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

['Lida Derevnina', 'The Sainsbury Laboratory', 'University Of East Anglia', 'Norwich', 'United Kingdom', 'Mauricio P. Contreras', 'Hiroaki Adachi', 'Jessica Upson', 'Angel Vergara Cruces', 'Department Of Biology']

Date: 2021-10

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.

Funding: This work was funded by the Gatsby Charitable Foundation (Core grant to TSL) and Biotechnology and Biological Sciences Research Council (BBSRC, UK) BB/P012574 and BB/V002937/1. S.K. also receives funding from the European Research Council (ERC NGRB and BLASTOFF projects). L.D. was funded by a Marie Sklodowska-Curie actions fellowship (BoostR), H.A. was funded by the Japan Society for the Promotion of Sciences Postdoctoral fellowship, A.V.C. was funded by a British Society for Plant Pathology summer bursary, J.U. was funded by the Gatsby Charitable Foundation PhD studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The NRC superclade is massively expanded in solanaceous plants, where it comprises up to half of the NLRome in some species [ 24 ]. Given this prevalence, we hypothesised that pathogens convergently evolved to secrete effectors that target the NRC network. In this study, we screened collections of effector proteins from 6 major parasites of solanaceous plants, i.e., the bacterium Pseudomonas syringae, the oomycete Phytophthora infestans, the cyst nematodes Globodera rostochiensis and Globodera pallida, and the aphids Myzus persicae and Acrythosiphon pisum, for their capacity to suppress the hypersensitive cell death triggered by the NRC-dependent sensor NLRs Prf and Rpi-blb2. These screens revealed 5 effectors as suppressors of components of the NRC network. Among these, the oomycete protein PITG-16705 (PBHR_012, henceforth, AVRcap1b) [ 32 ] and the cyst nematode protein SPRYSEC15 (where the term SPRYSEC will be referred to as SS from here on in) stood out for being able to specifically suppress the response triggered by autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they are able to counteract a subset of NRC helper proteins independently of their sensor NLR partners. Further studies revealed that AVRcap1b and SS15 suppress NRC2 and NRC3 through different mechanisms. While AVRcap1b suppression of NRC2 and NRC3 mediated immunity is dependent on the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a), SS15 directly binds the NB-ARC domain of NRC2 and NRC3 to perturb their function. We conclude that evolutionarily divergent plant pathogens have convergently evolved distinct molecular strategies to counteract central nodes of the NRC immune receptor network. Coevolution with pathogen effectors may, therefore, underpin the diversification of NRCs as functionally redundant nodes in a massively expanded NLR network.

Until very recently, the molecular mechanisms that underpin plant NLR activation and the subsequent execution of NTI pathways and hypersensitive cell death have remained unknown. The recent elucidation of the CC-NLR ZAR1 (HopZ-activated resistance 1) and the TIR-NLRs ROQ1 (Recognition of XopQ 1) and RPP1 (Recognition of Peronospora parasitica 1) structures have resulted in a new conceptual framework [ 27 – 30 ]. Activated NLRs oligomerize into a wheel-like multimeric complex known as the resistosome. In the case of ZAR1, activation induces conformational changes in the NB-ARC domain resulting in ADP release followed by dATP/ATP binding and oligomerization of ZAR1 and its partner host proteins into the pentameric resistosome structure [ 28 , 31 ]. ZAR1 resistosomes expose a funnel-shaped structure formed by the N-terminal α1 helices, which was proposed to perturb plasma membrane integrity to trigger hypersensitive cell death [ 28 , 29 ]. This α1 helix is defined by a molecular signature, the MADA motif, which is present in approximately 20% of angiosperm CC-NLRs, including NRCs [ 25 ]. This implies that the biochemical “death switch” mechanism of the ZAR1 resistosome probably applies to NRCs and other MADA motif containing NLRs across diverse plant taxa.

Plant genomes may encode anywhere between 50 and approximately 1,000 NLRs [ 17 , 22 ]. Some of these NLRs are functional singletons operating as single biochemical units, while others function in pairs or in more complex receptor networks [ 23 ]. Paired and networked NLRs consist of functionally specialised sensor NLRs that detect pathogen effectors and helper NLRs that translate this effector recognition into hypersensitive cell death and resistance. In the Solanaceae, a major phylogenetic clade of NLRs forms a complex immunoreceptor network in which multiple helper CC-NLRs, known as NLR required for cell death (NRCs), are necessary for a large number of sensor NLRs [ 24 ]. These sensor NLRs, encoded by R gene loci, confer resistance against diverse pathogens, such as viruses, bacteria, oomycetes, nematodes, and insects [ 24 ] ( S1B Fig ). Together, NRCs and their NLR partners form the NRC superclade, a well-supported phylogenetic cluster divided into an NRC helper clade and 2 larger clades that include all known NRC-dependent sensor NLRs [ 24 , 25 ]. The NRC superclade emerged over 100 million years ago (Mya) from an NLR pair that expanded to make up a significant fraction of the NLRome of asterid plant species [ 24 ]. The current model is that NRCs form redundant central nodes in this massively diversified bow tie network, with different NRCs exhibiting some specificity towards their sensor NLR partners. For example, whereas the bacterial resistance protein Prf requires NRC2 and NRC3 in a genetically redundant manner, the potato blight resistance protein Rpi-blb2 is dependent only on NRC4 [ 24 , 26 ] ( S1B Fig ).

NLRs are multidomain proteins with a nucleotide-binding domain shared with APAF-1, various R-proteins, and CED-4 (NB-ARC) and at least 1 additional domain [ 16 ]. The C-terminus of plant NLRs is generally a leucine-rich repeat (LRR) domain, but they can be sorted into phylogenetic subgroups with distinct N-terminal domains [ 16 – 18 ]. In angiosperms, NLRs form several major phylogenetic subgroups, including TIR-NLRs with an N-terminal Toll/interleukin-1 receptor (TIR) domain, CC-NLRs, with the Rx-type coiled-coil (CC) domain, CC R -NLRs with the RPW8-type CC (CC R ) domain, and the CC G10 -NLR subclade with a distinct type of CC (CC A or CC G10 ) domain. Of these, CC-NLRs are the most common type, forming the largest group of NLRs in angiosperms [ 16 , 18 , 19 ]. The TIR- and CC-type N-terminal domains are involved in downstream immune signalling, oligomerization, and cell death execution [ 20 ]. The central NB-ARC domain exhibits ATP binding and hydrolysis activities and functions as a molecular switch that determines the NLR inactive/active status [ 21 ]. Finally, the LRR domain can mediate effector perception and often engages in intramolecular interactions [ 8 , 21 ].

