soybeans. However, loss of expression of the incoming Avr1b-1 gene was frequently observed in KI transformants, which resulted in these transformants readily infecting Rps1b soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that Avr1b-1 may be unusually susceptible to transcriptional variation.

Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen Phytophthora sojae can overcome resistance conferred by the host resistance gene Rps1b through natural silencing of its corresponding effector gene, Avr1b-1. With the Phytophthora CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the Avr1b-1 gene was deleted from the P. sojae genome using a homology-directed recombination strategy that replaced Avr1b-1 with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on Rps1b plants, while infection of plants lacking Rps1b was not compromised. When a sgRNA-resistant version of Avr1b-1 was reintroduced into the Avr1b -1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed Avr1b-1 gene were unable to infect Rps1b -containing soybeans. However, loss of expression of the incoming Avr1b-1 gene was frequently observed in KI transformants, which resulted in these transformants readily infecting Rps1b soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that Avr1b-1 may be unusually susceptible to transcriptional variation. [ABSTRACT FROM AUTHOR]

Plasmopara viticola, the causal organism of grapevine downy mildew, secretes a vast array of effectors to manipulate host immunity. Previously, several cell death‐inducing PvRXLR effectors have been identified, but their functions and host targets are poorly understood. Here, we investigated the role of PvRXLR111, a cell death‐inducing RXLR effector, in manipulating plant immunity. When coexpressed with other PvRXLR effectors, PvRXLR111‐induced cell death was prevented. Transient expression of PvRXLR111 in Nicotiana benthamiana suppressed bacterial flagellin peptide flg22‐elicited immune responses and enhanced Phytophthora capsici infection. PvRXLR111 induction in Arabidopsis increased susceptibility to Hyaloperonospora arabidopsidis. PvRXLR111 expression in Pseudomonas syringae promoted bacterial colonization. By immunoprecipitation‐mass spectrometry analysis, yeast two‐hybrid, pull‐down, and bimolecular fluorescence complementation assays, it was shown that PvRXLR111 interacted with Vitis vinifera putative WRKY transcription factor 40 (VvWRKY40), which increased VvWRKY40 stability. Transient expression of VvWRKY40 in N. benthamiana inhibited flg22‐induced reactive oxygen species burst and enhanced P. capsici infection and silencing NbWRKY40 attenuated P. capsici colonization. These results suggest VvWRKY40 functions as a negative regulator in plant immunity and that PvRXLR111 suppresses host immunity by stabilizing VvWRKY40. [ABSTRACT FROM AUTHOR]

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

Summary: Pathogen effectors act as disease promoting factors that target specific host proteins with roles in plant immunity. Here, we investigated the function of the RxLR3 effector of the plant‐pathogen Phytophthora brassicae.Arabidopsis plants expressing a FLAG‐RxLR3 fusion protein were used for co‐immunoprecipitation followed by liquid chromatography‐tandem mass spectrometry to identify host targets of RxLR3. Fluorescently labelled fusion proteins were used for analysis of subcellular localisation and function of RxLR3.Three closely related members of the callose synthase family, CalS1, CalS2 and CalS3, were identified as targets of RxLR3. RxLR3 co‐localised with the plasmodesmal marker protein PDLP5 (PLASMODESMATA‐LOCALISED PROTEIN 5) and with plasmodesmata‐associated deposits of the β‐1,3‐glucan polymer callose. In line with a function as an inhibitor of plasmodesmal callose synthases (CalS) enzymes, callose depositions were reduced and cell‐to‐cell trafficking was promoted in the presence of RxLR3.Plasmodesmal callose deposition in response to infection was compared with wild‐type suppressed in RxLR3‐expressing Arabidopsis lines. Our results implied a virulence function of the RxLR3 effector as a positive regulator of plasmodesmata transport and provided evidence for competition between P. brassicae and Arabidopsis for control of cell‐to‐cell trafficking. [ABSTRACT FROM AUTHOR]

