miRNA regulation of plant innate immune receptors

Plant genomes contain large numbers of cell surface LRR and intracellular NB-LRR immune receptors encoded by resistance (R)-genes that recognize specific pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R-gene expression are not well understood. We identified miRNA progenitor genes (MIR), precursor transcripts (pre-miRNAs), and two miRNAs [nta-miR1 (22-nt) and nta-miR2 (21-nt)] that guide cleavage of the TIR-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-mR1 triggers RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)- and ribonuclease DICER-LIKE 4 (DCL4)-dependent biogenesis of 21-nt secondary siRNAs “in phase” with the 22-nt miR1 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR1 was dependent on an asymmetric bulge caused by mismatch in the nta-miR1 precursor. Interestingly, coexpression of N with nta-miR1 and nta-miR2 resulted in attenuation of N-mediated resistance to TMV indicating that these miRs have functional role in NB-LRR regulation. Using a bioinformatics approach, we identified seven additional families of 22-nt miRNAs and one 21-nt miRNA family from three Solanaceae species –tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R-genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in Solanaceae.