MicroRNAs as master regulators of the plant <i>NB-LRR</i> defense gene family via the production of phased, <i>trans</i>-acting siRNAs

TLDR

Legumes and other plants form symbiotic relationships with microbes, yet the mechanisms by which hosts promote beneficial interactions while suppressing harmful ones remain poorly understood, and trans‑acting siRNAs (tasiRNAs) that are processed by DCL4 into phased 21‑nt intervals negatively regulate target transcripts. We identified three highly abundant 22‑nt microRNA families that target conserved domains of NB‑LRR genes, triggering the production of trans‑acting siRNAs. Our data show that these miRNAs generate phased tasiRNAs that regulate over 60 % of Medicago NB‑LRR loci (and also in potato), with additional evidence of DCL2/SGS3 cleavage and two‑hit processing, revealing a complex miRNA‑tasiRNA network that likely evolved to facilitate symbiosis and positions miRNAs as master regulators of this gene family.

Abstract

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans -acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) “phased” intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRR s and trigger the production of trans -acting siRNAs. High levels of small RNAs were matched to &gt;60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRR s. 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 s that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.