A 5' tRNA-Ala derived small RNA regulates anti-fungal defense in plants

Apart from their primordial role in protein synthesis, tRNAs can be cleaved to produce tRNA-derived small RNAs (tsRNAs). The biological functions of tsRNAs in plants remain largely unknown. In this study, we developed RtcB-based RNA sequencing, a method that captures and distinguishes between 2',3'-cyclic-phosphate (cP)-terminated RNAs and 3'-OH-terminated RNAs, and profiled 5' tsRNAs and 5' tRNA halves in Arabidopsis thaliana. We found that Arabidopsis 5' tsRNAs and 5' tRNA halves predominantly contain a cP at the 3' end and require S-like RNase 1 (RNS1) and RNS3 for their production. One of the most abundant 5' tsRNA, 5' tsR-Ala, likely by associating with AGO1, negatively regulates Cytochrome P450 71A13 (CYP71A13) expression and camalexin biosynthesis to repress anti-fungal defense. 5' tsR-Ala is downregulated upon fungal infection. Our study provides a global view of 5' tsRNAs and 5' tRNA halves in Arabidopsis and unravels an important role of a 5' tsRNA in regulating anti-fungal defense. Overall design: To profile 5' tsRNAs and 5' tRNA halves in Arabidopsis seedlings, we developed a RtcB ligation-based sRNA sequencing (RtcB sRNA-seq) in this study. To understand the functions of 5' tsRNAs, we perform mRNA-seq of mutants for the biogenesis of 5' tsRNAs to indentify differentially expressed genes in these mutants