Most effectors studied to date suppress immune pathways induced by pathogen-associated molecular patterns (PAMPs). This so-called PAMP or pattern recognition receptor (PRR)-triggered immunity (PTI) response is mediated by cell surface PRRs [ 3 , 4 ] ( S1A Fig ). A subset of effectors, however, have avirulence (AVR) activity and inadvertently activate intracellular immune receptors of the nucleotide-binding, leucine-rich repeat (NLR) class of proteins, a response known as effector- or NLR-triggered immunity (ETI/NTI) [ 5 – 7 ]. In plants, recognition of AVR effectors by NLRs can occur either directly or indirectly following various mechanistic models [ 8 , 9 ]. NTI is usually accompanied by a localised form of programmed cell death known as the hypersensitive response (HR) that hinders disease progression [ 6 , 7 , 10 , 11 ]. NLR-mediated immunity activated by AVR effectors can also be suppressed by other effectors [ 12 ]. However, in sharp contrast to the widely studied PTI-suppressing effectors, the mechanisms by which effectors suppress NLR responses remain poorly understood [ 3 , 13 , 14 ]. Therefore, understanding how effectors suppress NLR functions should provide important insights into the black box of how these immune receptors activate cell death and innate immunity, one of the major unsolved questions in the field of plant pathology [ 15 ].

Our view of the pathogenicity mechanisms of plant pathogens and pests has significantly broadened over the years. Parasites as diverse as bacteria, oomycetes, nematodes, and aphids turned out to be much more sophisticated manipulators of their host plants than initially anticipated. Indeed, it is now well established that these parasites secrete an arsenal of proteins, termed effectors, which modulate plant responses, such as innate immunity, to enable host infection and colonisation. As a consequence, deciphering the biochemical activities of effectors to understand how parasites successfully colonise and reproduce has become a major conceptual paradigm in the field of molecular plant pathology [ 1 , 2 ]. In fact, effectors have emerged as molecular probes that can be utilised to unravel novel components and processes of the host immune system [ 2 ].

CoIP experiment between NbTOL9a and NRCs in the presence and absence of AVRcap1b. C-terminally 6xHA-tagged NbTOL9a was transiently coexpressed with NRC2, NRC3, and NRC4 proteins fused to a C-terminal 4xmyc tag in the presence of free GFP (EV:GFP) or N-terminally GFP-tagged AVRcap1b. C-terminally 4xmyc-tagged AVRcap1b was used as a positive control for association with NbTOL9a. IP was performed with agarose beads conjugated to MYC (MYC-IP) antibodies. Total protein extracts were immunoblotted with appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the left. Rubisco loading controls were conducted using Pierce staining. This experiment is representative of 2 independent replicates. coIP, co-immunoprecipitation; IP, immunoprecipitation; NRC, NLR required for cell death.

Given that NbTOL9a negatively regulates NRC2 and NRC3, we hypothesised that NbTOL9a associates with NRC2 and NRC3 to execute its immunomodulatory functions. Furthermore, since AVRcap1b requires NbTOL9a to fully suppress NRC3, AVRcap1b may act as a suppressor through altering NbTOL9a–NRC associations. To test these hypotheses, we coexpressed NbTOL9a::6xHA with NRC2::4xMyc, NRC3::4xMyc, and NRC4::4xMyc, either in the presence of GFP::AVRcap1b or free GFP in N. benthamiana leaves using agroinfiltration, and performed anti-Myc immunoprecipitation followed by western blot analyses. We included AVRcap1b::4xMyc as a positive control for association with NbTOL9a. While we were able to successfully detect association between NbTOL9a:6xHA and AVRcap1b::4xMyc, we did not observe any association between NbTOL9a:6xHA and any of the 3 NRC proteins tested, regardless of whether GFP::AVRcap1b was present or absent ( Fig 13 ). These results suggest that NbTOL9a-mediated negative regulation of NRC2 and NRC3 does not involve association between these 2 proteins in planta or, alternatively, involves protein–protein interactions that are too transient to be detected by coIP. Moreover, we conclude that AVRcap1b does not alter NbTOL9a–NRC associations (or lack thereof) to execute its immune suppression activities.

(A) Photo of representative N. benthamiana leaves showing HR after coexpression of RNAi::GUS and RNAi::NbTOL9a with NRC3 D480V + EV, NRC3 D480V + AVRcap1b, NRC4 D478V + EV, and NRC4 D478V + AVRcap1b. HR response was scored and photographed 5 days after agroinfiltration (left panel under white light, right panel autofluorescence under UV light) ( S12 Data ). (B) HR results are presented as dot plots, where the size of each dot is proportional to the number of samples with the same score (count). Results are based on 3 biological replicates. Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in ( S16 Fig ). HR, hypersensitive response.

Our finding that NbTOL9a exhibits a negative regulatory role in NRC2- and NRC3-mediated cell death led us to the hypothesis that AVRcap1b is co-opting NbTOL9a to execute its suppression activity. To test this, we coexpressed AVRcap1b with the autoimmune mutants NRC3 D480V or NRC4 D478V in N. benthamiana leaves that are expressing either RNAi::NbTOL9a (NbTOL9a silenced) or RNAi::GUS (negative control). Consistent with Fig 2 , overexpression of AVRcap1b suppressed the cell death triggered by NRC3 D480V but not by NRC4 D478V ( Fig 12 , S12 Data ). However, silencing of NbTOL9a compromised AVRcap1b suppression of NRC3 D480V autoimmunity and partially restored the cell death phenotype ( Fig 12 , S12 Data ). These results indicate that AVRcap1b genetically requires NbTOL9a to fully down-regulate NRC3 cell death activity and is likely co-opting this host protein to execute its suppression activities.

(A) Photo of representative N. benthamiana leaves showing HR after coexpression of EV and NbTOL9a (labelled above leaf panels) with MEK2 DD , NRC3 D480V , and NRC4 D478V . HR response was scored and photographed 5 days after agroinfiltration (left panel under white light, right panel autofluorescence under UV light). MEK2 DD was included as a positive control for cell death ( S11 Data ). (B) HR results are presented as dot plots, where the size of each dot is proportional to the number of samples with the same score (count). Three biological replicates were completed, indicated by columns for EV, NbTOL9a in each treatment (MEK2 DD , NRC3 D480V , NRC4 D478V ). Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in ( S15 Fig ). EV, empty vector; HR, hypersensitive response.

Next, we determined the effect of NbTOL9a overexpression by coexpressing it with the autoimmune NRCs in N. benthamiana leaves using agroinfiltration. Since NRC2 H480R autoimmune response is comparably weaker than NRC3 D480V , we focused on NRC3 D480V in this and subsequent experiments as it provides a more robust readout for cell death–based assays. NbTOL9a overexpression reduced the cell death response triggered by NRC3 D480V but did not affect NRC4 D478V or the constitutively active MEK2 DD , a mitogen-activated protein kinase kinase (MAPKK) involved in plant immune signalling, which we included as an additional control (Figs 11 and S15 and S11 Data ). Altogether, these 2 sets of experiments indicate that NbTOL9a modulates NRC2 and NRC3 activities in a manner consistent with a negative regulatory role in NRC2- and NRC3-mediated immunity.

