Oomycete Effectors Research Articles

Proteolytic processing of both RXLR and EER motifs in oomycete effectors.

Arg-any amino acid-Leu-Arg (RXLR) effectors are central oomycete virulence factors that suppress plantimmunity. Relatively little is known about how they are processed post-translationally before delivery into host cells. A range of molecular, cell and biochemical processes were used to investigate proteolytic processing of RXLR and Glu-Glu-Arg (EER) motifs in Phytophthora infestans effectors. Proteolytic cleavage at the RXLR motif occurred before secretion in all effectors tested, suggesting it is a general rule. Cleavage occurred between the leucine and the second arginine. There was no cleavage of a naturally occurring second RXLR motif in a structured region of Pi21388/AvrBlb1, or one introduced at a similar position in effector Pi04314, in keeping with the motif being positionally constrained, potentially to disordered regions closely following the signal peptide. Remarkably, independent proteolytic cleavage of the EER motif, often found immediately after the RXLR, was also observed, occurring immediately after the arginine. Full-length effectors expressed in host plant Nicotiana benthamiana revealed that, although secreted, they were poorly processed, suggesting that RXLR and EER cleavage does not occur in all eukaryotic cells. We conclude that, whether possessing both RXLR and EER, or either motif alone, these effectors are likely generally proteolytically processed before secretion.

A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

Oomycete pathogens deliver many effectors to enhance virulence or suppress plant immunity. Plant immune networks are interconnected, in which a few effectors can trigger a strong defense response when recognized by immunity-related proteins. How effectors activate plant defense response remains poorly understood. Here we report Phytophthora capsici effector RxLR23KM can induce plant cell death and plant immunity. RxLR23KM specifically binds to ERD15La, a regulator of abscisic acid and salicylic acid pathway, and the binding intensity depends on the amino acid residues (K93 and M320). NbNAC68, a downstream protein of ERD15La, can stimulate plant immunity that is compromised after binding with ERD15La. Silencing of NbNAC68 substantially prevents the activation of plant defense response. RxLR23KM binds to ERD15La, releasing NbNAC68 to activate plant immunity. These findings highlight a strategy of plant defense response that ERD15La as a central regulator coordinates RxLR23KM to regulate NbNAC68-triggered plant immunity.

Paths of Least Resistance: Unconventional Effector Secretion by Fungal and Oomycete Plant Pathogens.

Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional endoplasmic reticulum (ER)-Golgi pathway, while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In Magnaporthe oryzae, the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in M. oryzae requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, which is a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in M. oryzae has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide Phytophthora infestans cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Multiple deletions of candidate effector genes lead to the breakdown of partial grapevine resistance to downy mildew.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen.

Chloroplast protein StFC-II was manipulated by a Phytophthora effector to enhance host susceptibility.

Oomycete secretes a range of RxLR effectors into host cells to manipulate plant immunity by targeting proteins from several organelles. In this study, we report that chloroplast protein StFC-II is hijacked by a pathogen effector to enhance susceptibility. Phytophthora infestans RxLR effector Pi22922 is activated during the early stages of P. infestans colonization. Stable overexpression of Pi22922 in plants suppresses flg22-triggered reactive oxygen species (ROS) burst and enhances leaf colonization by P. infestans. A potato ferrochelatase 2 (FC-II, a nuclear-encoded chloroplast-targeted protein), a key enzyme for heme biosynthesis in chloroplast, was identified as a target of Pi22922 in the cytoplasm. The pathogenicity of Pi22922 in plants is partially dependent on FC-II. Overexpression of StFC-II decreases resistance of potato and Nicotiana benthamiana against P. infestans, and silencing of NbFC-II in N. benthamiana reduces P. infestans colonization. Overexpression of StFC-II increases heme content and reduces chlorophyll content and photosynthetic efficiency in potato leaves. Moreover, ROS accumulation both in chloroplast and cytoplasm is attenuated and defense-related genes are down-regulated in StFC-II overexpression transgenic potato and N. benthamiana leaves. Pi22922 inhibits E3 ubiquitin ligase StCHIP-mediated StFC-II degradation in the cytoplasm and promotes its accumulation in chloroplasts. In summary, this study characterizes a new mechanism that an oomycete RxLR effector suppresses host defenses by promoting StFC-II accumulation in chloroplasts, thereby compromising the host immunity and promoting susceptibility.

POOE: predicting oomycete effectors based on a pre-trained large protein language model.

