Loss of Dicer-like 5 induces temperature-sensitive genic male sterility in wheat: implications for hybrid crop development

We studied the environmental regulation of male sterility in durum wheat (Triticum turgidum ssp. durum) by generating dicer-like 5 (dcl5) mutants to explore the potential for small RNA-based hybrid breeding systems. We found that the loss of DCL5 depletes the production of both premeiotic and meiotic 24-nt phasiRNAs resulting in a TGMS phenotype with male sterility under standard growth conditions, while increased temperature partially restores male fertility. The presence of a single functional allele of DCL5 is sufficient to ensure complete fertility, highlighting the potential of a DCL5-based two-line system to produce fertile hybrid seeds as an alternative to the cytoplasmic male sterility and chemical hybridizing agents systems in wheat and other related crops. We demonstrate that the biogenesis of premeiotic 24-nt phasiRNA is independent of miRNA-mediated cleavage and a conserved motif plays a crucial role in initiating phasiRNA production by DCL5. Finally, single-cell RNA sequencing revealed co-expression of AGO1b, AGO4a, and AGO6 homeologs in cells likely involved in the biogenesis and function of 24-nt phasiRNA.