(A) Photo of representative N. benthamiana leaves showing HR after coexpression of NRC2 H480R , NRC3 D480V , NRC4 D478V , and NbZAR1 D481V with RNAi::GUS (control) and RNAi:NbTOL9a (labelled above leaf panels). To improve the robustness of the assay, we used increasing concentrations of A. tumefaciens expressing NRC2 H480R , NRC3 D480V , NRC4 D478V , and NbZAR1 D481V (OD 600 = 0.1, 0.25, or 0.5) ( S10 Data ). HR response was scored and photographed 5 days after agroinfiltration. (B) HR results are presented as dot plots, where the size of each dot is proportional to the number of samples with the same score (count). Three biological replicates were completed, indicated by columns for RNAi::GUS and RNAi::NbTOL9a, for each treatment combination. Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). The details of statistical analysis are presented in ( S14 Fig ). HR, hypersensitive response.

To gain additional insights into the role of NbTOL9a in NRC-mediated hypersensitive cell death, we altered NbTOL9a expression in N. benthamiana. First, we investigated the effect of silencing NbTOL9a on NRC autoimmunity. We generated a hairpin-silencing construct (RNAi::NbTOL9a) that mediates silencing of NbTOL9a in transient expression assays in N. benthamiana leaves ( S13 Fig ). We then coexpressed RNAi::NbTOL9a with NRC2 H480R and NRC3 D480V using agroinfiltration of N. benthamiana leaves to test the degree to which silencing of NbTOL9a affects NRC2- and NRC3-mediated cell death. To improve robustness of the assay, we used increasing concentrations of A. tumefaciens expressing NRC2 H480R and NRC3 D480V (OD 600 = 0.1, 0.25, or 0.5). Silencing of NbTOL9a at all tested OD 600 concentrations enhanced the cell death response triggered by NRC2 H480R and NRC3 D480V but did not affect NRC4 D478V or NbZAR1 D481V (NRC-independent NLR; [ 54 ]), compared to the RNAi::GUS silencing control (Figs 10 and S14 and S10 Data).

Computational analyses of the N. benthamiana genome revealed 4 TOL paralogs in addition to NbTOL9a, which we termed NbTOL9b (Nbv6.1trP9166), NbTOL3 (Nbv6.1trA40123), NbTOL6 (Nbv6.1trP73492), and NbTOL9c (Nbv6.1trA64113) following previously published nomenclature ( S5 Table , S12 Fig ) [ 53 ]. To validate the association between AVRcap1b and NbTOL proteins, we coexpressed GFP::AVRcap1b with C-terminally 6xHA-tagged fusions of the 5 TOL paralogs, in N. benthamiana leaves, and performed anti-GFP and anti-HA immunoprecipitations. AVRcap1b associated with NbTOL9a and, to a lesser extent, with NbTOL9b and NbTOL9c in the GFP pulldown (GFP IP). However, AVRcap1b only associated with NbTOL9a in the reciprocal HA pulldown (HA IP) ( Fig 9B ). In both experiments, NbTOL9a protein did not associate with the negative control GFP::PexRD54. These results indicate that AVRcap1b associates with members of the NbTOL family, exhibiting a stronger affinity for NbTOL9a. Based on this conclusion, we focused subsequent experiments on NbTOL9a.

(A) Schematic diagram of domain organisation of NbTOL9a, showing Y2H hits from the screen ( S2 Table ). (B) CoIP experiment between AVRcap1b and 5 NbTOL family proteins (NbTOL9a, NbTOL9b, NbTOL3, NbTOL6, and NbTOL9c). N-terminally GFP-tagged AVRcap1b was transiently coexpressed with all 5 NbTOL proteins fused to a C-terminal 6xHA tag. N-terminally GFP-tagged PexRD54 was used as a negative control. IP were performed with agarose beads conjugated to either GFP (GFP-IP) or HA (HA-IP) antibodies. Total protein extracts were immunoblotted with appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Rubisco loading controls were conducted using Pierce staining. This experiment is representative of 3 independent replicates. coIP, co-immunoprecipitation; IP, immunoprecipitation; NbTOL, N. benthamina Target of Myb 1-like protein; Y2H, yeast two-hybrid.

Given that AVRcap1b did not interact with NRCs, we reasoned that this effector targets another host protein that is involved in NLR immunity. To narrow down the list of 13 candidate interactors obtained from the Y2H screen, we subjected AVRcap1b to in planta coIP coupled with tandem mass spectrometry (IP-MS) using methods that are well established in our laboratory [ 48 – 50 ]. IP-MS experiments with GFP::AVRcap1b resulted in the identification of 8 unique AVRcap1b interactors that were not recovered with the control GFP::PexRD54, another P. infestans effector of a similar fold and size ( S11 Fig , S4 Table ). Three of the 8 interactors were different members of the Target of Myb 1-like protein (TOL) family of ENTH/VHS-GAT domain-containing proteins that function in membrane trafficking as part of the endosomal sorting complex required for transport (ESCRT) pathway [ 51 , 52 ] (Figs 9A and S10 and S4 Table ). One of these candidate TOLs, Nbv6.1trP4361, was independently recovered in the Y2H screen ( S2 Table ). Therefore, we decided to further investigate the corresponding protein (hereafter referred to as NbTOL9a) as a candidate host target of AVRcap1b.

N-terminally 4xHA-tagged SS15 was transiently coexpressed in N. benthamiana with C-terminally 4xMyc tagged-NRC2, NRC3, NRC4, NRC2 H480R , NRC3 D480V , and NRC4 D478V . IP was performed with agarose beads conjugated to Myc (Myc IP) antibodies. Total protein extracts (Input) and proteins obtained by coIP were immunoblotted with appropriate antisera labelled on the right. Approximate molecular weights (kDa) of the proteins are shown on the right. Rubisco loading controls were conducted using Pierce staining. This experiment is representative of 2 independent replicates. coIP, co-immunoprecipitation; IP, immunoprecipitation.

We next investigated the extent to which SS15 associates with activated NRC2 and NRC3. We coexpressed 4xHA::SS15 with C-terminally 4xMyc-tagged autoimmune forms of NRC2 H480R and NRC3 D480V in N. benthamiana using agroinfiltration and subjected the protein extracts to anti-Myc immunoprecipitation and western blot analyses. We found that SS15 co-immunoprecipitated with NRC2 H480R , indicating that this effector associates with activated forms of NRC2 ( Fig 8 ). Our results for NRC3 D480V , however, were inconclusive, since this protein displayed poor accumulation and could not be detected in western blot analyses under these conditions. Despite this, our overall conclusion is that SS15 binds both inactive and activated forms of NRC2.

