Potato miR394 targets StA/N-INVE and StLCR to negatively regulate late blight resistance.

MicroRNAs (miRNAs) are small noncoding RNAs in eukaryotes. Plant endogenous miRNAs play pivotal roles in regulating plant development and defense responses. MicroRNA394 (miR394) has been reported to regulate plant development, abiotic stresses and defense responses. Previous reports showed that miR394 responded to P. infestans inoculation in potato, indicating that miR394 may be involved in defense responses. In this study, we further investigated its role in potato defense against P. infestans. Stable expression of miR394 in tobacco and potato enhances the susceptibility to P. infestans, which is accompanied with the reduced accumulation of ROS and down-regulation of the PTI (pattern-triggered immunity) marker genes. Besides well-known target StLCR, miR394 also targets StA/N-INVE, which encodes a chloroplast Alkaline/Neutral Invertases (A/N-INVE). Both StLCR and StA/N-INVE positively regulate late blight resistance, while miR394 degrades them. Interestingly, StA/N-INVE is located in the chloroplast, indicating that miR394 may manipulate chloroplast immunity. Degradation of StA/N-INVE may affect the chloroplast function and hence lead to the compromised ROS (reactive oxygen species) burst and reduced retrograde signaling from the chloroplast to the nucleus and cytoplasm. In summary, this study provides new information that miR394 targets and degrades StA/N-INVE and StLCR, which are positive regulators, to enhance potato susceptibility to P. infestans.

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

A Phytophthora infestans RXLR effector targets a potato ubiquitin-like domain-containing protein to inhibit the proteasome activity and hamper plant immunity.

Ubiquitin-like domain-containing proteins (UDPs) are involved in the ubiquitin-proteasome system because of their ability to interact with the 26S proteasome. Here, we identified potato StUDP as a target of the Phytophthora infestans RXLR effector Pi06432 (PITG_06432), which supresses the salicylic acid (SA)-related immune pathway. By overexpressing and silencing of StUDP in potato, we show that StUDP negatively regulates plant immunity against P. infestans. StUDP interacts with, and destabilizes, the 26S proteasome subunit that is referred to as REGULATORY PARTICLE TRIPLE-A ATP-ASE (RPT) subunit StRPT3b. This destabilization represses the proteasome activity. Proteomic analysis and Western blotting show that StUDP decreases the stability of the master transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) in SA biosynthesis. StUDP negatively regulates the SA signalling pathway by repressing the proteasome activity and destabilizing StSARD1, leading to a decreased expression of the SARD1-targeted gene ISOCHORISMATE SYNTHASE 1 and thereby a decrease in SA content. Pi06432 stabilizes StUDP, and it depends on StUDP to destabilize StRPT3b and thereby supress the proteasome activity. Our study reveals that the P. infestans effector Pi06432 targets StUDP to hamper the homeostasis of the proteasome by the degradation of the proteasome subunit StRPT3b and thereby suppresses SA-related immunity.

A Phytophthora capsici RXLR effector manipulates plant immunity by targeting RAB proteins and disturbing the protein trafficking pathway.

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.

Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance.

Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.

A potato late blight resistance gene protects against multiple Phytophthora species by recognizing a broadly conserved RXLR-WY effector.

Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens.

Short Linear Motifs (SLiMs) in "Core" RxLR Effectors of Phytophthora parasitica var. nicotianae: a Case of PpRxLR1 Effector.

