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.