The p-loop motif within the NB-ARC domain of NLR proteins is crucial for ATP binding and hydrolysis, a biochemical step that is essential for NLR oligomerisation and activation [ 47 ]. To determine whether SS15 associates with p-loop mutants of the NRCs, we performed coIP experiments in N. benthamiana with the NRC p-loop mutants NRC2 K188R , NRC3 K191R , and NRC4 K190R [ 24 ]. These experiments revealed that SS15 associates with the p-loop mutants of NRC2 (NRC2 K188R ) and NRC3 (NRC3 K191R ) ( S10 Fig ). Since an intact p-loop is not required for SS15-NRC association, our findings suggest that SS15 can probably enter in complex with inactive forms of NRC2 and NRC3.

To further examine the association between SS15 and the NB-ARC domains of NRCs, we purified these proteins for in vitro assays using Escherichia coli as a heterologous expression system. We successfully obtained homogeneous SS15 and NRC3 NB-ARC domain (NRC3 NB-ARC ) proteins but were unable to obtain purified NRC2 NB-ARC or NRC4 NB-ARC domains due to solubility and stability issues. We subjected purified SS15 and NRC3 NB-ARC to gel filtration and found that these proteins elute at 239 ml and 227 ml, which correspond to 42.52 and 27.75 kDa, respectively ( Fig 7 ). To determine whether the 2 proteins form a complex in vitro, we mixed cells expressing individual proteins and copurified SS15 and NRC3 NB-ARC for gel filtration assays (see Protein–protein interaction studies: Protein purification from E. coli and in vitro protein–protein interaction studies). The copurified mixture of SS15 and NRC3 NB-ARC resulted in a peak shift with an elution volume at 211 ml, which corresponds to 84.02 kDa. Further validation by SDS-PAGE of the fractions under the new peak confirmed the presence of both proteins ( Fig 7 ). In our gel filtration assays, the protein molecular weight calibration led to overestimates of the predicted molecular masses of the proteins, both alone and in complex (NRC3 NB-ARC is 41 kDa, SS15 is 24.5 kDa, and NRC3 NB-ARC –SS15 complex is 65.5 kDa). However, the results indicate that monomeric forms of each state exist in solution and that the 2 proteins probably enter in a 1:1 complex under these experimental conditions. Taken together, these results suggest that SS15 forms a complex with NRC NB-ARC in vitro.

The Y2H results prompted us to investigate the association between AVRcap1b, SS15, and NRCs using co-immunoprecipitation (coIP) of proteins expressed in N. benthamiana, which should reveal the association between the effectors and full-length NRC proteins in a more physiologically relevant condition. To achieve this, we coexpressed each of AVRcap1b::6xHA and 4xHA::SS15 with NRC2::4xMyc, NRC3::4xMyc, and NRC4::4xMyc in N. benthamiana leaves using agroinfiltration and subjected the protein extracts to anti-Myc immunoprecipitation and western blot analyses. These experiments revealed that SS15 co-immunoprecipitated with NRC2 and NRC3, but not with NRC4 ( Fig 6B ). While we did detect association between NRC4 and SS15 in Y2H, we could only detect a weak NRC4 signal in some biological replicates, suggesting that SS15 may also associate with NRC4 in planta but with markedly lower affinity compared to NRC2 and NRC3 ( S9 Fig ). This result aligns with our observation that SS15 is unable to suppress NRC4 in cell death assays. In the case of AVRcap1b, the coIP experiments were consistent with the Y2H screens as we did not detect an association between AVRcap1b and any of the 3 NRCs ( Fig 6B ).

To further investigate the interactions between SS15 and NRCs, we sought out to delimit the binding domain within the NLRs. Since the prey fragment hits from the Y2H screen covered the CC-NB-ARC domain of NRC3 and NRC4 ( Fig 6A ), we generated CC and NB-ARC domain truncations and tested them in additional Y2H experiments for interaction with SS15. These assays revealed that SS15 binds to the NB-ARC but not the CC domains of NRC2, NRC3, and NRC4 ( S8 Fig ). Consistent with earlier observations, AVRcap1b did not interact with either the CC or NB-ARC domains of the NRCs in these Y2H experiments ( S8 Fig ).

(A) Schematic diagram of domain organisation of NRC proteins, showing Y2H hits from the Hybrigenics services screen ( S3 Table ). (B) CoIP experiment of C-terminally 4xmyc-tagged NRC2, NRC3, and NRC4 with C-terminally HA-tagged AVRcap1b::6xHA and N-terminally tagged 4xHA:SS15 (labelled above). Proteins obtained by coIP with MYC beads (MYC IP) and total protein extracts (input) were immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Rubisco loading control was carried out using Pierce staining. The experiment was performed more than 3 times under different pulldown conditions with similar results. CC, coiled-coil; coIP, co-immunoprecipitation; HD1, helical domain 1; LRR, leucine-rich repeat; NBD, nucleotide-binding domain; NRC, NLR required for cell death; WHD, winged helix domain; Y2H, yeast two-hybrid.

To investigate how AVRcap1b and SS15 suppress NRC activities, we set out to identify their host interactors. We used the effectors as baits in unbiased yeast two-hybrid (Y2H) screens against a N. benthamiana–mixed tissue cDNA library (ULTImate Y2H, Hybrigenics Services, Paris, France). AVRcap1b was screened against a combined approximately 140 million clones, which resulted in 13 candidate interacting proteins from a total of 35 positive clones ( S2 Table ). SS15 was screened against approximately 61 million clones resulting in 10 candidate proteins from a total of 202 positive clones ( S3 Table ). Remarkably, NRC3 and NRC4 were among the recovered SS15 protein interactors, which is notable given that NLR proteins are rarely recovered from Y2H screens. The NRC3 and NRC4 fragments that were recovered from the Y2H screen matched the CC-NB-ARC domains indicating that SS15 may bind the N-terminal half of NRC proteins ( Fig 6A , S3 Table ). NRC2 was not recovered as an interactor in the Y2H screen for SS15; however, we cannot rule out that this might be due to poor accumulation of this NLR or its subdomains in the yeast strains used.

