A conserved small RNA-generating gene cluster undergoes sequence diversification and contributes to plant immunity.

Small RNA-mediated gene silencing contributes to plant immunity. The secondary small interfering RNA (siRNA) pathway promotes defense by silencing target genes in invading fungal and oomycete pathogens. Many secondary siRNAs are derived from transcripts potentially encoding pentatricopeptide repeat (PPR) proteins. Here, we report that siRNA production is an ancient function of a conserved clade of PPR genes that undergo extensive within-species diversification. In Arabidopsis thaliana, siRNA-source PPRs are physically clustered on Chromosome 1. These sequences are diversified through gene duplication followed by sequence diversification and accumulation of high-impact variations including pseudogenization, leading to the accumulation of a diverse siRNA pool. These features are consistent with the engagement of PPR-siRNAs in a co-evolutionary arms race with the pathogens. This study defines siRNA-producing PPRs as a class of defense genes and highlights the potential of PPR-siRNA-based engineering as a strategy to enhance disease resistance.