Helper NLR proteins NRC2a/b and NRC3 but not NRC1 are required for Pto‐mediated cell death and resistance in Nicotiana benthamiana

Plants defend against pathogens using both cell surface and intracellular immune receptors (Dodds & Rathjen, 2010; Win et al., 2012). Plant cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs), which respond to pathogen-derived apoplastic molecules (Boller & Felix, 2009; Thomma et al., 2011). By contrast, plant intracellular immune receptors are typically nucleotide-binding leucine-rich repeat (NB-LRR or NLR) proteins, which respond to translocated effectors from a diversity of pathogens (Eitas & Dangl, 2010; Bonardi et al., 2012). These receptors engage in microbial perception either by directly binding pathogen molecules or indirectly by sensing pathogen-induced perturbations (Win et al., 2012). However, signaling events downstream of pathogen recognition remain poorly understood. In addition to their role in microbial recognition, some NLR proteins contribute to signal transduction and/or amplification (Gabriels et al., 2007; Bonardi et al., 2011; Cesari et al., 2014). An emerging model is that NLR proteins often function in pairs, with 'helper' proteins required for the activity of 'sensors' that mediate pathogen recognition (Bonardi et al., 2011, 2012). Among previously reported NLR helpers, NRC1 (NB-LRR protein required for hypersensitive-response (HR)-associated cell death 1) stands out for having been reported as a signaling hub required for the cell death mediated by both cell surface immune receptors such as Cf-4, Cf-9, Ve1 and LeEix2, as well as intracellular immune receptors, namely Pto, Rx and Mi-1.2 (Gabriels et al., 2006, 2007; Sueldo, 2014; Sueldo et al., 2015). However, these studies did not take into account the Nicotiana benthamiana genome sequence, and it remains questionable whether NRC1 is indeed required for the reported phenotypes. Functional analyses of NRC1 were performed using virus-induced gene silencing (VIGS) (Gabriels et al., 2007), a method that is popular for genetic analyses in several plant systems, particularly the model solanaceous plant N. benthamiana (Burch-Smith et al., 2004). However, interpretation of VIGS can be problematic as the experiment can result in off-target silencing (Senthil-Kumar & Mysore, 2011). In addition, heterologous gene fragments from other species (e.g. tomato) have been frequently used to silence homologs in N. benthamiana, particularly in studies that predate the sequencing of the N. benthamiana genome (Burton et al., 2000; Liu et al., 2002b; Lee et al., 2003; Gabriels et al., 2006, 2007; Senthil-Kumar et al., 2007; Oh et al., 2010). In the NRC1 study, a fragment of a tomato gene corresponding to the LRR domain was used for silencing in N. benthamiana (Gabriels et al., 2007). Given that a draft genome sequence of N. benthamiana has been generated (Bombarely et al., 2012) and silencing prediction tools have become available (Fernandez-Pozo et al., 2015), we can now design better VIGS experiments and revisit previously published studies. Two questions arise about the NRC1 study. First, is there a NRC1 ortholog in N. benthamiana? Second, are the reported phenotypes caused by silencing of NRC1 in N. benthamiana? In this study, we investigated NRC1-like genes in solanaceous plants using a combination of genome annotation, phylogenetics, gene silencing and genetic complementation experiments. We discovered three paralogs of NRC1, which we termed NRC2a, NRC2b and NRC3, are required for hypersensitive cell death and resistance mediated by Pto, but are not essential for the cell death triggered by Rx and Mi-1.2. NRC2a/b and NRC3 weakly contribute to the hypersensitive cell death triggered by Cf-4. Our results highlight the importance of applying genetic complementation assays to validate gene function in RNA silencing experiments. Tomato NRC1 (Solyc01g090430) was used to identify homologs in the predicted protein databases (Nicotiana benthamiana Genome v0.4.4 predicted protein, Tomato proteins ITAG release 2.40, and