(A) Toothpick inoculation method previously described by Wu and colleagues allowed examination of the spread of trailing necrotic lesions due to the partial resistance mediated by Rx. EV, NRC2, NRC3, or NRC4 were silenced individually or in combination in Rx-transgenic N. benthamiana plants by TRV. (B) EV or AVRcap1b and (C) EV or SS15 were expressed, via agroinfiltration, in leaves of the NRC-silenced plants 1 day before PVX inoculation. PVX-GFP (pGR106-GFP) was inoculated using the toothpick inoculation method, as per panel (A). Photographs were taken 12–16 days after PVX inoculation. The size of the necrotic lesions was measured using Fiji (previously ImageJ) ( S8 Data ). Data acquired from different biological replications are presented in different colours. Statistical differences among the samples were analysed with mixed model ANOVA and a Tukey HSD test (p-value < 0.01), where the fixed effect is the silencing treatment (TRV:EV, TRV:NRC2/3, TRV:NRC4, TRV:NRC2/3/4) with either EV or AVRcap1b and the random effect is the experimental replicate. Significant differences between the conditions are indicated with asterisks (***, p-value < 0.0001) ( S9 Data ). Representative phenotypes observed for each treatment are presented below the boxplots. Immunoblot analysis of GFP accumulation of pGR106-GFP toothpick inoculated sites in the presence of (D) AVRcap1b and (E) SS15 in Rx-transgenic or WT N. benthamiana plants. AVR, avirulence; CBB, Coomassie brilliant blue; EV, empty vector; HSD, honestly significant difference; NRC, NLR required for cell death; PVX, Potato virus X; TRV, Tobacco rattle virus; WT, wild type.

We previously showed that cosilencing of NRC2, NRC3, and NRC4 not only compromised Rx-mediated hypersensitive cell death but also abolished extreme resistance to PVX, leading to trailing necrotic lesions indicative of virus spread [ 24 ]. To determine the extent to which suppression mediated by AVRcap1b and SS15 translates into reduced viral infection, we tested the effectors ability to compromise Rx-mediated resistance to PVX. We transiently expressed AVRcap1b or SS15 in Rx-transgenic N. benthamiana plants that were silenced for NRCs either individually (TRV:NRC4) or in combination (TRV:NRC2/3, TRV:NRC2/3/4). We then infected the leaves by agroinfection with Agrobacterium tumefaciens carrying PVX (pGR106::PVX::GFP) using a toothpick inoculation method and documented the formation of trailing necrotic lesions at the inoculated spots (see PVX infection assays (agroinfection)) [ 24 , 43 , 46 ] ( Fig 5A ). Consistent with previous experiments [ 24 ], we observed trailing necrosis in the NRC triple silenced Rx leaves (TRV:NRC2/3/4) regardless of the presence or absence of AVRcap1b or SS15 ( Fig 5B and 5C , S8 and S9 Data). We also observed trailing necrosis when AVRcap1b or SS15 was expressed in NRC4-silenced (TRV:NRC4) Rx leaves, indicating that both effectors compromised Rx-mediated resistance when NRC4 is depleted ( Fig 5B and 5C , S8 and S9 Data). In contrast, Rx-mediated resistance to PVX was not compromised by AVRcap1b or SS15 in NRC2/3-silenced (TRV:NRC2/3) leaves. Virus accumulation in the different treatments was validated by western blot detection of GFP protein driven by the subgenomic promoter of PVX::GFP ( Fig 5D and 5E ). Perturbation of PVX resistance was more markedly affected by SS15 compared to AVRcap1b based on lesion size and PVX accumulation ( Fig 5C–5E ). These results indicate that the AVRcap1b and SS15 effectors not only suppress NLR-mediated cell death but also counteract the disease resistance phenotype mediated through NRC2 and NRC3.

(A) A schematic representation of the silencing and infiltration strategy undertaken. Photo of representative N. benthamiana leaves showing HR after coexpression of (B) EV or AVRcap1b and (D) EV or SS15 in TRV:EV, TRV:NRC2/3, and TRV:NRC4 silenced Rx-transgenic N. benthamiana plants, where EV was used as a negative control. Silencing treatments (TRV:EV, TRV:NRC2/3, and TRV:NRC4) are indicated above leaf panels. Plants were photographed 5 days after agroinfiltration. Cell death response was scored 5 days after agroinfiltration ( S7 Data ) (C) EV and AVRcap1b and (E) EV and SS15 and presented as dot plots, where dot colour represents either EV (blue), AVRcap1b (green), or SS15 (orange) and dot size is proportional to the number of samples with the same score (count). The experiment was independently repeated 3 times. Each replicate is represented by different columns within each silencing treatment for either EV, AVRcap1b, or SS15. Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S7 Fig . EV, empty vector; HR, hypersensitive response; VIGS, virus-induced gene silencing.

The NRC-dependent sensor NLR Rx, which confers extreme resistance to Potato virus X (PVX) by recognising viral coat protein (CP), is dependent on NRC2, NRC3, and NRC4 in a genetically redundant manner [ 42 – 45 ]. The genetic redundancy of NRCs may explain why the Rx-mediated cell death is not suppressed by AVRcap1b and SS15 in N. benthamiana ( Fig 3 ). Since AVRcap1b and SS15 suppress the cell death activity of NRC2 and NRC3 but not NRC4, we reasoned that these 2 effectors should be able to suppress Rx-mediated cell death in the absence of NRC4. To challenge this hypothesis, we knocked down NRCs in Rx-transgenic N. benthamiana plants using Tobacco rattle virus (TRV)-induced gene silencing, either individually (TRV:NRC4) or in combination (TRV:NRC2/3) ( Fig 4A ). Three weeks after inoculation with TRV, we coexpressed AVRcap1b or SS15 with CP in NRC-silenced leaves. These experiments showed that both AVRcap1b and SS15 suppress Rx-mediated cell death in NRC4-silenced leaves, but not in NRC2/3-silenced leaves nor in the TRV:EV control (Figs 4B–4E and S7 and S7 Data ). These results further validate our earlier finding that AVRcap1b and SS15 can specifically suppress the activities of NRC2 and NRC3 but not NRC4.

Representative leaf panels showing HR phenotypes of (A) EV and AVRcap1b or (C) EV and SS15 coinfiltrated with a range of sensor NLRs (labelled on the bottom of panels B and D). Photographs were taken 5 days post-agroinfiltration. (B, D) HR results are presented as dot plots, where the size of each dot is proportional to the number of samples with the same score (count). A total of 8 technical replicates were completed for each treatment ( S6 Data ). Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S6 Fig . EV, empty vector; HR, hypersensitive response; NLR, nucleotide-binding domain and leucine-rich repeat; NRC, NLR required for cell death.

To further challenge our finding that AVRcap1b and SS15 target NRC2 and NRC3 but not NRC4, we screened the 2 effectors for suppression of 6 disease resistance sensor NLRs (SW5b, Gpa2, R1, Rx, Bs2, and R8) that were previously assigned to the NRC network [ 24 , 25 ]. We transiently coexpressed AVRcap1b and SS15 with these NRC-dependent sensor NLRs in addition to the previously tested Prf and Rpi-blb2 in N. benthamiana leaves using agroinfiltration. Of these, 5 NLRs were coexpressed with their cognate AVR effectors, whereas we used a constitutively active version of SW5b (SW5b D857V ). We also included Rpi-vnt1 as an NRC-independent NLR protein negative control [ 24 ]. Interestingly, while SW5b was previously shown to signal through NRC2, NRC3, and NRC4 in a redundant manner [ 24 ], SW5b D857V signalled through NRC2 and NRC3 only. These experiments revealed that AVRcap1b and SS15 suppress SW5b D857V and Gpa2 in addition to Prf, but none of the other 5 NLRs (Rpi-blb2, R8, R1, Rx, and Bs2). Our findings do, however, contrast to a previous study that showed that SS15 was able to partially suppress Rx-mediated cell death [ 35 ]. Additionally, neither AVRcap1b nor SS15 suppressed cell death mediated by the NRC-independent NLR Rpi-vnt1 (Figs 3 and S6 and S6 Data ). These results are consistent with the model that AVRcap1b and SS15 suppress NRC2 and NRC3 but not NRC4, given that, similar to Prf, both SW5b D857V and Gpa2 are dependent on NRC2 and NRC3, whereas the other tested NLRs signal through NRC4 specifically or redundantly with NRC2 and NRC3 [ 24 , 25 ].

