Secondary si RNA s from Medicago NB ‐ LRR s modulated via mi RNA –target interactions and their abundances

Summary Small RNA s are a class of non‐coding RNA s that are of great importance in gene expression regulatory networks. Different families of small RNA s are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary si RNA s (phasi RNA s); these require RDR 6, DCL 4, and (typically) a micro RNA (mi RNA ) trigger for their biogenesis. Protein‐encoding genes are an important source of phasi‐ RNA s. The model legume Medicago truncatula generates phasi RNA s from many PHAS loci, and we aimed to investigate their biogenesis and mechanism by which mi RNA s trigger these molecules. We modulated mi RNA abundances in transgenic tissues showing that the abundance of phasi RNA s correlates with the levels of both mi RNA triggers and the target, precursor transcripts. We identified sets of phasi RNA s or PHAS loci that predominantly and substantially increase in response to mi RNA overexpression. In the process of validating targets from mi RNA overexpression tissues, we found that in the mi RNA ‐ mRNA target pairing, the 3′ terminal nucleotide (the 22nd position), but not the 10th position, is important for phasi RNA production. Mutating the single 3′ terminal nucleotide dramatically diminishes phasi RNA production. Ectopic expression of Medicago NB ‐ LRR ‐targeting mi RNA s in Arabidopsis showed that only a few NB ‐ LRR s are capable of phasi RNA production; our data indicate that this might be due to target inaccessibility determined by sequences flanking target sites. Our results suggest that target accessibility is an important component in mi RNA –target interactions that could be utilized in target prediction, and the evolution of mRNA sequences flanking mi RNA –target sites may be impacted.