Peer Review File Manuscript Title: A wheat resistosome defines common principles of immune receptor channels Reviewer Comments & Author Rebuttals

Plant intracellular NLRs recognize pathogen effectors to trigger plant ETI. The group solved the first structure of the plant NLR complex, termed ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated ETI. In this manuscript, the authors reported the cryo-EM structure of wheat NLR Sr35 in complex with the effector AvrSr35. The structure of wheat Sr35 and Arabidopsis ZAR1 resistosomes show a striking similarity. Like ZAR1 resistosome, AvrSr35-Sr35 complex exhibits non-selective cation channel activities. Both Sr35 and ZAR1 are CNLs. The studies reveal the evolutionary conservation of CNL resistosomes in activating plant immunity. Furthermore, this study reports an arginine cluster in the LRR domain that co-evolves and forms intramolecular interaction with CC EDVID motif. C-terminal LRR of Sr35 recognizes AVrSr35. Interestingly, structure-based domain swapping and mutational analysis of orphan CNLs bearing homology with Sr35 generate gain-of-function of CNLs that recognize AvrSr35, suggesting the potential of structure-based engineering of NLRs for crop improvement. The studies not only reveal the conservation of plant CNL resistosomes, but also provide novel insights into plant NLR activation and potential application in crop improvement. The manuscript provides a conceptional advance in understanding plant NLR activation. It was well written with high-quality data.