Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173-initiated trans-acting cascade silencing.

Small RNAs such as microRNAs (miRNAs) and small interfering (si)RNAs are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22-nucleotide long miRNAs trigger biogenesis of cascades of trans-acting secondary small interfering RNAs, involved in various developmental and stress responses. Here we show that in Himalayan Arabidopsis thaliana accessions having natural mutations in miR158 locus exhibit robust cascade silencing in pentatricopeptide (PPR)-like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural variants of MIR158 having deletions or insertions led to improper processing of miR158 precursors thereby blocking synthesis of mature miR158. Reduced miR158 led to increased level of its target, a pseudo-pentatricopeptide gene that is otherwise targeted by ta-siRNAs generated by miR173 cascade in other accessions. Using small RNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to build up of pseudo-PPR derived tertiary small RNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2 a Na + -K + /H + antiporter protein thereby regulating transpiration and stomatal conductance. Overall, we deciphered a new module of sRNA network, miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2 in plant adaptation.