Oomycetes of the genus Phytophthora encompass several of the most successful plant pathogens described to date. The success of infection by Phytophthora species is attributed to the pathogens' ability to secrete effector proteins that alter the host's physiological processes. Structural analyses of effector proteins mainly from bacterial and viral pathogens have revealed the presence of intrinsically disordered regions that host short linear motifs (SLiMs). These motifs play important biological roles by facilitating protein-protein interactions as well as protein translocation. Nonetheless, SLiMs in Phytophthora species RxLR effectors have not been investigated previously and their roles remain unknown. Using a bioinformatics pipeline, we identified 333 candidate RxLR effectors in the strain INRA 310 of Phytophthora parasitica. Of these, 71 (21%) were also found to be present in 10 other genomes of P. parasitica, and hence, these were designated core RxLR effectors (CREs). Within the CRE sequences, the N terminus exhibited enrichment in intrinsically disordered regions compared to the C terminus, suggesting a potential role of disorder in effector translocation. Although the disorder content was reduced in the C-terminal regions, it is important to mention that most SLiMs were in this terminus. PpRxLR1 is one of the 71 CREs identified in this study, and its genes encode a 6-amino acid (aa)-long SLiM at the C terminus. We showed that PpRxLR1 interacts with several host proteins that are implicated in defense. Structural analysis of this effector using homology modeling revealed the presence of potential ligand-binding sites. Among key residues that were predicted to be crucial for ligand binding, L102 and Y106 were of interest since they form part of the 6-aa-long PpRxLR1 SLiM. In silico substitution of these two residues to alanine was predicted to have a significant effect on both the function and the structure of PpRxLR1 effector. Molecular docking simulations revealed possible interactions between PpRxLR1 effector and ubiquitin-associated proteins. The ubiquitin-like SLiM carried in this effector was shown to be a potential mediator of these interactions. Further studies are required to validate and elucidate the underlying molecular mechanism of action. IMPORTANCE The continuous gain and loss of RxLR effectors makes the control of Phytophthora spp. difficult. Therefore, in this study, we endeavored to identify RxLR effectors that are highly conserved among species, also known as "core" RxLR effectors (CREs). We reason that these highly conserved effectors target conserved proteins or processes; thus, they can be harnessed in breeding for durable resistance in plants. To further understand the mechanisms of action of CREs, structural dissection of these proteins is crucial. Intrinsically disordered regions (IDRs) that do not adopt a fixed, three-dimensional fold carry short linear motifs (SLiMs) that mediate biological functions of proteins. The presence and potential role of these SLiMs in CREs of Phytophthora spp. have been overlooked. To our knowledge, we have effectively identified CREs as well as SLiMs with the potential of promoting effector virulence. Together, this work has advanced our comprehension of Phytophthora RxLR effector function and may facilitate the development of innovative and effective control strategies.

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

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.

Interactions of tagatose with the sugar metabolism are responsible for Phytophthora infestans growth inhibition.

Tagatose is a rare sugar metabolised by a limited number of microorganisms that inhibits a large spectrum of phytopathogens. In particular, tagatose inhibited Phytophthora infestans growth and negatively affected mitochondrial processes. However, the possible effects of tagatose on P. infestans metabolism have not yet been investigated. The aim of this study was to analyse the impact of this rare sugar on the sugar metabolism in P. infestans, in order to better understand its mode of action. Tagatose inhibited the growth of P. infestans with a precise reprogramming of the carbohydrate metabolism that involved a decrease of glucose, glucose-1-phosphate and mannose content and ß-glucosidase activity. The combination of tagatose with common sugars led to three different responses and highlighted antagonistic interactions. In particular, glucose partially attenuated the inhibitory effects of tagatose, while fructose fully impaired tagatose-mediated growth inhibition and metabolite changes. Moreover, sucrose did not attenuate tagatose effects, suggesting that the inhibition of sucrose catabolism and the alteration of glucose-related pathways contributed to the growth inhibition caused by tagatose to P. infestans. The interactions of tagatose with the common sugar metabolism were found to be a key mode of action against P. infestans growth, which may represent the basis for the further development of tagatose as an eco-friendly fungicide.

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

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

Proteinaceous effector discovery and characterization in filamentous plant pathogens.

The complicated interplay of plant-pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets.

Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.

A proper PiCAT2 level is critical for sporulation, sporangium function, and pathogenicity of Phytophthora infestans.