EDS1 (Enhanced Disease Susceptibility 1) plays a crucial role in both effector‐triggered immunity activation and plant basal defence. However, whether pathogen effectors can target EDS1 or an EDS1‐related pathway to manipulate immunity is rarely reported. In this study, we identified a Phytophthora capsici Avirulence Homolog (Avh) RxLR (Arg‐any amino acid‐Leu‐Arg) effector PcAvh103 that interacts with EDS1. We demonstrated that PcAvh103 can facilitate P. capsici infection and is required for pathogen virulence. Furthermore, genetic evidence showed that PcAvh103 contributes to virulence through targeting EDS1. Finally, PcAvh103 specifically interacts with the lipase domain of EDS1 and can promote the disassociation of EDS1–PAD4 (Phytoalexin Deficient 4) complex in planta. Together, our results revealed that the P. capsici RxLR effector PcAvh103 targets host EDS1 to suppress plant immunity, probably through disrupting the EDS1–PAD4 immune signalling pathway. [ABSTRACT FROM AUTHOR]

Litchi downy blight, caused by the phytopathogenic oomycete Peronophythora litchii, results in tremendous economic loss in litchi production every year. To successfully colonize the host cell, Phytophthora species secret hundreds of RXLR effectors that interfere with plant immunity and facilitate the infection process. Previous work has already predicted 245 candidate RXLR effector‐encoding genes in P. litchii, 212 of which have been cloned and tested for plant cell death‐inducing activity in this study. We found three such RXLR effectors could trigger plant cell death through transient expression in Nicotiana benthamiana. Further experiments demonstrated that PlAvh142 could induce cell death and immune responses in several plants. We also found that PlAvh142 localized in both the cytoplasm and nucleus of plant cells. The cytoplasmic localization was critical for its cell death‐inducing activity. Moreover, deletion either of the two internal repeats in PlAvh142 abolished the cell death‐inducing activity. Virus‐induced gene silencing assays showed that cell death triggered by PlAvh142 was dependent on the plant transduction components RAR1 (require for Mla12 resistance), SGT1 (suppressor of the G2 allele of skp1) and HSP90 (heat shock protein 90). Finally, knockout of PlAvh142 resulted in significantly attenuated P. litchii virulence on litchi plants, whereas the PlAvh142‐overexpressed mutants were more aggressive. These data indicated that PlAvh142 could be recognized in plant cytoplasm and is an important virulence RXLR effector of P. litchii. [ABSTRACT FROM AUTHOR]

Abstract Background Diverse plant pathogens deliver effectors into plant cells to alter host processes. Oomycete pathogen encodes a large number of putative RxLR effectors which are likely to play a role in manipulating plant defense responses. The secretome of Plasmopara viticola (downy mildew of grapevine) contains at least 162 candidate RxLR effectors discovered in our recent studies, but their roles in infection and pathogenicity remain to be determined. Here, we characterize in depth one of the putative RxLR effectors, PvRxLR16, which has been reported to induce cell death in Nicotiana benthamiana in our previous study. Results The nuclear localization, W/Y/L motifs, and a putative N-glycosylation site in C-terminal of PvRxLR16 were essential for cell death-inducing activity. Suppressor of G-two allele of Skp1 (SGT1), heat shock protein 90 (HSP90) and required for Mla12 resistance (RAR1), but not somatic embryogenesis receptor-like kinase (SERK3), were required for the cell death response triggered by PvRxLR16 in N. benthamiana. Some mitogen-activated protein kinases and transcription factors were also involved in the perception of PvRxLR16 by N. benthamiana. PvRxLR16 could also significantly enhance plant resistance to Phytophthora capsici and the nuclear localization was required for this ability. However, some other PvRxLR effectors could suppress defense responses and disease resistance induced by PvRxLR16, suggesting that it may not trigger host cell death or immune responses during physiological infection under natural conditions. Conclusion These data demonstrate that PvRxLR16 may be recognized by endogenous proteins in nucleus to trigger immune responses in N. benthamiana, which in turn can be suppressed by other PvRxLR effectors.

Downy mildew is one of the most serious diseases of grapevine (Vitis spp). The causal agent of grapevine downy mildew, Plasmopara viticola, is an obligate biotrophic oomycete. Although oomycete pathogens such as P. viticola are known to secrete RxLR effectors to manipulate host immunity, there have been few studies of the associated mechanisms by which these may act. Here, we show that a candidate P. viticola RxLR effector, PvAvh74, induces cell death in Nicotiana benthamiana leaves. Using agroinfiltration, we found that nuclear localization, two putative N-glycosylation sites, and 427 amino acids of the PvAvh74 carboxyl terminus were necessary for cell-death-inducing activity. Using virus-induced gene silencing (VIGS), we found that PvAvh74-induced cell death in N. benthamiana requires EDS1, NDR1, SGT1, RAR1, and HSP90, but not BAK1. The MAPK cascade components MEK2, WIPK, and SIPK were also involved in PvAvh74-induced cell death in N. benthamiana. Transient expression of PvAvh74 could suppress Phytophthora capsici colonization of N. benthamiana, which suggests that PvAvh74 elicits plant immune responses. Suppression of P. capsici colonization also was dependent on nuclear localization of PvAvh74. Additionally, PvAvh74-triggered cell death could be suppressed by another effector, PvAvh8, from the same isolate. This work provides a framework to further investigate the interactions of PvAvh74 and other RxLR effectors with host immunity.