Potato ITAG release 1 predicted proteins) on Solanaceae Genomics Network (SGN). Top hits of BLASTP search results were collected for further analyses. NRC2 homologs in potato were missing in Potato ITAG release 1 predicted proteins database. Therefore, two NRC2 sequences of potato identified in Potato PGSC DM v3.4 protein sequences were included in the analyses. The phylogenetic tree of NRC homologs was built using Mega6-Beta2 (Tamura et al., 2013) with Neighbor-joining and Maximum-likelihood methods with bootstrap values based on 1000 iterations. Chromosome assignments of NRC homologs were based on tomato and potato genomes. Cloning of tomato NRC homologs was performed with Gateway cloning kit (Invitrogen, Thermo Scientific, Waltham, MA, USA) following the manufacturer's instruction. Primer pairs used in the cloning are listed as follows: SlNRC1-F/R (5′-CACCATGGTTGATGTAGGGGTTGAATTTC-3′ and 5′-CTAAGAAGCTGTCTGTACATCAGAATC-3′), SlNRC2-F/R (5′-CACCATGGCGAACGTAGCAGTGGAATTTC-3′ and 5′-TCAGAGATCAGGAGGGAATATGGAAAG-3′), and SlNRC3-F/R (5′-CACCATGGCGGATGTAGCAGTAAAGTTCTTA-3′ and 5′-TTACAATCCAAGATCATGAGGGAAT-3′). The amplified fragments from tomato cDNA were cloned into pENTR/D-TOPO (Invitrogen) and then introduced into the pK7WG2 destination vector (Karimi et al., 2002) by Gateway LR recombination enzyme (Invitrogen). Nicotiana benthamiana NRC2a, NRC2b and NRC3 were amplified with the corresponding primer pairs (NbNRC2a-F/R: 5′-CACCATGGCGAACGTTGCGGTGGAGTTTCTGG-3′ and 5′-TCAGAGATCGGGAGGGAATATAGAGAGCTT-3′; NbNRC2b-F/R: 5′-ATGGCGAACGTTGCGGTGGA-3′ and 5′-AATTGGTCTCTAAGCTTAGAGATCGGGAGGGAATATAGAG-3′; NbNRC3-F/R: 5′-AATTGGTCTCTAATGGCAGATGCAGTAGTGAATTTTCTGGTG-3′ and 5′-ATTGGTCTCGAAGCTTACTGTGTGGCCTTGGATCCAGCTTC-3′) from cDNA and cloned into pCR8/GW/TOPO (Invitrogen) by TA cloning. The fragments were then used for further amplification and subcloning into pICH86988 with Golden Gate cloning (Weber et al., 2011). The synthetic fragments of NbNRC2a/b and NbNRC3 were designed manually to introduce synonymous substitution in every codon when possible, and the syntheses of these fragments were performed by Genewiz (South Plainfield, NJ, USA). The synthetic fragments were then subcloned into pICH86988 together with the remaining NbNRC2a, NbNRC2b or NbNRC3 fragment to generate full-length NbNRC variants. VIGS was performed in N. benthamiana as described by Liu et al. (2002a). Suspensions of Agrobacterium tumefaciens strain GV3101 harboring TRV RNA1 (pYL155) and TRV RNA2 (pYL279) (Liu et al., 2002a) with corresponding fragments from indicated genes were mixed in a 2 :1 ratio in infiltration buffer (10 mM MES (2-[N-morpholino]ethanesulfonic acid), 10 mM magnesium chloride (MgCl2), and 150 μM acetosyringone, pH 5.6) to a final OD600 of 0.3. Two-week-old N. benthamiana plants were infiltrated with A. tumefaciens for VIGS assays, upper leaves were used 2–3 wk later for further agroinfiltrations. For silencing of NRC homologs in N. benthamiana, 5′ coding region of each gene (NbNRC2a/b, 1-429b; NbNRC2c, 1-426bp; NbNRC3, 1-444bp) were cloned into TRV RNA2 vector. For co-silencing of NbNRC2a/b and NbNRC3, the fragments were fused by overlap PCR and cloned into TRV RNA2 vector. The following primers were designed for generating the TRV2-SlNRC1 construct based on the SlNRC1 fragment that was used for silencing by Gabriels et al. (2007): 5′-CACCTTAAAGTCATTCCGAAACATGTTGG-3′ and 5′-TCGAGAGAACATACTCAGTGCAGC-3′. The silencing constructs for SGT1 and SERK3 were described previously (Peart et al., 2002; Heese et al., 2007). In planta transient agroinfiltration assays were performed according to methods described previously (Bos et al., 2006). Four to five-week-old N. benthamiana plants (i.e. 2–3 wk after virus inoculation) were used to test for cell death in VIGSed leaves. The concentration of A. tumefaciens strains carrying the expression constructs were adjusted in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone, pH 5.6). The final concentrations of A. tumefaciens (OD600) used in cell death assays in VIGSed leaves are indicated as follows: