Genetically clustered antifungal phytocytokines and receptor protein family members cooperate to trigger plant immune signaling

Abstract

Phytocytokines regulate plant immunity by cooperating with cell surface proteins. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. The 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 molecular physiology assays to functionally characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Despite a low sequence identity, Salix purpurea RISP1 (SpRISP1) shows properties and activities similar to PtRISP1. Both PtRISP1 and SpRISP1 induced a reactive oxygen species (ROS) burst and phosphorylation of mitogen-activated protein kinases (MAPKs) in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 also triggers a rapid stomatal closure in poplar. Altogether, these results indicate that plants evolved phytocytokines with direct antimicrobial activities, and that the genes encoding these phytocytokines co-evolved and physically cluster with genes encoding LRR-RPs required to initiate immune signaling.