Leaf rust (Puccinia triticina) mediated RNAi in wheat (Triticum aestivum L.) prompting host susceptibility

Significance of microRNAs in regulating gene expression in higher eukaryotes as well as in pathogens like fungi to suppress host defense is a well-established phenomenon. The present study focuses on leaf rust fungi Puccinia triticina (Pathotype 77-5) mediated RNAi to make wheat (Triticum aestivum L.) more susceptible. To reach such conclusions, we first confirmed the presence of argonaute (AGO) and dicer-like protein (DCL) family sequences in Puccinia. Bioinformatic tools were applied to retrieve the sequences from Puccinia genome followed by cloning and sequencing from P. triticina pathotype 77-5 cDNA to obtain the specific sequences. Their homologs were searched in other 14 Puccinia races to relate them with pathogenesis. Further, precursor sequences for three miRNA-like RNA molecules (milRs) were cloned from P. triticina cDNA. Their target genes like MAP kinase were successfully predicted and validated through degradome mapping and qRT-PCR. Gradual increase in milR2 (milR and milR*) expression over progressive time point of infection and positive expression for all the milRs within 77-5 urediniospores confirmed a complete host- independent RNAi activity by P. triticina.