Signaling, Localization, and Interaction of Sr35, a Wheat CC-NLR Protein that Confers Resistance to Ug99 Stem Rust

Plant diseases on food crops can threaten global food security. Plants resist pathogen infection with an elaborate innate immune system to detect and respond to pathogen features and proteins. Specifically, plant intracellular innate immune receptors or nucleotide-binding domain leucine rich-repeat receptor (NLR) proteins are capable of recognizing pathogen effector proteins, which are deployed by pathogens to subvert host immunity. Wheat is a major food crop of global importance, and wheat yields are negatively affected by fungal pathogens, including stem rust caused by Puccinia graminis f. sp. tritici. Sr35 is a source of resistance to stem rust, and it confers near immunity to Ug99 and related races of stem rust. Sr35 encodes for a coiled-coil (CC)-type NLR protein that recognizes a stem rust fungal effector protein AvrSr35. Upon Sr35 activation by AvrSr35, cell death is observed, which is thought to help limit pathogen infection. Here, we dissect Sr35 protein to identify the domains involved in AvrSr35 recognition and cell death induction in both the model plant Nicotiana benthamiana and barley. We found that the CC-NB-ARC domains of Sr35 are important for cell death induction, whereas the LRR domain appears to function in AvrSr35 recognition. We also identified the subcellular localization of Sr35 protein in N. benthamiana, and we found it to be localized to the endoplasmic reticulum, where it is peripherally associated with the membrane and facing the cytoplasm. Finally, we investigated the nature of Sr35-AvrSr35 interaction. We did not find evidence for a direct interaction by yeast 2-hybrid (Y2H) assays. We also did not find evidence for Sr35 intramolecular interactions by Y2H. However, we did find that the auto-active Sr35 CC-NB-ARC D503V truncation variant was able to oligomerize by Y2H, tying oligomerization to activation of Sr35 protein. This work provides molecular insight into the function of a societally important plant immune protein