Abstract
Nucleotide‐binding (NB) leucine‐rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled‐coil (CC) NLR that confers broad‐spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1–159) domain has cell‐death‐inducing activity, whereas the CC‐containing truncations, including CC‐NB and CC‐NB‐LRR, do not induce cell‐death responses. Coimmunoprecipitation (Co‐IP) assay showed that the eCC domain self‐associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V‐mediated full‐length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell‐death induction. Two mutations (R73Q and E80K) interfered with D491V‐mediated Pm21 autoactivation without affecting the cell‐death‐inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell‐death‐inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell‐death signalling and disease‐resistance signalling, which are potentially independent of each other.
Highlights
Plants prevent pathogen invasion via a two-layered immune system (Dodds and Rathjen, 2010; Dangl et al, 2013)
The entire green fluorescent protein (GFP) protein, which was used as the negative control, was not coprecipitated with the Myc-fused eCC domain (Figure 1d). These results suggest that the active eCC domain of Pm21 self-associates in planta
To explore the effect of the MHD motif on the eCC activity, we introduced an aspartate-to-valine substitution in the MHD motif of the CC-NB fragment and FL Pm21 protein, generating two variants CC-NB (D491V) and FL (D491V) fused with the GFP tag in the C terminus
Summary
Plants prevent pathogen invasion via a two-layered immune system (Dodds and Rathjen, 2010; Dangl et al, 2013). The crystal structure of ZAR1 shows that once activated, ZAR1 oligomerizes with other host proteins into an immune complex termed the ZAR1 resistosome, in which the oligomerization of the CC domains forms a funnel-shaped structure that is necessary for the immune function of ZAR1 (Wang et al, 2019b) Consistent with this result, evidence from several studies supports an important role of CC domain self-association in cell-death-inducing activity (Maekawa et al, 2011; Cesari et al, 2016; El Kasmi et al, 2017). Total proteins were extracted from N. benthamiana leaves at 20 hpi and detection was done by western blot with anti-GFP antibody results revealed that, compared with the wheat-resistant cultivar Yangmai 18, the 10 mutants showed phenotypes sensitive to Bgt (Figure S3) These data suggest that some specific residues in the CC domain are essential for Pm21 resistance function. As some mutations that abolish resistance to powdery mildew do not affect the cell-death-inducing activity, it is suggested that Pm21-governed cell-death signalling and disease-resistance signalling are potentially independent of each other
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