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

Abstract

Reproductive, male-enriched small RNAs are present in flowering plants and animals, yet their role in plants remains underexplored. We generated <i>dicer-like 5</i> (<i>dcl5</i>) mutants in durum wheat (<i>Triticum turgidum</i> ssp. <i>durum</i> 2n = 4× = 28; AABB), revealing temperature-sensitive genic male sterility. Loss of <i>DCL5</i> 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 <i>DCL5</i> 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 <i>dcl5</i> 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 <i>dcl5</i> 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 <i>Argonaute</i> (<i>AGO1b</i>, <i>AGO4a,</i> and <i>AGO6</i>) homeologs in 24-nt phasiRNA-producing cells identifies candidate effectors and suggests a role for 24-nt phasiRNAs in transcriptional gene silencing.