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 ligation-based small RNA (sRNA) sequencing, a method that captures and distinguishes between 3′-2′,3′-cyclic-phosphate (cP)/phosphate (P)-terminated sRNAs and 3′-OH-terminated sRNAs, 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′ tsRNAs, 5′ tsR-Ala, by associating with AGO1, negatively regulates Cytochrome P450 71A13 (CYP71A13) expression and camalexin biosynthesis to repress anti-fungal defense. Interestingly, 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.