Comprehensive analysis of hairpin small RNAs involved in resistance and pathogenesis during wheat-Puccinia triticina interactions.

Wheat leaf rust, caused by the fungus Puccinia triticina (Pt), severely affects the grain quality and quantity of bread wheat (Triticum aestivum L.). Hairpin small(s)RNAs, like micro(mi)RNAs and their variants [including isomiRNAs (isomiRs) and microRNA-like RNAs (milRNAs)], along with their corresponding target genes, bestow leaf rust disease resistance, development and progression from both interacting species. However, the regulatory networks remain inadequately understood. Thirteen differentially expressed novel miRNAs, including two isomiRs and three milRNAs were discerned from induced reads of wheat sRNA libraries, and a further 5,393 and 1,275 candidate target genes were predicted in wheat and Pt, respectively. Functional annotation divulged that wheat-originated miRNAs/isomiRs were involved in resistance, while Pt-derived milRNAs imparted pathogenesis. The identified milRNAs- Tae-Pt-milR5, Tae-Pt-milR12, and Tae-Pt-milR14b and their cleavage sites on Pt target gene MEP5 were confirmed through degradome library screening, suggesting cross-kingdom translocation of Pt virulent genes in wheat host. Co-expression analysis of miRNAs/isomiRs-target genes provided insights into combating leaf rust disease, while co-expression analysis of milRNAs-target gene pairs reflected the extent of pathogenicity exerted by Pt with varied expression levels at the analyzed time points. The analysis pinpointed leaf rust-responsive candidate hairpin sRNAs- Tae-miR8, Tae-Pt-miR12, Tae-Pt-miR14a, and Tae-Pt-miR14b in wheat and Tae-Pt-milR12 in Pt. This study provides new insights into the hairpin sRNAs involved in the resistance and pathogenesis of wheat and Pt, respectively. Furthermore, crucial hairpin sRNAs and their promising targets for future biotechnological interventions to augment stress resilience have been identified.