A Plant Immune Receptor Adopts a Two-Step Recognition Mechanism to Enhance Viral Effector Perception

Abstract

Plant intracellular nucleotide binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen surveillance. Most plant NLRs characterized so far were found to use a single domain/sensor to recognize pathogen effectors. Here we report that the Sw-5b NLR immune receptor uses two distinct domains to detect the viral movement protein NSm encoded by tospovirus. In addition to its leucine-rich repeat (LRR) domain that has been previously reported, the N-terminal Solanaceae domain (SD) of Sw-5b also interacts with NSm and a conserved 21-amino-acid region of NSm (NSm 21). The specific interaction between Sw-5b SD and NSm is required for releasing the inhibitory effect of coiled-coil domain on the NB-ARC-LRR region. Furthermore, we found that the binding of NSm affects the nucleotide binding activity of the NB-ARC-LRR in vitro, while Sw-5b NB-ARC-LRR is activated only when NSm and NSm 21 levels are high. Interestingly, Sw-5b SD could significantly enhance the ability of the NB-ARC-LRR to detect low levels of NSm effector and facilitate its activation and induction of defense response. An Sw-5b SD mutant that is disrupted in NSm recognition failed to enhance the ability of the NB-ARC-LRR to sense low levels of NSm and NSm 21. Taken together, our results suggest that Sw-5b SD functions as an extra sensor and the NB-ARC-LRR as an activator, and that Sw-5b NLR adopts a two-step recognition mechanism to enhance viral effector perception.

Evolution of NLR resistance genes with non-canonical N-terminal domains in wild tomato species

“…In consideration of their physical clusters in chromosomes, it seems an ancestral NL tandemly proliferated to increase the copy number. If the N-terminal domain and CC are essential for auto-inhibition of NLRs as found for Sw-5 in G6[8,61–63], it is yet unclear how this ancestral NL achieved it without the two domains and was eventually selected by evolution. Although some attempted to elucidate their functions, more diverse studies will be required[64].…”

“…Recently, additional integrated domains (IDs) which may function as baits for effector recognition have been identified in NLRs of flowering plants[4, 5]. In Solanaceae, a novel domain named Solanaceae Domain (SD) was found to be integrated at the N-terminus of CNLs and function for oligomerization or interaction with a decoy or effectors from pathogens[6–8].…”

Rescue of Tomato spotted wilt tospovirus entirely from cDNA clones, establishment of the first reverse genetics system for a segmented (-)RNA plant virus

“…Total protein was extracted from 1 g Agrobacterium -infiltrated leaf patches, healthy or TSWV-infected N. benthamiana systemic leaves in a 1 mL extraction buffer [10 % (v/v) glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10 mM dithiothreitol, 2 % (w/v) polyvinylpolypyrrolidone, 0.5 % (v/v) Triton X-100 and 1× protease inhibitors cocktail] (7). Protein samples were separated by SDS-PAGE gels, transferred to PVDF membranes (GE Healthcare, UK), blocked with 5 % skim milk solution and incubated with a polyclonal antiserum specific to the TSWV N, NSm, NSs, Gn, Gc, GFP, mCherry or T7 RNA pol at room temperature for 1 h or overnight at 4 °C and washed three times.…”

Perspectives on intracellular perception of plant viruses

“…Therefore, Sw‐5b recognizes a small PAMP‐like peptide within a pathogen‐encoded effector to activate broad‐spectrum resistance, analogous to the mechanism of PRR‐mediated recognition of PAMPs. In addition to the NB‐LRR region, the N‐terminal SD also interacts with NSm 21 (Li et al , ). Interestingly, the SD domain plays an important role in enhancing the ability of NB‐LRR region to recognize low levels of NSm or NSm 21 and to induce robust HR (Li et al , ) (Fig.…”

“…Consistent with this earlier observation, recent studies reported that other pathogen‐recognizing NLRs (called sensor NLRs) also require other NLRs or a network of NLRs (called helper NLRs) for immune signalling (Wu et al , ). Sw‐5b and Rx1 require NRC2/3/4 NLRs for the induction of TSWV‐NSm and PVX‐CP‐mediated HR‐PCD, respectively (Li et al , ; Wu et al , ). Recently, EDS1 has been shown to associate with NRG1 (Qi et al ., ).…”