Pto, 0.6 (de Vries et al., 2006); AvrPto, 0.1 (de Vries et al., 2006); Cf-4, 0.4 (Liebrand et al., 2012), AVR4, 0.4 (Van der Hoorn et al., 2000); Rx, 0.2 (Tameling & Baulcombe, 2007); CP, 0.1 (Tameling & Baulcombe, 2007); Mi-1.2T557S, 0.8 (Lukasik-Shreepaathy et al., 2012), INF1, 0.3 (Bos et al., 2006) and SlNRC/NbNRC variants, 0.6. The HR cell death phenotype was scored at 7 d post infiltration (dpi), according to a previously described scale, which was modified from zero (no HR observed) to seven (confluent necrosis) (Segretin et al., 2014). To detect the accumulation of proteins in complementation assays, tomato and N. benthamiana NRCs are subcloned into pK7WGF2 or pICH86966 with N-terminal GFP fusion (Karimi et al., 2002; Weber et al., 2011). Three days after agroinfiltration in control or NRC-silenced leaves, total plant proteins were extracted and analyzed by western blot analyses according to previously described methods (Oh et al., 2009). Commercial anti-GFP (Invitrogen) and anti-rabbit antibody conjugated to horseradish peroxidase (Sigma-Aldrich) were used as primary and secondary antibody. Plant total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). 2 μg RNA of each sample was subject to first strand cDNA synthesis using Ominiscript RT Kit. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using DreamTaq (Thermo Scientific) with 25 to 30 amplification cycles followed by electrophoresis with 2% agarose gel stained with Ethidium bromide. Primer pairs used in the PCR reaction are listed as follows: NbNRC2a/b-RT-F/R (5′-AGTGGATGAGAGTGTGGGTG-3′ and 5′-AAGCAGGGATCTCAAAGCCT-3′), NbNRC2c-RT-R/F (5′-TCAAAACATGCCGTGTTCAT-3′ and 5′-CCTGCGGGTTTTGTACTGAT-3′) and NbNRC3-RT-F/R (5′-CCTCGAAAAGCTGAAGTTGG-3′ and 5′-TGTCCCCTAAACGCATTTTC-3′). Primers for internal control NbEF1α were as described previously (Segonzac et al., 2011). VIGS was used to silence NRC2a/b and NRC3 in both wild type and Pto/Prf transgenic (R411B) N. benthamiana plants (Balmuth & Rathjen, 2007). Bacteria growth assay were performed as previously described with minor modifications (Balmuth & Rathjen, 2007). The Pseudomonas syringae DC3000 ΔhopQ1-1 culture (Wei et al., 2007) was adjusted to OD600 of 0.2 and then diluted 10 000-fold with 10 mM MgCl2. Five-week-old N. benthamiana with VIGS control or NRC2a/b/3-silencing were inoculated with the bacterial culture using needleless syringe. Four replicate plants were sampled using 0.33 cm2 cork borer at each time points, and then the sample were homogenized in 10 mM MgCl2 for serial dilution and plating. Experiments were repeated three times with similar results. Polyclonal anti-myc antibody A-14 (Santa Cruz Biotechnology, Dallas, TX, USA) was used for detecting accumulation of Prf:5myc. The nomenclatures of the genes mentioned in this article are based on the orthology to NRC1 and results of phylogenetic analysis as shown in Fig. 1. Species names are indicated as two-letter prefixes to the gene names; Sl is used for tomato (Solanum lycopersicum), St is used for potato (Solanum tuberosum) and Nb is used for N. benthamiana. DNA sequence data from this article can be found in the SGN or GenBank/EMBL databases under the following accession numbers: SlNRC1 (Solyc01g090430, NP_001234202), SlNRC2 (Solyc10g047320), SlNRC3 (XP_004238948.1), NbNRC2a (NbS-00018282), NbNRC2b (NbS00026706), NbNRC2c (NbS000-31134), NbNRC3 (NbS00011087), StNRC3 (Sotub05g007690). Sequences of SlNRC1, SlNRC2, SlNRC3 and NbNRC3 were confirmed by cDNA sequencing, and are identical to the sequences in the database with accession numbers listed above. Sequence of NbNRC2a and NbNRC2b was re-annotated by sequencing the coding region and submitted to NCBI under accession numbers KT936525, KT936526. To identify putative homologs of NRC1 in N. benthamiana, potato, and tomato genomes, we performed a BLASTP (Altschul et al., 1990) search against the predicted protein databases in SGN using the polypeptide sequence of tomato NRC1 (Solyc01g090430) as a query. Phylogenetic analyses and sequence comparisons of the top protein hits