Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence

Abstract Fungal small RNAs (sRNAs) hijack the plant RNA silencing pathway to manipulate host gene expression, named cross-kingdom RNA interference (ckRNAi). It is currently unknown how conserved and significant ckRNAi is for microbial virulence. Here, we found for the first time that sRNAs of a pathogen representing the oomycete kingdom invade the host plant’s Argonaute (AGO)/RNA-induced silencing complex. To demonstrate the functionality of the plant-invading oomycete Hyaloperonospora arabidopsidis sRNAs ( Hpa sRNAs), we designed a novel CRISPR endoribonuclease Csy4/GUS repressor reporter to visualize in situ pathogen-induced target suppression in Arabidopsis thaliana host plant. By using 5’ RACE-PCR we demonstrated Hpa sRNAs-directed cleavage of plant mRNAs. The significant role of Hpa sRNAs together with At AGO1 in virulence was demonstrated by plant atago1 mutants and by transgenic Arabidopsis expressing a target mimic to block Hpa sRNAs, that both exhibited enhanced resistance. Individual Hpa sRNA plant targets contributed to host immunity, as Arabidopsis gene knockout or Hpa sRNA-resistant gene versions exhibited quantitative enhanced or reduced susceptibility, respectively. Together with previous reports, we found that ckRNAi is conserved among oomycete and fungal pathogens.