Plastid tRNA-derived small RNAs associate with AGO1 and accumulate upon sulfur deprivation in Chlamydomonas

Transfer RNA-derived small RNAs (tDRs) represent a diverse class of noncoding RNAs with emerging regulatory roles across all domains of life. In the green alga Chlamydomonas reinhardtii, next-generation sequencing revealed abundant, specific tDRs, with a predominance of those derived from plastid-encoded tRNAs. Most of these tDRs map to the 5’ ends of mature tRNAs and are 29-33 nucleotides in length, suggesting that they arise from cleavage at the anticodon loop or stem. We confirmed the plastid origin of some tDRs by engineering the chloroplast genome to replace the plastid-encoded tRNA^Asn(GTT) gene with a nuclear counterpart. As reported in land plants, DICER-LIKE endonucleases play a limited role in tDR biogenesis in Chlamydomonas. However, a distinct subset of abundant tDRs, originating from both nucleus- and chloroplast-encoded tRNAs, associates with ARGONAUTE1 (AGO1). Using Gaussia luciferase reporter assays, we demonstrated that AGO1-associated tDRs can mediate RNAi-like gene silencing, and that tDRs originating from plastid-encoded tRNAs can function outside the chloroplast. Additionally, the tDR population in Chlamydomonas undergoes dynamic changes in response to nutrient availability. While nitrogen starvation causes a general decrease in both mature tRNA and tDR levels, sulfur deprivation specifically induces the accumulation of certain tDRs. Furthermore, strains in which the plastid tRNA^Asn(GTT) gene was replaced by a nuclear counterpart exhibited phenotypic defects under sulfur-deprived conditions, suggesting a potential regulatory role for plastid-derived tDRs in nutrient stress responses. Collectively, these findings expand our understanding of tDR functions, particularly those of chloroplast origin, in photosynthetic eukaryotes.