indicated that the NRC family is composed of at least three subclades (NRC1-3) belonging to clade CNL-14 as described by Andolfo et al. (2014). This NRC/CNL-14 clade is distinct from a previously described clade CCR/CNL-RPW8, which includes helper NLRs ADR1 and NRG1 (Collier et al., 2011; Andolfo et al., 2014). Surprisingly, a N. benthamiana ortholog was missing in the NRC1 subclade and a tomato ortholog was also missing in the NRC3 subclade. To determine whether the missing sequences are due to misannotation in the tomato and N. benthamina genomes, we searched all the available nucleotide and protein databases of N. benthamiana and tomato in SGN with representative NRC sequences. We failed to identify sequences that show high similarity to NRC1 in N. benthamiana, even after blast searches against scaffolds and contigs sequences in both SGN and the N. benthamiana genome database (http://www.benthgenome.com) (Naim et al., 2012; Nakasugi et al., 2014). We, therefore, concluded that NRC1 is probably missing in the N. benthamiana genome although it may have been somehow omitted from the assembly. By contrast, using TBLASTN searches, we detected a misannotated tomato gene in contig SL2.40ct02653 with high similarity to potato NRC3. Based on sequence comparisons, this gene has three exons and two introns; the first two exons were annotated as Solyc05g009630 whereas the third exon was missing in the annotation (Supporting Information Fig. S1a). To validate the sequence and expression of tomato NRC3, we designed primers based on our predicted full-length sequence and performed PCR using tomato cDNA and genomic DNA as template. We successfully amplified a fragment from genomic DNA and cDNA (Fig. S1b). The amplified cDNA fragment was cloned and sequenced. The identity between tomato NRC3 and potato NRC3 is 95%, consistent with our interpretation that the encoding gene is the NRC3 ortholog in tomato (Fig. S1c). Phylogenetic analyses that include the newly identified tomato NRC3 revealed that the sequences in the NRC family fall into three subclades that are supported by robust bootstrap values (Fig. 1). Pairwise comparisons indicated that protein sequences from the same subclade have at least 78% sequence identity (Figs 1, S2; Table S1). According to the genome information of potato and tomato, sequences in these three clades are located on three different chromosomes (Fig. 1), consistent with the view that genes in the same NRC subclade are orthologous. We exploited the N. benthamiana genome sequence and associated gene silencing target prediction tool (SGN VIGS tool; http://vigs.solgenomics.net) to analyze the specificity of the NRC1 VIGS fragment that was used in the NRC1 VIGS experiments (Gabriels et al., 2007). We found that the tomato NRC1 (SlNRC1) fragment, which matches the LRR domain, would most probably target the N. benthamiana genes NbNRC2a/b and NbNRC2c, and possibly NbNRC3. Based on pairwise sequence comparisons, this SlNRC1-LRR fragment has 70–80% sequence identity to NbNRC2a/b/c and NbNRC3 (Fig. S3). This prompted us to test the degree to which silencing of the individual NRC2a/b, NRC2c or NRC3 genes could suppress the cell death mediated by different immune receptors. To design more specific constructs for silencing individual NbNRC paralogs, we analyzed the NbNRC sequences with the VIGS tool. The 5′ coding regions of each gene provided the highest specificity and were selected to design new gene silencing constructs. Nicotiana benthamiana plants were subjected to VIGS and challenged with the cell death triggered by immune receptors Pto, Rx, and Mi-1.2. Silencing of NRC2a/b or NRC3 moderately but significantly reduced the cell death mediated by Pto but not Rx and Mi-1.2 (Fig. 2a). Semi-quantitative RT-PCR indicated that the VIGS constructs reduced the expression of the targeted gene with no detectable effects on the other paralogs (Fig. 2b). Next, we combined the two NRC2a/b and NRC3 fragments in one construct with the aim of obtaining more robust phenotypes. Interestingly, the double-silencing construct