Oomycetes are fungus-like eukaryotic microorganisms which can cause catastrophic diseases in many plants. Successful infection of oomycetes depends highly on their effector proteins that are secreted into plant cells to subvert plant immunity. Thus, systematic identification of effectors from the oomycete proteomes remains an initial but crucial step in understanding plant-pathogen relationships. However, the number of experimentally identified oomycete effectors is still limited. Currently, only a few bioinformatics predictors exist to detect potential effectors, and their prediction performance needs to be improved. Here, we used the sequence embeddings from a pre-trained large protein language model (ProtTrans) as input and developed a support vector machine-based method called POOE for predicting oomycete effectors. POOE could achieve a highly accurate performance with an area under the precision-recall curve of 0.804 (area under the receiver operating characteristic curve = 0.893, accuracy = 0.874, precision = 0.777, recall = 0.684, and specificity = 0.936) in the fivefold cross-validation, considerably outperforming various combinations of popular machine learning algorithms and other commonly used sequence encoding schemes. A similar prediction performance was also observed in the independent test. Compared with the existing oomycete effector prediction methods, POOE provided very competitive and promising performance, suggesting that ProtTrans effectively captures rich protein semantic information and dramatically improves the prediction task. We anticipate that POOE can accelerate the identification of oomycete effectors and provide new hints to systematically understand the functional roles of effectors in plant-pathogen interactions. The web server of POOE is freely accessible at http://zzdlab.com/pooe/index.php. The corresponding source codes and data sets are also available at https://github.com/zzdlabzm/POOE.IMPORTANCEIn this work, we use the sequence representations from a pre-trained large protein language model (ProtTrans) as input and develop a Support Vector Machine-based method called POOE for predicting oomycete effectors. POOE could achieve a highly accurate performance in the independent test set, considerably outperforming existing oomycete effector prediction methods. We expect that this new bioinformatics tool will accelerate the identification of oomycete effectors and further guide the experimental efforts to interrogate the functional roles of effectors in plant-pathogen interaction.

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.

EffectorO: Motif-Independent Prediction of Effectors in Oomycete Genomes Using Machine Learning and Lineage Specificity.

Oomycete plant pathogens cause a wide variety of diseases, including late blight of potato, sudden oak death, and downy mildews of plants. These pathogens are major contributors to loss in numerous food crops. Oomycetes secrete effector proteins to manipulate their hosts to the advantage of the pathogen. Plants have evolved to recognize effectors, resulting in an evolutionary cycle of defense and counter-defense in plant-microbe interactions. This selective pressure results in highly diverse effector sequences that can be difficult to computationally identify using only sequence similarity. We developed a novel effector prediction tool, EffectorO, that uses two complementary approaches to predict effectors in oomycete pathogen genomes: i) a machine learning-based pipeline that predicts effector probability based on the biochemical properties of the N-terminal amino-acid sequence of a protein and ii) a pipeline based on lineage specificity to find proteins that are unique to one species or genus, a sign of evolutionary divergence due to adaptation to the host. We tested EffectorO on Bremia lactucae, which causes lettuce downy mildew, and Phytophthora infestans, which causes late blight of potato and tomato, and predicted many novel effector candidates while recovering the majority of known effector candidates. EffectorO will be useful for discovering novel families of oomycete effectors without relying on sequence similarity to known effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Playing with FIRE: How an RXLR Oomycete Effector Fuels Disease by Hijacking 14-3-3 Proteins.

The plant pathogen Phytophthora palmivora causes rot disease in several monocots and dicots. The plant 14-3-3 proteins are targets of different types of effector molecules secreted by the pathogens. An RXLR-type effector FIRE (14-3-3 interacting RXLR effector) and its target 14-3-3 proteins that localize to haustoria have been identified, pointing to a potential site of interaction. The pathogen hijacks the host 14-3-3 proteins through FIRE-mediated interaction and lowers the immunity for disease progression. The effector FIRE and 14-3-3 interaction deciphered in this study could pave the way for genetic modification of plants with altered 14-3-3 protein for broad host resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

FIRE Mimics a 14-3-3-Binding Motif to Promote Phytophthora palmivora Infection.

The oomycete Phytophthora palmivora infects a wide range of tropical crops worldwide. Like other filamentous plant pathogens, it secretes effectors to colonize plant tissues. Here, we characterize FIRE, an RXLR effector that contains a canonical mode I 14-3-3 phospho-sensor-binding motif that is conserved in effectors of several Phytophthora species. FIRE is phosphorylated in planta and interacts with multiple 14-3-3 proteins. Binding is sensitive to the R18 14-3-3 inhibitor. FIRE promotes plant susceptibility and co-localizes with its target around haustoria. This work uncovers a new type of oomycete effector target mechanism. It demonstrates that substrate mimicry for 14-3-3 proteins is a cross-kingdom effector strategy used by both prokaryotic and eukaryotic plant pathogens to suppress host immunity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Molecular characterisation and structural assessment of an RXLR effector from Phytophthora palmivora, the coconut bud rot pathogen

