Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR‐like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non‐pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen’s host.     

Lessons learned from type III effector transgenic plants

The Gram negative bacterial phytopathogen Pseudomonas syringae employs a molecular syringe termed the type III secretion system (TTSS) to deliver an array of type III secreted effector (TTSE) proteins into plant cells. The major function ascribed to type III effectors of P. syringae is their ability to suppress plant immunity. Because individual pathovars of P. syringae can possess over 30 TTSEs, functional redundancy can provide a hurdle to ascribing functions by TTSE-deletion or -overexpression in such TTSE-rich backgrounds. Approaches to overcome functional redundancy have included the deletion of multiple TTSEs from individual pathovars as well as engineering the plant commensal P. fluorescens strain to express the P. syringae TTSS and deliver P. syringae TTSEs. As we describe here, transgenic Arabidopsis plants expressing individual TTSEs have also been used to overcome problems of functional redundancy and provide invaluable insights into TTSE virulence functions.Key words: pathogen, virulence, effector, plant immunity, HopF2_Pto, RIN4     

Phytophthora infestans RXLR Effector PexRD2 Interacts with Host MAPKKKε to Suppress Plant Immune Signaling

Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity–related signaling.     

Oomycete RXLR effectors: delivery, functional redundancy and durable disease resistance

To manipulate host defences, plant pathogenic oomycetes secrete and translocate RXLR effectors into plant cells. Recent reports have indicated that RXLR effectors are translocated from the extrahaustorial matrix during the biotrophic phase of infection and that they are able to suppress PAMP-triggered immunity. Oomycete genomes contain potentially hundreds of highly diverse RXLR effector genes, providing the potential for considerable functional redundancy and the consequent ability to readily shed effectors that are recognised by plant surveillance systems without compromising pathogenic fitness. Understanding how these effectors are translocated, their precise roles in virulence, and the extent to which functional redundancy exists in oomycete RXLR effector complements, are major challenges for the coming years.     

Distinct Pseudomonas type‐III effectors use a cleavable transit peptide to target chloroplasts

The pathogen Pseudomonas syringae requires a type‐III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type‐III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type‐III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector‐triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP‐triggered immune responses compared with wild‐type plants. An N‐terminal region of HopK1 shares similarity with the corresponding region in the well‐studied type‐III effector AvrRps4; however, their C‐terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N‐terminal regions of these type‐III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4‐induced HR did not. Our results suggest that a primary virulence target of these type‐III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type‐III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity.     

Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ～44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.     

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins

Background

Plants have two related immune systems to defend themselves against pathogen attack. Initially, pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses.

Results

We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling.

Conclusions

Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0492-1) contains supplementary material, which is available to authorized users.     

Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens

Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors—virulence proteins delivered into infected tissues to modulate host functions—contribute to pathogen virulence remains poorly understood. Melampsora larici‐populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein‐tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.     

Screening soybean cyst nematode effectors for their ability to suppress plant immunity

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pathogens of soybeans. SCN is an obligate and sedentary parasite that transforms host plant root cells into an elaborate permanent feeding site, a syncytium. Formation and maintenance of a viable syncytium is an absolute requirement for nematode growth and reproduction. In turn, sensing pathogen attack, plants activate defence responses and may trigger programmed cell death at the sites of infection. For successful parasitism, H. glycines must suppress these host defence responses to establish and maintain viable syncytia. Similar to other pathogens, H. glycines engages in these molecular interactions with its host via effector proteins. The goal of this study was to conduct a comprehensive screen to identify H. glycines effectors that interfere with plant immune responses. We used Nicotiana benthamiana plants infected by Pseudomonas syringae and Pseudomonas fluorescens strains. Using these pathosystems, we screened 51 H. glycines effectors to identify candidates that could inhibit effector-triggered immunity (ETI) and/or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We identified three effectors as ETI suppressors and seven effectors as PTI suppressors. We also assessed expression modulation of plant immune marker genes as a function of these suppressors.     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.     

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.     

454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.     

The Pseudomonas syringae type III effector HopG1 targets mitochondria,alters plant development and suppresses plant innate immunity

The bacterial plant pathogen Pseudomonas syringae uses a type III protein secretion system to inject type III effectors into plant cells. Primary targets of these effectors appear to be effector‐triggered immunity (ETI) and pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). The type III effector HopG1 is a suppressor of ETI that is broadly conserved in bacterial plant pathogens. Here we show that HopG1 from P. syringae pv. tomato DC3000 also suppresses PTI. Interestingly, HopG1 localizes to plant mitochondria, suggesting that its suppression of innate immunity may be linked to a perturbation of mitochondrial function. While HopG1 possesses no obvious mitochondrial signal peptide, its N‐terminal two‐thirds was sufficient for mitochondrial localization. A HopG1–GFP fusion lacking HopG1's N‐terminal 13 amino acids was not localized to the mitochondria reflecting the importance of the N‐terminus for targeting. Constitutive expression of HopG1 in Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Lycopersicon esculentum (tomato) dramatically alters plant development resulting in dwarfism, increased branching and infertility. Constitutive expression of HopG1 in planta leads to reduced respiration rates and an increased basal level of reactive oxygen species. These findings suggest that HopG1's target is mitochondrial and that effector/target interaction promotes disease by disrupting mitochondrial functions.     

The TIR-NBS protein TN13 associates with the CC-NBS-LRR resistance protein RPS5 and contributes to RPS5-triggered immunity in Arabidopsis

Nucleotide-binding site (NBS)–leucine-rich repeat (LRR) domain receptor (NLR) proteins play important roles in plant innate immunity by recognizing pathogen effectors. The Toll/interleukin-1 receptor (TIR)-NBS (TN) proteins belong to a subtype of the atypical NLRs, but their function in plant immunity is poorly understood. The well-characterized Arabidopsis thaliana typical coiled-coil (CC)-NBS-LRR (CNL) protein Resistance to Pseudomonas syringae 5 (RPS5) is activated after recognizing the Pseudomonas syringae type III effector AvrPphB. To explore whether the truncated TN proteins function in CNL-mediated immune signaling, we examined the interactions between the Arabidopsis TN proteins and RPS5, and found that TN13 and TN21 interacted with RPS5. However, only TN13, but not TN21, was involved in the resistance to P. syringae pv. tomato (Pto) strain DC3000 carrying avrPphB, encoding the cognate effector recognized by RPS5. Moreover, the regulation of Pto DC3000 avrPphB resistance by TN13 appeared to be specific, as loss of function of TN13 did not compromise resistance to Pto DC3000 hrcC⁻ or Pto DC3000 avrRpt2. In addition, we demonstrated that the CC and NBS domains of RPS5 play essential roles in the interaction between TN13 and RPS5. Taken together, our results uncover a direct functional link between TN13 and RPS5, suggesting that TN13 acts as a partner in modulating RPS5-activated immune signaling, which constitutes a previously unknown mechanism for TN-mediated regulation of plant immunity.     

Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant–pathogen interactions in gymnosperm forest trees, including kauri.