that targets both NRC2a/b and NRC3 dramatically suppressed Pto-mediated cell death close to background levels (Fig. 2a,c). Rx and Mi-1.2-mediated cell death remained unaffected with single or double NRC-silencing constructs, whereas silencing SGT1 compromised Rx-, Mi-1.2 as well as Pto-mediated cell death (Fig. 2a). These results suggest that NRC2a/b and NRC3 may be functionally redundant in Pto-mediated cell death. Gabriels et al. (2006, 2007) reported that silencing in N. benthamiana, with a tomato NRC1 fragment matching part of the LRR, reduced the cell death induced by AVR4 and INF1, which are recognized extracellularly by RLPs (Rivas & Thomas, 2005; Du et al., 2015). We tested whether silencing of NRC2a/b, NRC2c or NRC3 genes impairs cell death triggered by these proteins. Silencing of NRC2a/b and/or NRC3 weakly reduced the Cf-4/AVR4 cell death but did not affect INF1-triggered cell death, whereas cell death from both Cf-4/AVR4 and INF1 were reduced in SERK3 silencing (Fig. 2b). Our NbNRC silencing experiments did not fully match the results of Gabriels et al. (2006, 2007) given that we did not observe effects on cell death mediated by Rx, Mi-1.2, and INF1 with any of the tested constructs (Fig. 2a). This prompted us to perform VIGS with the original fragment used in Gabriels et al. (2006, 2007) (Fig. S4, see also nucleotide alignment of this fragment with NbNRCs in Fig. S3). These VIGS experiments revealed moderate effects on Pto and Mi-1.2 mediated cell death but no detectable alteration of cell death mediated by Rx, Cf-4 and INF1 (Fig. S4). The discrepancy between our results and those of Gabriels et al. (2006, 2007) are striking but could still be due to differences in experimental set up and materials used in the experiments. To summarize, we observed robust reduction of Pto-mediated cell death after silencing NRC2a/b and NRC3, as a result we decided to focus on these genes in follow-up experiments. To clarify which tomato NRC homologs are implicated in Pto-mediated cell death, we performed complementation experiments in N. benthamiana plants silenced for endogenous NRC genes (Figs 1, 2). Our experiment was motivated by the observation that the tomato NRC sequences are probably divergent enough from the N. benthamiana ones to be resilient to silencing. These experiments revealed that SlNRC3 partially rescued Pto-elicited cell death, SlNRC2 showed weak complementation activity but SlNRC1 did not rescue Pto-mediated cell death (Fig. S5a). To test whether silencing affects the expression of SlNRC1, SlNRC2, and SlNRC3, we generated GFP-tagged SlNRC1, SlNRC2, and SlNRC3 and assessed protein accumulation in NRC2a/b/3 silenced leaves. Protein level of SlNRC variants were reduced but were still detectable in NRC2a/b/3-silenced leaves (Fig. S5b). Based on these results and the observation that the NRC1 ortholog is missing in N. benthamiana, we reasoned that NRC1 is not the gene responsible for Pto-elicited hypersensitive response in N. benthamiana. We aimed to determine which N. benthamiana NRC homologs are implicated in the cell death elicited by Pto given that the complementation assays were performed with tomato NRC genes (Fig. S5). We focused on NRC2a/b and NRC3, as silencing of NRC2c did not yield reduction of cell death (Figs 2, S2). To achieve this, we generated a synthetic version of NbNRC2a, NbNRC2b, and NbNRC3, termed NbNRC2asyn, NbNRC2bsyn, and NbNRC3syn, with shuffled synonymous codon sequences and that should be divergent enough to evade VIGS (Figs 3a, S6a, S7a). Expression of NbNRC2asyn, NbNRC2bsyn, or NbNRC3syn in NRC2a/b/3-silenced N. benthamiana leaves rescued the cell death mediated by Pto, whereas the original versions of NbNRC2a, NbNRC2b, or NbNRC3 gene failed to complement (Figs 3b, S6b, S7b). To confirm that the synthetic variants of NbNRC2a, NbNRC2b, and NbNRC3 evade VIGS, we generated GFP-tagged NbNRC2a, NbNRC2b, and NbNRC3 variants and assessed protein accumulation in NRC2a/b/3 silenced leaves (Figs 3c, S6c, S7c). NbNRC2asyn, NbNRC2bsyn, and NbNRC3syn accumulated to a similar