Phytophthora species are phytopathogenic oomycetes that damage a wide variety of crops. Phytophthora delivers effectors, which are secretory proteins, into the host cells. Effectors promote infection by reprogramming the host cellular machinery and are key determinants of oomycete virulence. The major class of Phytophthora effector proteins contains the RXLR motif. In this study, we have carried out the molecular and structural characterisation of an RXLR effector (RXLR6744) from a virulent P. palmivora isolated from bud rot disease-affected coconut palm. The open reading frame (ORF) of the RXLR6744, amplified using RT-PCR, had a length of 411 bp. The gene was found to encode a predicted protein of 136 amino acids and had a molecular weight of 15.52 kDa. Phylogenetic analysis of the amino acid sequence revealed that it was closely related to RXLR proteins from P. palmivora (causing black pod disease in cocoa) and related species P. megakarya. Topology analysis revealed that the protein was composed of six α-helices. The structural prediction was undertaken by computer-aided homology modelling. From the Ramachandran plot analysis, it could be observed that the majority (96.3%) of amino acids were present in the preferred region, 3.7 per cent of amino acid residues were present in the allowed region, and no residues were observed in the disallowed region. The structure showed an average quality of 94.4 per cent, indicating it to be a high-quality structure. This study provides the detailed characterisation of an RXLR effector from P. palmivora. It will aid the elucidation of its role in pathogenesis and facilitate further refined investigations of the structure/function relationships of oomycete effectors.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

A Phytophthora effector promotes homodimerization of host transcription factor StKNOX3 to enhance susceptibility.

Oomycete pathogens secrete hundreds of cytoplasmic RxLR effectors to modulate host immunity by targeting diverse plant proteins. Revealing how effectors manipulate host proteins is pivotal to understanding infection processes and to developing new strategies to control plant disease. Here we show that the Phytophthora infestans RxLR effector Pi22798 interacts in the nucleus with a potato class II knotted-like homeobox (KNOX) transcription factor, StKNOX3. Silencing the ortholog NbKNOX3 in Nicotiana benthamiana reduces host colonization by P. infestans, whereas transient and stable overexpression of StKNOX3 enhances infection. StKNOX3 forms a homodimer which is dependent on its KNOX II domain. The KNOX II domain is also essential for Pi22798 interaction and for StKNOX3 to enhance P. infestans colonization, indicating that StKNOX3 homodimerization contributes to susceptibility. However, critically, the effector Pi22798 promotes StKNOX3 homodimerization, rather than heterodimerization to another KNOX transcription factor StKNOX7. These results demonstrate that the oomycete effector Pi22798 increases pathogenicity by promoting homodimerization specifically of StKNOX3 to enhance susceptibility.

Transgenic Soybeans Expressing Phosphatidylinositol-3-Phosphate-Binding Proteins Show Enhanced Resistance Against the Oomycete Pathogen Phytophthora sojae

Oomycete and fungal pathogens cause billions of dollars of damage to crops worldwide annually. Therefore, there remains a need for broad-spectrum resistance genes, especially ones that target pathogens but do not interfere with colonization by beneficial microbes. Motivated by evidence suggesting that phosphatidylinositol-3-phosphate (PI3P) may be involved in the delivery of some oomycete and fungal virulence effector proteins, we created stable transgenic soybean plants that express and secrete two different PI3P-binding proteins, GmPH1 and VAM7, in an effort to interfere with effector delivery and confer resistance. Soybean plants expressing the two PI3P-binding proteins exhibited reduced infection by the oomycete pathogen Phytophthora sojae compared to control lines. Measurements of nodulation by nitrogen-fixing mutualistic bacterium Bradyrhizobium japonicum, which does not produce PI3P, revealed that the two lines with the highest levels of GmPH1 transcripts exhibited reductions in nodulation and in benefits from nodulation. Transcriptome and plant hormone measurements were made of soybean lines with the highest transcript levels of GmPH1 and VAM7, as well as controls, following P. sojae- or mock-inoculation. The results revealed increased levels of infection-associated transcripts in the transgenic lines, compared to controls, even prior to P. sojae infection, suggesting that the plants were primed for increased defense. The lines with reduced nodulation exhibited elevated levels of jasmonate-isoleucine and of transcripts of a JAR1 ortholog encoding jasmonate-isoleucine synthetase. However, lines expressing VAM7 transgenes exhibited normal nodulation and no increases in jasmonate-isoleucine. Overall, together with previously published data from cacao and from P. sojae transformants, the data suggest that secretion of PI3P-binding proteins may confer disease resistance through a variety of mechanisms.

Virulence strategies of an insect herbivore and oomycete plant pathogen converge on host E3 SUMO ligase SIZ1.