To ascertain whether the identified effectors suppress the activity of the sensor NLRs (Prf or Rpi-blb2) or the underlying helper NLRs (NRC2, NRC3, and NRC4), we generated constitutively active (autoimmune) NRCs by mutating the methionine–histidine–aspartate (MHD) motif resulting in the NRC autoactive variants NRC2 H480R , NRC3 D480V , and NRC4 D478V [ 40 ] ( Fig 2B ). When transiently expressed in N. benthamiana, all 3 NRC mutants caused cell death in the absence of a pathogen AVR effector ( Fig 2C ). Next, we coexpressed the suppressors AVRcap1b, SS15, PITG-15278, SS10, and SS34, along with the EV control, with NRC2 H480R , NRC3 D480V , and NRC4 D478V in N. benthamiana leaves ( S4 Fig , S4 Data ). PITG-15278, SS10, and SS34 did not suppress any of the autoactive NRCs, indicating that they target the sensor NLR Rpi-blb2 or the interaction between Rpi-blb2 and NRC4 ( S4 Fig ). Interestingly, both AVRcap1b and SS15 suppressed the cell death induced by autoimmune NRC2 and NRC3, therefore recapitulating their specific suppression of the sensor NLRs Prf (Figs 2C and 2D and S5 , S5 Data ). In contrast, AVRcap1b and SS15 did not affect the autoactivity of NRC4 (Figs 2C and 2D and S5 ). These results indicate that the suppression activity of AVRcap1b and SS15 is independent of the sensor NLR Prf and is specific to NRC2 and NRC3. In light of these results, we focused on AVRcap1b and SS15 as they are likely acting on the NRC2 and NRC3 helper nodes of the NRC network and thus hold more potential to shed light on the molecular mechanisms underpinning helper NLR activation and downstream signalling.

(A) Domain organisation of the RXLR-WY/LWY domain containing P. infestans effector AVRcap1b and the B30.2/SPRY ( IPR001870 ) domain containing G. rostochiensis effector SS15, where SP = signal peptide, R = RxLR motif, U = uncharacterized domain. (B) Schematic representation of NRC2, NRC3, and NRC4 and the mutated sites in the MHD motif. Substituted residues are shown in red in the multiple sequence alignment. (C) Representative N. benthamiana leaves showing HR after coexpression of EV, AVRcap1b, or SS15, indicated above leaf panels, with autoimmune NRC2 H480R , NRC3 D480V , and NRC4 D478V mutants. Plants were photographed 5 days after agroinfiltration. (D) HR was scored 5 days post-agroinfiltration ( S5 Data ). The results are presented as a dot plot, where the size of each dot is proportional to the number of samples with the same score (count) within each biological replicate. The experiment was independently repeated 3 times each with 6 technical replicates. The columns of each tested condition (labelled on the bottom of the plot) correspond to results from different biological replicates. Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S4 Fig . CC, coiled-coil; EV, empty vector; HR, hypersensitive response; LRR, leucine-rich repeat; MHD, methionine–histidine–aspartate; NB-ARC, nucleotide-binding domain shared with APAF-1, various R-proteins, and CED-4; WT, wild type.

(A) A schematic representation of cell death assay strategy used. Effectors and EV were transformed into Agrobacterium tumefaciens and transiently coexpressed in N. benthamiana plants with either Pto/AvrPto or Rpi-blb2/AVRblb2. EV was used as a negative control. HR was scored based on a modified 0–7 scale (Segretin and colleagues) between 5–7 days post-infiltration. (B) A scatterplot of the average HR score of EV versus the average HR score of each tested effector (n = 165); Pto/AvrPto (left panel) Rpi-blb2/AVRblb2 (right panel). Effectors that have suppression activity are represented as outliers within the plot. Results are based on 6 technical replicates ( S1 – S3 Data). Δ Two alleles of PITG-15278 (P. infestans strain T30-4 and 17777) suppressed Rpiblb2-mediated cell death. PITG-15278 T30-4 is used as a representative in subsequent experiments. (C) Representative N. benthamiana leaves infiltrated with appropriate constructs photographed 5–7 days after infiltration. The R/AVR pair tested, Prf (Pto/AvrPto) or Rpi-blb2/AVRblb2, are labelled above the leaf panel and effectors labelled on leaf image. (D) HR was scored 5 days post-agroinfiltration ( S4 Data ). The results are presented as a dot plot, where the size of each dot is proportional to the number of samples with the same score (count) within each biological replicate. The experiment was independently repeated 3 times with 6 technical replicates. The columns for either EV or for each individual effector correspond to results from different biological replicates. Dot colours represent pathogen species as indicated in (B). Statistical tests were implemented using the besthr R library [ 41 ]. We performed bootstrap resampling tests using a lower significance cutoff of 0.025 and an upper cutoff of 0.975. Mean ranks of test samples falling outside of these cutoffs in the control samples bootstrap population were considered significant. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S2 Fig . AVR, avirulence; EV, empty vector; HR, hypersensitive response.

To determine the extent to which pathogens have evolved to target the NRC network, we screened candidate effectors from 6 diverse pathogen species for their ability to suppress the hypersensitive cell death triggered by the disease resistance proteins Prf (responds to Pto/AvrPto; NRC2/3 dependent) and Rpi-blb2 (responds to AVRblb2; NRC4 dependent) [ 24 , 26 ]. The effectors and empty vector (EV, control) were transiently expressed with Pto/AvrPto or Rpi-blb2/AVRblb2 in leaves of the model experimental plant Nicotiana benthamiana and assessed for the absence of cell death, which is indicative of a suppression phenotype ( Fig 1A ). Our screen included 165 effector candidates from oomycetes (P. infestans, 63), bacteria (P. syringae, 26), cyst nematodes (G. rostochiensis, 23 and G. pallida, 3), and aphids (M. persicae, 47 and A. pisum, 3). The panel of effectors used in the screen was comprised of candidates with previously reported involvement in immune suppression and effectors that were readily available in the lab. Among the 165 effectors tested, 2 effectors (AVRcap1b and SS15) suppressed Prf-mediated cell death, and 3 effectors (PITG-15278, SS10, and SS34) suppressed Rpi-blb2–mediated cell death (Figs 1B–1D and S2 , S1 – S4 Data). AVRcap1b and PITG-15278 are RXLR-WY/LWY domain containing effectors from P. infestans [ 33 , 34 ]. SS10, SS15, and SS34 are SPRY domain containing effectors from G. rostochiensis [ 35 – 37 ] ( Fig 2A , S1 Table ). Interestingly, HopAB2 (also known as AvrPtoB), which is well known to suppress Prf-mediated cell death [ 38 ], was not a robust suppressor in our assays, and only suppressed Prf-mediated cell death in older leaves of N. benthamiana ( S3 Fig ), leaf age as defined by [ 39 ].