In plants, subcellular fluctuations in Ca2+ ion concentration are among the earliest responses to biotic and abiotic stresses. Calmodulin, which is a ubiquitous Ca2+ ion sensor in eukaryotes, plays a major role in translating these Ca2+ signatures to cellular responses by interacting with numerous proteins located in plasma membranes, cytoplasm, organelles and nuclei. In this report, we show that one of the Phytophthora RXLR effector, Avrblb2, interacts with calmodulin at the plasma membrane of the plant cells. Using deletion and single amino acid mutagenesis, we found that calmodulin binds to the effector domain of Avrblb2. In addition, we show that most known homologs of Avrblb2 in three different Phytophthora species interact with different isoforms of calmodulin. Type of amino acids at position 69 in Avrblb2, which determines Rbi-blb2 resistance protein-mediated defense responses, is not involved in the Avrblb2-calmodulin interaction. Using in planta functional analyses, we show that calmodulin binding to Avrblb2 is required for its recognition by Rpi-blb2 to incite hypersensitive response. These findings suggest that Avrblb2 by interacting with calmodulin interfere with plant defense associated Ca2+ signaling in plants.

Resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes, denoted Pl. Twenty-two Pl genes have been identified and genetically mapped so far. However, over the past 50 years, wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H. annuus ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a P. halstedii RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published Pl genes that we also mapped physically on the same reference genome. The new genes were designated Pl23–Pl32 according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower.

Summary: Phytophthora pathogens manipulate host innate immunity by secreting numerous RxLR effectors, thereby facilitating pathogen colonization. Predicted single and tandem repeats of WY domains are the most prominent C‐terminal motifs conserved across the Phytophthora RxLR superfamily. However, the functions of individual WY domains in effectors remain poorly understood.The Phytophthora sojae effector PSR1 promotes infection by suppressing small RNA biogenesis in plant hosts. We identified one single WY domain following the RxLR motif in PSR1. This domain was required for RNA silencing suppression activity and infection in Nicotiana benthamiana, Arabidopsis and soybean.Mutations of the conserved residues in the WY domain did not affect the subcellular localization of PSR1 but abolished its effect on plant development and resistance to viral and Phytophthora pathogens. This is at least in part due to decreased protein stability of the PSR1 mutants in planta.The identification of the WY domain in PSR1 allows predicts that a family of PSR1‐like effectors also possess RNA silencing suppression activity. Mutation of the conserved residues in two members of this family, PpPSR1L from P. parasitica and PcPSR1L from P. capsici, perturbed their biological functions, indicating that the WY domain is critical in Phytophthora PSR1 and PSR1‐like effectors. [ABSTRACT FROM AUTHOR]

Downy mildew is one of the most serious diseases of grapevine (Vitis spp). The causal agent of grapevine downy mildew, Plasmopara viticola , is an obligate biotrophic oomycete. Although oomycete pathogens such as P. viticola are known to secrete RxLR effectors to manipulate host immunity, there have been few studies of the associated mechanisms by which these may act. Here, we show that a candidate P. viticola RxLR effector, PvAvh74, induces cell death in Nicotiana benthamiana leaves. Using agroinfiltration, we found that nuclear localization, two putative N -glycosylation sites, and 427 amino acids of the PvAvh74 carboxyl terminus were necessary for cell-death-inducing activity. Using virus-induced gene silencing (VIGS), we found that PvAvh74-induced cell death in N. benthamiana requires EDS1, NDR1, SGT1, RAR1, and HSP90, but not BAK1. The MAPK cascade components MEK2, WIPK, and SIPK were also involved in PvAvh74-induced cell death in N. benthamiana. Transient expression of PvAvh74 could suppress Phytophthora capsici colonization of N. benthamiana , which suggests that PvAvh74 elicits plant immune responses. Suppression of P. capsici colonization also was dependent on nuclear localization of PvAvh74. Additionally, PvAvh74-triggered cell death could be suppressed by another effector, PvAvh8, from the same isolate. This work provides a framework to further investigate the interactions of PvAvh74 and other RxLR effectors with host immunity. [ABSTRACT FROM AUTHOR]

