Biogenesis Mechanism And Biological Functions Of 5' TsRNA In Arabidopsis

Transfer RNA(tRNA)is an adaptor molecule that is crucial for protein synthesis.tRNAs fold into a cloverleaf-like secondary structure,which comprises the acceptor stem,dihydrouridine(D)stem-loop,anticodon stem-loop and T?C stem-loop(?represents pseudouridine).D and T?C loops,as their names indicate,bear D and ?modifications,respectively.tRNAs can be cleaved to produce tRNA-derived small RNAs(tsRNAs).5' tsRNAs cleaved in the D loop are widely conserved in eukaryotes.However,the mechanisms of 5' tsRNA biogenesis and the functions remain unclear.In this study,we find that 5' tsRNAs predominantly contain a 2',3'-cyclic phosphate at the 3' termini(3'-cP).RNase T2 family proteins,S-like RNase 1(RNS1)and RNS3 are required for the production of 5' tsRNAs.-1 and +1 positions of the tRNA cleavage sites are often G and U.Us are often modified to D.DUS1 to DUS5 are five dihydrouridine synthase genes in Arabidopsis.In dus1 and dus5 mutants,the accumulation of 5' tsRNAs is reduced.Our results suggest that D modification may promote 5' tsRNA production.We further find that 5' tsR-Ala,the most abundant 5'tsRNA,can be incorporated into ARGONAUTE 1(AGO1)and targets cytochrome P450 monooxygenases 71A13(CYP71A13).CYP71A13 catalyzes a key intermediate step in camalexin(3-thiazol-2'-yl-indole)biosynthesis.Camalexin,a major phytoalexin in Arabidopsis,confers resistance to necrotrophic fungal pathogens including Botrytis cinerea.In rns1?rns3?dus1?dus5 and dus1 dus5 mutants as well as 5' tsR-Ala knock-down lines,CYP71A13 and camalexin levels are increased and all the mutants are more resistant to B.cinerea,suggesting that 5' tsR-Ala negatively regulates plant resistance against B.cinerea through downregulating CYP71A13.Intriguingly,5'tsR-Ala is down-regulated in response to B.cinerea,likely through the down-regulation of D modification,suggesting that D is a dynamic modification that can be regulated.We establish the link between D modification and 5' tsRNA biogenesis under normal and fungi-infected conditions.Our study provides insights into the mechanism of 5' tsRNA biogenesis and reveals a role of 5' tsRNAs in plant anti-fungal defense.