Identification of novel microRNAs and their target genes associated with stress-tolerant phytohormones in wheat (Triticum aestivum L.) during leaf rust pathogenesis

Phytohormones serve as the ace of regulatory mechanisms in plants during gene expression under different environmental conditions. They are also involved in combating diseases during plant-pathogen interactions. MicroRNAs are a group of small, non-coding and post-transcriptional regulatory RNAs that potentially function in stress tolerance as they target specific mRNAs and promote endonucleolytic cleavage or translational inhibition. Here, we aim to identify potential novel miRNAs that could assist in mitigating leaf rust resistance in wheat to combat rust epidemics. A total of 64 novel miRNAs were identified from Next Generation Sequencing datasets of small RNA libraries prepared from resistant and susceptible wheat near-isogenic lines (NILs) under mock and leaf rust pathogen (Puccinia triticina Eriks.) infected conditions. Eight miRNAs (present post leaf rust infection) complementary RNA cognate targets were predicted through psRNATarget. Ontological annotations and combined pathway analysis were applied to identify their specific functions in different phytohormone biosynthesis or signaling pathways. The miRNA target interaction was successfully validated through degradome mapping. RT-qPCR revealed that most miRNAs (TamiRs) were downregulated in the resistant NIL introgressed with Lr24 gene with an antagonistic upregulation of their phytohormone-associated target genes (AOC, BAGT, BSK3, FAR, GABA, HSF, JUNG, and RPD). However, target gene, RPP showed no such correlation in the presence of microRNA or Lr24 and was upregulated throughout the timepoints of disease progression in both the NILs. This study deciphers the regulatory linkage between the identified novel miRNAs and their phytohormonal target transcripts during leaf rust disease in wheat to assist in rust-resistant breeding programs.