Summary: The grapevine downy mildew pathogen Plasmopara viticola secretes a set of RXLR effectors (PvRXLRs) to overcome host immunity and facilitate infection, but how these effectors function is unclear. Here, the biological function of PvRXLR131 was investigated via heterologous expression. Constitutive expression of PvRXLR131 in Colletotrichum gloeosporioides significantly enhanced its pathogenicity on grapevine leaves. Constitutive expression of PvRXLR131 in Arabidopsis promoted Pseudomonassyringae DC3000 and P.syringae DC3000 (hrcC‐) growth as well as suppressed defence‐related callose deposition. Transient expression of PvRXLR131 in Nicotiana benthamiana leaves could also suppress different elicitor‐triggered cell death and inhibit plant resistance to Phytophthora capsici. Further analysis revealed that PvRXLR131 interacted with host Vitis vinifera BRI1 kinase inhibitor 1 (VvBKI1), and its homologues in N. benthamiana (NbBKI1) and Arabidopsis (AtBKI1). Moreover, bimolecular fluorescence complementation analysis revealed that PvRXLR131 interacted with VvBKI1 in the plasma membrane. Deletion assays showed that the C‐terminus of PvRXLR131 was responsible for the interaction and mutation assays showed that phosphorylation of a conserved tyrosine residue in BKI1s disrupted the interaction. BKI1 was a receptor inhibitor of growth‐ and defence‐related brassinosteroid (BR) and ERECTA (ER) signalling. When silencing of NbBKI1 in N. benthamiana, the virulence function of PvRXLR131 was eliminated, demonstrating that the effector activity is mediated by BKI1. Moreover, PvRXLR131‐transgenic plants displayed BKI1‐overexpression dwarf phenotypes and suppressed BR and ER signalling. These physiological and genetic data clearly demonstrate that BKI1 is a virulence target of PvRXLR131. We propose that P. viticola secretes PvRXLR131 to target BKI1 as a strategy for promoting infection. [ABSTRACT FROM AUTHOR]

Summary: Phytophthora pathogens secrete many effector proteins to manipulate host innate immunity. PsAvh238 is a Phytophthora sojae N‐terminal Arg‐X‐Leu‐Arg (RXLR) effector, which evolved to escape host recognition by mutating one nucleotide while retaining plant immunity‐suppressing activity to enhance infection. However, the molecular basis of the PsAvh238 virulence function remains largely enigmatic.By using coimmunoprecipitation and liquid chromatography‐tandem mass spectrometry analysis, we identified the 1‐aminocyclopropane‐1‐carboxylate synthase (ACS) isoforms, the key enzymes in ethylene (ET) biosynthesis, as a host target of PsAvh238.We show that PsAvh238 interacts with soybean ACSs (GmACSs) in vivo and in vitro. By destabilizing Type2 GmACSs, PsAvh238 suppresses Type2 ACS‐catalyzed ET biosynthesis and facilitates Phytophthora infection. Silencing of Type2 GmACSs, and inhibition of ET biosynthesis or signaling, increase soybean susceptibility to P. sojae infection, supporting a role for Type2 GmACSs and ET in plant immunity against P. sojae. Moreover, wild‐type P. sojae but not the PsAvh238‐disrupted mutants, inhibits ET induction and promotes P. sojae infection in soybean.Our results highlight the ET biosynthesis pathway as an essential part in plant immunity against P. sojae and a direct effector target. [ABSTRACT FROM AUTHOR]

