microRNAs as Master Regulators of the Plant NB-LRR Defense Gene Family via the Production of Phased, Trans-acting siRNAs

Legumes and many non-leguminous plants enter symbiotic interactions with microbes, and it’s poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide “phased” intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt miRNA families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to over 60% of all ~540 encoded Medicago NB-LRRs; in potato, a model for mycorrhizal interactions, phased siRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent “two-hit” phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRR that potentially evolved to facilitate symbiotic interactions, and demonstrate miRNAs as master regulators of a large gene family, a new paradigm for miRNA function.