Genetically-clustered antifungal phytocytokines and receptor proteins function together to trigger plant immune signaling

Summary Phytocytokines regulate plant immunity via cell-surface receptors. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP- ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and functional assays to characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Two divergent RISPs, PtRISP1 and Salix purpurea RISP1 (SpRISP1), induced a reactive oxygen species (ROS) burst and mitogen- activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 triggers a rapid stomatal closure in poplar, and both PtRISP1 and SpRISP1 directly inhibit rust pathogen growth. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with their cognate receptors.