Discussion

The aim of this study was to address the hypothesis that solanaceous parasites have evolved effector proteins that target the NRC network of NLR immune receptors. We confirmed this hypothesis by carrying out an effectoromics screen, which yielded 5 effectors that can compromise the NRC network: SS10, SS15, and SS34 from the cyst nematode G. rostochiensis and AVRcap1b and PITG-15278 from the potato late blight pathogen P. infestans. These 5 effectors can suppress the hypersensitive cell death induced in N. benthamiana by either Prf or Rpi-blb2, 2 NRC-dependent sensor NLRs that function as bona fide disease resistance proteins. Interestingly, these effectors appear to function at different points in the NRC network (Fig 14). While SS10, SS34, and PITG-15278 suppress cell death mediated by Rpi-blb2, they do not interfere with an autoimmune mutant of the downstream helper NRC4. SS15 and AVRcap1b, however, can robustly suppress autoimmune mutants of NRC2 and NRC3, indicating that they act at the level of the NRC helpers or their downstream pathways. We found that SS15 directly binds the NB-ARC domain of NRC2 and NRC3, while AVRcap1b associates with NbTOL9a and requires this host protein to fully suppress NRC3. We conclude that cyst nematodes and P. infestans convergently evolved sequence unrelated effectors that target key nodes of the NRC network or their downstream components to suppress host immune signalling. Our paper also highlights the value of using effectors as probes to dissect key regulatory components of immunity and to study the complex interactions between NLR receptors and the networks they form.

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TIFF original image Download: Fig 14. Evolutionary divergent pathogens have evolved to target multiple layers of the Solanaceae NLR network. P. infestans and G. rostochiensis, 2 evolutionary distinct pathogens, have evolved effectors that suppress signalling mediated by the NRC network. Two effectors from the cyst nematode pathogen, G. rostochiensis (SS10 and SS34), and 1 effector from the blight pathogen, P. infestans (PITG-15278) suppress the function of the NRC4-dependent sensor NLR, Rpi-blb2. The cyst nematode effector, SS15, binds to the NB-ARC domain of both inactive and activated forms of NRC2 and NRC3. By binding the NB-ARC domain of NRC2 and NRC3, SS15 is able to suppress their function. The P. infestans effector, AVRcap1b, suppresses the function of NRC2 and NRC3, and in the case of NRC3, AVRcap1b suppression requires the ESCRT-related protein NbTOL9a. ESCRT, endosomal sorting complex required for transport; ETI/NTI, effector/NLR-triggered immunity; NB-ARC, nucleotide-binding domain shared with APAF-1, various R-proteins, and CED-4; NLR, nucleotide-binding domain and leucine-rich repeat; NRC, NLR required for cell death. https://doi.org/10.1371/journal.pbio.3001136.g014

Our findings help to explain why plants have evolved NLR receptor networks with complex architectures. We previously postulated that NLR networks, such as the NRC network, help maintain the robustness of the immune system in light of external perturbations [55]. A key feature of the NRC network is that NRCs act as central nodes that function downstream of multiple disease resistance proteins (NLR sensors) that form a massively expanded phylogenetic clade in the Solanaceae. The NRC nodes have overlapping NLR sensor specificities and display varying degrees of redundancy [24]. Given that NRCs are critical for immune signalling of multiple disease resistance proteins, they would be ideal targets for pathogen effectors. Our finding that 2 distantly related pathogens, an oomycete and a cyst nematode, evolved effectors to suppress NRC signalling through distinct mechanisms, supports this hypothesis. It is tempting to speculate that NRC redundancy has, therefore, emerged as a strategy to enhance the plant’s capacity to evade immune suppression. For example, although both SS15 and AVRcap1b are robust suppressors of NRC2 and NRC3, they are not able to suppress their paralog NRC4. This redundancy would allow the host to mount an effective immune response even in the presence of NRC2 and NRC3 suppressors. However, it should be noted that while we did observe binding between SS15 and NRC4 in Y2H screens, their association was very weak or not detectable in planta. It is possible that physiological conditions in the plant do not favour association between SS15 and NRC4, which is consistent with our observations that SS15 cannot suppress NRC4 signalling. Interestingly, the majority of NRC-dependent potato blight R genes, e.g., R1, R8, and Rpi-blb2, signal through NRC4 [24]. NRC4 may therefore have evolved as the helper NLR that predominantly functions with late blight R genes because it evades suppression by P. infestans. However, Rpi-amr1 was recently shown to require NRC2 and NRC3 for resistance to P. infestans, indicating that the interactions between sensor NLRs, helper NRCs, and pathogen effectors are extremely complex [56]. Nonetheless, this work supports the hypothesis that NLR networks make plant immune systems more resilient through redundant signalling architectures. The extent to which pathogen effectors target NLR networks and the molecular mechanisms by which they do so, however, are still not fully understood.

An emerging paradigm in plant immunity is that NTI and PTI signalling employ common modules and reinforce each other, blurring the division between these 2 classes of plant immunity [57–60]. It is possible that NTI suppressing effectors, such as AVRcap1b and SS15, simultaneously compromise both NTI and PTI by targeting shared immune signalling nodes. Therefore, identification of NTI suppressors may translate into new insights regarding basal resistance and help us further decipher the intricate nature of the plant immune system.