level to control treatments whereas the original NbNRC2a, NbNRC2b, and NbNRC3 were undetectable in silenced leaves, indicating that the shuffled codons enabled VIGS evasion (Figs 3c, S6c, S7c). These experiments clearly demonstrate that NbNRC2a/b and NbNRC3 are the causal N. benthamiana genes that mediate hypersensitive death following Pto perception of AvrPto. In the previous study, Gabriels et al. (2007) showed that silencing of NRC1 did not affect Pto-mediated resistance. Since we have now identified that NRC2a/b and NRC3 are the genes required for Pto-mediated cell death, we decided to test whether silencing of NRC2a/b and NRC3 compromise Pto-mediated resistance. Pto/Prf transgenic N. benthamiana has higher resistance to P. syringae DC3000 compared to wild type plants (Balmuth & Rathjen, 2007). Hence, we silenced NRC2a/b and NRC3 in Pto/Prf transgenic N. benthamiana (R411B), and inoculated P. syringae DC3000 ΔhopQ1-1 by syringe infiltration. Bacterial growth assay revealed that Pto-mediated resistance is compromised in NRC2a/b and NRC3 silenced leaves (Fig. 4a), demonstrating that NRC2a/b and NRC3 are required for Pto-mediated resistance. The results of western blot analyses indicated that the accumulation of Prf is not affect by NRC2a/b and NRC3 silencing (Fig. 4b). In summary, we revisited the role of NRC1 as a helper NLR protein and discovered that NRC2a/b and NRC3, rather than NRC1, are the causal proteins required for Pto-mediated cell death in N. benthamiana. Therefore, the previously proposed model of NRC1 as a signaling hub for multiple immune receptors postulated by Gabriels et al. (2007) needs to be revised. In fact, the N. benthamiana genome appears to lack an ortholog of tomato NRC1 (Fig. 1). Furthermore, although NRC2a/b and NRC3 are required for the hypersensitive cell death induced by Pto, silencing of these genes did not affect the response elicited by Rx and Mi-1.2. The previous finding of Gabriels et al. (2007) that silencing of NRC1 suppresses Rx and Mi-1.2 –mediated cell death may be due to other NRC1-like sequences in N. benthamiana. We did observe that NRC silencing reduced the cell death induced by Cf-4 as reported earlier (Gabriels et al., 2007). However, the effect of NRC2/NRC3 silencing is not as dramatic as in the case of Pto-mediated cell death (Fig. 2). Our findings emphasize the importance of following RNA silencing experiments with genetic complementation assays to minimize the risk of misinterpreting data due to off-target effects (Kumar et al., 2006; Jonchere & Bennett, 2013; Pliego et al., 2013). Genetic complementation can be performed using genes from a different species or using a silencing-resilient synthetic version of the gene with shuffled codon sequences. We recommend that genetic complementation be applied to RNA silencing experiment whenever possible to avoid gene misidentification. The authors thank Florian Jupe for discussion on the identification of NRC homologs in solanaceous plants, and Lida Derevnina for carefully reading the manuscript. This project was funded by the Biotechnology and Biological Science Research Council (BBSRC), the European Research Council (ERC), and the Gatsby Charitable Foundation. C-H.W., K.B., T.O.B. and S.K. planned and designed the research. C-H.W. and M.S.B. performed experiments, C-H.W. and S.K. analyzed data and wrote the manuscript. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Fig. S1 Cloning of tomato NRC3. Fig. S2 Protein sequence alignment of NRC homologs of N. benthamiana. Fig. S3 Pairwise alignment of SlNRC1-LRR fragment with NbNRC2a and NbNRC3. Fig. S4 Virus-induced gene silencing (VIGS) assay with SlNRC1-LRR fragment. Fig. S5 Tomato NRC3 mediates Pto-induced cell death in N. benthamiana. Fig. S6 Synthetic NbNRC2a rescues Pto-mediated cell death in NRC-silenced N. benthamiana. Fig. S7 Synthetic NbNRC2b rescues Pto-mediated cell death in NRC-silenced N. benthamiana. Table S1 Pairwise protein sequences comparison of tomato and N. benthamiana NRC homologs Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.