Catalase is present in prokaryotic and eukaryotic organisms and is important for the protective effects of the antioxidant system against free radicals. Many studies have confirmed that catalase is required for the growth, development, and pathogenesis of bacteria, plants, animals, and fungi. However, there has been relatively little research on the catalases in oomycetes, which form an important group of fungus-like eukaryotes that produce zoosporangia. In this study, we detected two Phytophthora infestans genes encoding catalases, but only PiCAT2 exhibited catalase activity in the sporulation stage and was highly produced during asexual reproduction and in the late infection stage. Compared with the wild-type strain, the PiCAT2-silenced P. infestans transformants were more sensitive to abiotic stress, were less pathogenic, and had a lower colony expansion rate and lower PiMPK7, PiVPS1, and PiGPG1 expression levels. In contrast, the PiCAT2-overexpressed transformants were slightly less sensitive to abiotic stress. Interestingly, increasing and decreasing PiCAT2 expression from the normal level inhibited sporulation, germination, and infectivity, and down-regulated PiCdc14 expression, but up-regulated PiSDA1 expression. These results suggest that PiCAT2 is required for P. infestans mycelial growth, asexual reproduction, abiotic stress tolerance, and pathogenicity. However, a proper PiCAT2 level is critical for the formation and normal function of sporangia. Furthermore, PiCAT2 affects P. infestans sporangial formation and function, pathogenicity, and abiotic stress tolerance by regulating the expression of cell cycle-related genes (PiCdc14 and PiSDA1) and MAPK pathway genes. Our findings provide new insights into catalase functions in eukaryotic pathogens.

Metabolic Model of the Phytophthora infestans-Tomato Interaction Reveals Metabolic Switches during Host Colonization.

The oomycete pathogen Phytophthora infestans causes potato and tomato late blight, a disease that is a serious threat to agriculture. P. infestans is a hemibiotrophic pathogen, and during infection, it scavenges nutrients from living host cells for its own proliferation. To date, the nutrient flux from host to pathogen during infection has hardly been studied, and the interlinked metabolisms of the pathogen and host remain poorly understood. Here, we reconstructed an integrated metabolic model of P. infestans and tomato (Solanum lycopersicum) by integrating two previously published models for both species. We used this integrated model to simulate metabolic fluxes from host to pathogen and explored the topology of the model to study the dependencies of the metabolism of P. infestans on that of tomato. This showed, for example, that P. infestans, a thiamine auxotroph, depends on certain metabolic reactions of the tomato thiamine biosynthesis. We also exploited dual-transcriptome data of a time course of a full late blight infection cycle on tomato leaves and integrated the expression of metabolic enzymes in the model. This revealed profound changes in pathogen-host metabolism during infection. As infection progresses, P. infestans performs less de novo synthesis of metabolites and scavenges more metabolites from tomato. This integrated metabolic model for the P. infestans-tomato interaction provides a framework to integrate data and generate hypotheses about in planta nutrition of P. infestans throughout its infection cycle.IMPORTANCE Late blight disease caused by the oomycete pathogen Phytophthora infestans leads to extensive yield losses in tomato and potato cultivation worldwide. To effectively control this pathogen, a thorough understanding of the mechanisms shaping the interaction with its hosts is paramount. While considerable work has focused on exploring host defense mechanisms and identifying P. infestans proteins contributing to virulence and pathogenicity, the nutritional strategies of the pathogen are mostly unresolved. Genome-scale metabolic models (GEMs) can be used to simulate metabolic fluxes and help in unravelling the complex nature of metabolism. We integrated a GEM of tomato with a GEM of P. infestans to simulate the metabolic fluxes that occur during infection. This yields insights into the nutrients that P. infestans obtains during different phases of the infection cycle and helps in generating hypotheses about nutrition in planta.

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

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

The oomycete microbe-associated molecular pattern Pep-13 triggers SERK3/BAK1-independent plant immunity.