While the precise molecular mechanism that the cyst nematode effector SS15 utilises to suppress NRC2 and NRC3 remains to be determined, our experiments provide some important insights. SS15 binds both inactive (Figs 6B and S9 and S10) and constitutively active forms of NRCs (Fig 8), presumably through their NB-ARC domain (Fig 7). There are only a few examples of effectors that directly bind NLRs to suppress their activities. NleA, an effector from human enteropathogenic E. coli was shown to suppress the NOD-like receptor (NLR) NLRP3 by directly binding to it, thereby interfering with deubiquitination, which is critical for inflammasome activation [61]. NleA associates with both ubiquitinated and nonubiquitinated NLRP3 by interacting with the PYD and LRR domains [62]. While the exact mechanism utilised by NleA is currently unknown, the authors theorise that binding of NleA to the PYD and LRR domains may be responsible for inhibition of NLRP3 inflammasome formation by preventing NLRP3 interaction with other downstream signalling partners or blocking access of the deubiquitinating enzyme to the polyubiquitinated NLR [62]. P. infestans IPI-O4 is a plant pathogen effector reported to bind NLRs to suppress host immunity. Chen and colleagues [63] and Karki and colleagues [64] showed that the P. infestans effector IPI-O4 compromises the HR mediated by the NLR disease resistance protein RB (also known as Rpi-blb1) by directly binding to its N-terminal CC domain, possibly to compete with binding by the AVR effector AVRblb1, a homolog of IPI-O4. Therefore, IPI-O4 acts directly on the sensor NLR and is more reminiscent of the 3 Rpi-blb2 suppressors we report here than to the NRC suppressors AVRcap1b and SS15 (Fig 13).

The recent elucidation of the ZAR1, RPP1, and ROQ1 structures have revealed that structural remodelling of the NB-ARC domain, a region involved in NLR activation, is essential for resistosome formation [27–30]. The mechanism utilised by SS15 to suppress NRC-mediated immunity may involve tampering with NB-ARC structural rearrangements. In mammalian systems, for example, a compound known as MCC950 directly binds both the inactive and activated forms of NLRP3 to inhibit its function [65,66]. MCC950 binds the central NACHT domain, the mammalian equivalent of the NB-ARC domain of plant NLRs, to interfere with ATP hydrolysis and prevent conformational changes that are critical for NLRP3 activation and subsequent inflammasome assembly. This ultimately drives NLRP3 towards a closed and inactive conformation [65,66]. Based on our findings, we propose that SS15 could be acting as an NLR inhibitor that directly perturbs NRC activities by binding the NB-ARC domain and forcing NRC2 and NRC3 into an inactivated state, possibly through mechanisms analogous to MCC950. Further studies investigating the extent to which SS15 perturbs structural remodelling of the NB-ARC domain of NRCs will provide mechanistic insights into how this effector is able to suppress NRC2 and NRC3.

Unlike SS15, AVRcap1b from the potato late blight pathogen P. infestans indirectly suppresses the function of autoimmune NRC2 and NRC3 and therefore likely targets host proteins downstream of these cell death executor NLRs. We identified NbTOL9a, a member of the TOL protein family, as a host target of AVRcap1b and showed that this host protein acts as a negative modulator of NRC3-mediated hypersensitive cell death. TOL proteins are key components of the ESCRT machinery and have well characterised roles in intracellular protein trafficking [52,67,68]. They act as ubiquitin receptors in the early steps of the ESCRT trafficking pathway by interacting with ubiquitinated cargo via their ENTH/VHS and GAT domains [51–53]. Conlan and colleagues [69] identified a TOL family member that is proximal to the P. syringae effector AvrPto, when this effector protein was transiently expressed in N. benthamiana leaf tissue. In addition, silencing of this TOL protein resulted in decreased growth of P. syringae pv. tabaci on N. benthamiana, which is in line with our observation that TOL proteins can act as negative regulators of plant immune signalling. The fact that AvrPto is proximal to TOLs further links the NRC network to TOL proteins, since AvrPto is recognised by the NRC2- and NRC3-dependent sensor NLR Prf. Together, these findings strengthen our hypothesis that TOLs can act as negative regulators of this complex immune signalling network. The precise mechanism TOLs utilise to modulate plant immunity, however, is still unknown. In the case of NbTOL9a, this negative regulation does not seem to involve protein–protein interactions between NbTOL9a and NRCs. In mammalian systems, the ESCRT pathway is involved in negatively regulating several forms of programmed cell death, including necroptosis and pyroptosis, by repairing damaged sections of the plasma membrane [70–72]. It is possible that AVRcap1b is co-opting NbTOL9a to hijack a similar immunomodulatory trafficking pathway in the plant cell to counteract NRC-mediated HR cell death and suppress immunity. This model would be consistent with the observation that vesicle trafficking is massively reprogrammed by P. infestans effectors during infection [49,73–75].

Interestingly, even though AVRcap1b and SS15 are robust NTI suppressors, they can activate immunity on certain Solanaceae species. AVRcap1b, for example, can activate immunity on accessions of Solanum capsicibaccatum carrying the NLR disease resistance gene Rpi-cap1—hence the moniker AVR [32,76]. SS15, on the other hand, is recognised by a yet to be described protein in Nicotiana tabacum resulting in HR cell death [35]. The fact that both AVRcap1b and SS15 act as both triggers and suppressors of NLR immunity further highlights the complex coevolutionary dynamics that exist between effectors and NLRs/NLR networks and the need for studies that take into account these intricate epistatic interactions. Additionally, understanding the interplay between AVRcap1b, TOLs, and NRCs will help advance our knowledge of the regulatory mechanisms that govern plant immunity and determine the outcome of multipartite host–pathogen interactions mediated by a complex immune receptor network.

The field of NLR biology has seen significant progress over the past few years, and yet the genetic components and immune signalling pathways downstream of NLR activation remain obscure [77]. Moreover, the precise molecular mechanisms that underpin the activation of paired and networked NLRs and subsequent cell death response are still unknown. Here, we have gained insights into the molecular strategies that plant pathogens utilise to counteract host immune function. We identified 5 effectors (SS15, AVRcap1b, PITG-15278, SS10, and SS34) that compromise components of the NRC network. We focused on SS15 and AVRcap1b, as these effectors counteract NRCs—MADA-type CC-NLRs—which form central nodes within the immune receptor network. SS15 and AVRcap1b, therefore, have the unique potential to uncover valuable mechanistic details regarding the activation of MADA-type CC-NLR resistosomes and their downstream signalling elements. The fact that these distantly related pathogens, an oomycete and a cyst nematode, have independently evolved effectors to counteract NRC2 and NRC3 further highlights the critical role NRCs play in mediating immunity to solanaceous parasites. Beyond SS15 and AVRcap1, there is still much we can learn by studying PTIG-15278, SS10, and SS34, the 3 Rpi-blb2 suppressors identified in this study. Further research may allow us to determine the target(s) and mechanism(s) utilised by these 3 effectors to suppress host immunity. Recently, effectors from the oomycete pathogen Phytophthora capsici and the aphid pest M. persicae were shown to converge on host E3 SUMO ligase SIZ1 to suppress plant immunity [78]. Taken together with our work, this suggests that microbial pathogens, herbivorous insects, and parasitic nematodes may share more common virulence mechanisms than anticipated. Studying immunosuppressors holds the potential to advance our understanding of the functional principles and evolutionary dynamics that underpin plant immune receptor networks. This knowledge can then be leveraged to guide new approaches for breeding disease resistance to maximise crop protection, for example, by engineering NLRs that evade pathogen suppression.

[END]

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