Abolished miR158 activity leads to 21-nucleotide tertiary phasiRNA biogenesis that targets NHX2 in Arabidopsis thaliana

Small RNAs including microRNAs (miRNAs) are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Some small RNAs, mostly 22-nucleotide long trigger biogenesis of secondary small interfering RNAs (siRNAs). Those siRNAs having distinctive phased configuration are known as phased siRNAs (phasiRNAs) and act either in cis or trans enhancing silencing cascade. Here, we report natural variants of MIR158 having deletions or insertions led to negligible or reduced expression of miR158. The deletion/insertion events affected processing of primary transcript of miR158 to precursor and to mature miR158. We show that miR158 targets a pseudo-pentatricopeptide gene and its abolished activity led to 21-nucleotide tertiary phasiRNA generation from its target. The biogenesis of these phasiRNAS is triggered by TAS2 derived two siRNAs. Accordingly, small RNA analyses of these natural variants, mutants and over-expression lines of MIR158 or its target exhibited enhanced or reduced phasiRNA biogenesis. Finally, we functionally validated the highest expressed tertiary phasiRNA that targets NHX2 thereby regulating transpiration and stomatal conductance. Overall, we deciphered a new module of small RNA network, miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2, suggesting an additional layer of gene regulation and larger role of pseudogene in plants.