Summary Pathogens and pests secrete proteins (effectors) to interfere with plant immunity through modification of host target functions and disruption of immune signalling networks. The extent of convergence between pathogen and herbivorous insect virulence strategies is largely unexplored.We found that effectors from the oomycete pathogen, Phytophthora capsici, and the major aphid pest, Myzus persicae target the host immune regulator SIZ1, an E3 SUMO ligase. We used transient expression assays in Nicotiana benthamiana as well as Arabidopsis mutants to further characterize biological role of effector–SIZ1 interactions in planta. We show that the oomycete and aphid effector, which both contribute to virulence, feature different activities towards SIZ1. While M. persicae effector Mp64 increases SIZ1 protein levels in transient assays, P. capsici effector CRN83_152 enhances SIZ1‐E3 SUMO ligase activity in vivo.SIZ1 contributes to host susceptibility to aphids and an oomycete pathogen. Knockout of SIZ1 in Arabidopsis decreased susceptibility to aphids, independent of SNC1, PAD4 and EDS1. Similarly SIZ1 knockdown in N. benthamiana led to reduced P. capsici infection.Our results suggest convergence of distinct pathogen and pest virulence strategies on an E3 SUMO ligase to enhance host susceptibility.

EffectorP 3.0: Prediction of Apoplastic and CytoplasmicEffectors in Fungi and Oomycetes.

Many fungi and oomycete species are devasting plant pathogens. These eukaryotic filamentous pathogens secrete effector proteins to facilitate plant infection. Fungi and oomycete pathogens have diverse infection strategies and their effectors generally do not share sequence homology. However, they occupy similar host environments, either the plant apoplast or plant cytoplasm, and, therefore, may share some unifying properties based on the requirements of these host compartments. Here, we exploit these biological signals and present the first classifier (EffectorP 3.0) that uses two machine-learning models: one trained on apoplastic effectors and one trained on cytoplasmic effectors. EffectorP 3.0 accurately predicts known apoplastic and cytoplasmic effectors in fungal and oomycete secretomes with low estimated false-positive rates of 3 and 8%, respectively. Cytoplasmic effectors have a higher proportion of positively charged amino acids, whereas apoplastic effectors are enriched for cysteine residues. The combination of fungal and oomycete effectors in training leads to a higher number of predicted cytoplasmic effectors in biotrophic fungi. EffectorP 3.0 expands predicted effector repertoires beyond small, cysteine-rich secreted proteins in fungi and RxLR-motif containing secreted proteins in oomycetes. We show that signal peptide prediction is essential for accurate effector prediction, because EffectorP 3.0 recognizes a cytoplasmic signal also in intracellular, nonsecreted proteins.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An oomycete effector targets a plant RNA helicase involved in root development and defense.

Oomycete plant pathogens secrete effector proteins to promote disease. The damaging soilborne legume pathogen Aphanomyces euteiches harbors a specific repertoire of Small Secreted Protein effectors (AeSSPs), but their biological functions remain unknown. Here we characterize AeSSP1256. The function of AeSSP1256 is investigated by physiological and molecular characterization of Medicago truncatula roots expressing the effector. A potential protein target of AeSSP1256 is identified by yeast-two hybrid, co-immunoprecipitation, and fluorescent resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) assays, as well as promoter studies and mutant characterization. AeSSP1256 impairs M. truncatula root development and promotes pathogen infection. The effector is localized to the nucleoli rim, triggers nucleoli enlargement and downregulates expression of M. truncatula ribosome-related genes. AeSSP1256 interacts with a functional nucleocytoplasmic plant RNA helicase (MtRH10). AeSSP1256 relocates MtRH10 to the perinucleolar space and hinders its binding to plant RNA. MtRH10 is associated with ribosome-related genes, root development and defense. This work reveals that an oomycete effector targets a plant RNA helicase, possibly to trigger nucleolar stress and thereby promote pathogen infection.

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

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

Oomycete intracellular effectors: specialised weapons targeting strategic plant processes.

Oomycete phytopathogens have adapted to colonise plants using effectors as their molecular weapons. Intracellular effectors, mostly proteins but also small ribonucleic acids, are delivered by the pathogens into the host cell cytoplasm where they interfere with normal plant physiology. The diverse host processes emerging as 'victims' of these 'specialised bullets' include gene transcription and RNA-mediated silencing, cell death, protein stability, protein secretion and autophagy. Some effector targets are directly involved in defence execution, while others participate in fundamental metabolisms whose alteration collaterally affects defences. Other effector targets are susceptibility factors (SFs), that is host components that make plants vulnerable to pathogens. SFs are mostly negative regulators of immunity, but some seem necessary to sustain or promote pathogen colonisation.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.