Abstract Reactive oxygen species (ROS) play a vital role in plant immune response, but the genes involved in the regulation of ROS are scantily reported. Phytophthora pathogens produce a large number of effectors to promote infection, but the modes of action adopted are largely unknown. Here, we report that RxLR207 could activate ROS-mediated cell death in Nicotiana benthamiana and was essential for virulence of P. capsici. We found that this effector targeted BPA1 (binding partner of ACD11) and four members of BPLs (BPA1-Like proteins) in Arabidopsis , and the bpa1 and bpl mutants had enhanced ROS accumulation and cell death under biotic or abiotic stresses. Furthermore, we showed that BPA1 and several BPLs functioned redundantly in plant immunity to P. capsici. We discovered that BPA1 and all six BPLs interacted with ACD11, and stabilization of ACD11 was impaired in the bpa1 , bpl2 , bpl3 , and bpl4 mutants. RxLR207 could promote the degradation of BPA1, BPL1, BPL2, and BPL4 to disrupt ACD11 stabilization in a 26S proteasome-dependent manner. Taken together, these findings indicate the important roles of Arabidopsis BPA1 and its homologs in ROS homeostasis and defense response, highlighting the usefulness of a pathogen effector-directed approach as a promising strategy for the discovery of novel plant immune regulators. The Phytophthora capsici virulence effector RxLR207 can induce ROS-mediated cell death for the transition from biotrophy to necrotrophy during pathogen infection and promote the degradation of ACD11 in a 26S proteasome-dependent manner by interfering with ACD11 binding partners, which are novel regulators of ROS-mediated defense response. [ABSTRACT FROM AUTHOR]

In plants, subcellular fluctuations in Ca2+ ion concentration are among the earliest responses to biotic and abiotic stresses. Calmodulin, which is a ubiquitous Ca2+ ion sensor in eukaryotes, plays a major role in translating these Ca2+ signatures to cellular responses by interacting with numerous proteins located in plasma membranes, cytoplasm, organelles and nuclei. In this report, we show that one of the Phytophthora RXLR effector, Avrblb2, interacts with calmodulin at the plasma membrane of the plant cells. Using deletion and single amino acid mutagenesis, we found that calmodulin binds to the effector domain of Avrblb2. In addition, we show that most known homologs of Avrblb2 in three different Phytophthora species interact with different isoforms of calmodulin. Type of amino acids at position 69 in Avrblb2, which determines Rbi-blb2 resistance protein-mediated defense responses, is not involved in the Avrblb2-calmodulin interaction. Using in planta functional analyses, we show that calmodulin binding to Avrblb2 is required for its recognition by Rpi-blb2 to incite hypersensitive response. These findings suggest that Avrblb2 by interacting with calmodulin interfere with plant defense associated Ca2+ signaling in plants. [ABSTRACT FROM AUTHOR]

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

Summary: Over the last 40 years, new sunflower downy mildew isolates (Plasmopara halstedii) have overcome major gene resistances in sunflower, requiring the identification of additional and possibly more durable broad‐spectrum resistances. Here, 354 RXLR effectors defined in silico from our new genomic data were classified in a network of 40 connected components sharing conserved protein domains. Among 205 RXLR effector genes encoding conserved proteins in 17 P. halstedii pathotypes of varying virulence, we selected 30 effectors that were expressed during plant infection as potentially essential genes to target broad‐spectrum resistance in sunflower. The transient expression of the 30 core effectors in sunflower and in Nicotiana benthamiana leaves revealed a wide diversity of targeted subcellular compartments, including organelles not so far shown to be targeted by oomycete effectors such as chloroplasts and processing bodies. More than half of the 30 core effectors were able to suppress pattern‐triggered immunity in N. benthamiana, and five of these induced hypersensitive responses (HR) in sunflower broad‐spectrum resistant lines. HR triggered by PhRXLRC01 co‐segregated with Pl22 resistance in F3 populations and both traits localized in 1.7 Mb on chromosome 13 of the sunflower genome. Pl22 resistance was physically mapped on the sunflower genome recently sequenced, unlike all the other downy mildew resistances published so far. PhRXLRC01 and Pl22 are proposed as an avirulence/resistance gene couple not previously described in sunflower. Core effector recognition is a successful strategy to accelerate broad‐spectrum resistance gene identification in complex crop genomes such as sunflower. Significance Statement: Five RXLR‐type effectors of the sunflower downy mildew pathogen conserved in 17 pathotypes of varying virulence, triggered plant immunity in six broad‐spectrum resistant sunflower lines, including two unpublished resistances to all pathotypes. Effector recognition accelerated plant resistance gene identification and fine mapping in complex crop genomes such as sunflower, for which no resistance gene has so far been characterized. [ABSTRACT FROM AUTHOR]

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

Resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes, denoted Pl. Twenty-two Pl genes have been identified and genetically mapped so far. However, over the past 50 years, wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H. annuus ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a P. halstedii RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published Pl genes that we also mapped physically on the same reference genome. The new genes were designated Pl23–Pl32 according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower. [ABSTRACT FROM AUTHOR]