DICER-LIKE 5 loss causes thermosensitive male sterility in durum wheat and reveals an AU-rich motif guiding 24-nt phasiRNA biogenesis

Reproductive, male-enriched small RNAs are present in flowering plants and animals, yet their role in plants remains underexplored. We generated dicer-like 5 ( dcl5 ) mutants in durum wheat ( Triticum turgidum ssp. durum 2n = 4× = 28; AABB), revealing temperature-sensitive genic male sterility. Loss of DCL5 depleted premeiotic and meiotic 24-nt phasiRNA production, correlating with sterility under standard growth conditions and partial fertility recovery at higher temperatures. A single functional DCL5 allele restored complete fertility, presenting a promising alternative to current methods for hybrid production. We demonstrate that premeiotic 24-nt phasiRNA biogenesis is independent of miRNA-mediated cleavage and driven by a conserved motif initiating DCL5 activity. In the dcl5 mutant developing under sterility-inducing conditions, developmental defects are observed during pollen maturation, rather than at peak 24-nt phasiRNA accumulation in premeiotic and meiotic anthers. Although no visible morphological abnormalities were apparent during early development, single-cell RNA sequencing revealed that dcl5 mutant cells exhibit transcriptional profiles distinct from those of wild-type cells, when premeiotic 24-nt phasiRNAs are accumulating at the early developmental stage. Finally, the coexpression of Argonaute ( AGO1b , AGO4a, and AGO6 ) homeologs in 24-nt phasiRNA-producing cells identifies candidate effectors and suggests a role for 24-nt phasiRNAs in transcriptional gene silencing.