KEY MESSAGE: Oomycetes MAMP Pep-13 can trigger SERK3/BAK1-independent PTI. Silencing of SERK3/BAK1 in solanaceous plants resulted in reduced expression of brassinosteroid marker genes and enhanced PTI transcriptional responses to Pep-13 treatment. To prevent disease, pattern recognition receptors (PRRs) are responsible for detecting microbe-associated molecular patterns (MAMPs) to switch on plant innate immunity. SOMATIC EMBROYOGENESIS KINASE 3 (SERK3)/BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) is a well-characterized receptor-like kinase (RLK) that serves as a pivotal co-receptor with PRRs to activate immunity following recognition of MAMPs including flg22, EF-Tu, INF1 and XEG1. However, the requirement for SERK3/BAK1 in many pattern-triggered immune (PTI) signaling pathways is not yet known. Pep-13 is an oomycete MAMP that consists of a highly conserved motif (an oligopeptide of 13 amino acids) shared in Phytophthora transglutaminases. Quantitative RT-PCR analysis reveals that the transcripts of three PTI marker genes (WRKY7, WRKY8 and ACRE31) rapidly accumulate in response to three different MAMPs: flg22, chitin and Pep-13. Whereas silencing of SERK3/BAK1 in Nicotiana benthamiana or potato compromised transcript accumulation in response to flg22, it did not attenuate WRKY7, WRKY8 and ACRE31 up-regulation in response to chitin or Pep-13. This indicates that Pep-13 triggers immunity in a SERK3/BAK1-independent manner, similar to chitin. Surprisingly, silencing of SERK3/BAK1 led to significantly increased accumulation of PTI marker gene transcripts following Pep-13 or chitin treatment, compared to controls. This was accompanied by reduced expression of brassinosteroid (BR) marker genes StSTDH, StEXP8 and StCAB50 and StCHL1, which is a negative regulator of PTI, supporting previous reports that SERK3/BAK1-dependent BR signaling attenuates plant immunity. We provide Pep-13 as an alternative to chitin as a trigger of SERK3/BAK1-independent immunity.

Phytophthora infestans effector SFI3 targets potato UBK to suppress early immune transcriptional responses.

The potato blight agent Phytophthora infestans secretes a range of RXLR effectors to promote disease. Recent evidence indicates that some effectors suppress early pattern-triggered immunity (PTI) following perception of microbe-associated molecular patterns (MAMPs). Phytophthora infestans effector PiSFI3/Pi06087/PexRD16 has been previously shown to suppress MAMP-triggered pFRK1-Luciferase reporter gene activity. How PiSFI3 suppresses immunity is unknown. We employed yeast-two-hybrid (Y2H) assays, co-immunoprecipitation, transcriptional silencing by RNA interference and virus-induced gene silencing (VIGS), and X-ray crystallography for structure-guided mutagenesis, to investigate the function of PiSFI3 in targeting a plant U-box-kinase protein (StUBK) to suppress immunity. We discovered that PiSFI3 is active in the host nucleus and interacts in yeast and in planta with StUBK. UBK is a positive regulator of specific PTI pathways in both potato and Nicotiana benthamiana. Importantly, it contributes to early transcriptional responses that are suppressed by PiSFI3. PiSFI3 forms an unusual trans-homodimer. Mutation to disrupt dimerization prevents nucleolar localisation of PiSFI3 and attenuates both its interaction with StUBK and its ability to enhance P. infestans leaf colonisation. PiSFI3 is a 'WY-domain' RXLR effector that forms a novel trans-homodimer which is required for its ability to suppress PTI via interaction with the U-box-kinase protein StUBK.

A MADS-box transcription factor regulates a central step in sporulation of the oomycete Phytophthora infestans.

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.

The Phytophthora infestans Haustorium Is a Site for Secretion of Diverse Classes of Infection-Associated Proteins.

The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection.IMPORTANCE There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection.

Phytophthora infestans RXLR effector SFI5 requires association with calmodulin for PTI/MTI suppressing activity.

Pathogens secrete effector proteins to interfere with plant innate immunity, in which Ca2+ /calmodulin (CaM) signalling plays key roles. Thus far, few effectors have been identified that directly interact with CaM for defence suppression. Here, we report that SFI5, an RXLR effector from Phytophthora infestans, suppresses microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) by interacting with host CaMs. We predicted the CaM-binding site in SFI5 using in silico analysis. The interaction between SFI5 and CaM was tested by both in vitro and in vivo assays. MTI suppression by SFI5 and truncated variants were performed in a tomato protoplast system. We found that both the predicted CaM-binding site and the full-length SFI5 protein interact with CaM in the presence of Ca2+ . MTI responses, such as FRK1 upregulation, reactive oxygen species accumulation, and mitogen-activated protein kinase activation were suppressed by truncated SFI5 proteins containing the C-terminal CaM-binding site but not by those without it. The plasma membrane localization of SFI5 and its ability to enhance infection were also perturbed by loss of the CaM-binding site. We conclude that CaM-binding is required for localization and activity of SFI5. We propose that SFI5 suppresses plant immunity by interfering with immune signalling components after activation by CaMs.