Complex feedback regulations govern the expression of miRNA396 and its GRF target genes.

The beet cyst nematode, Heterodera schachtii, is a sedentary root parasite that induces the formation of a specialized root feeding structure, the syncytium. We previously have shown that coordinated regulation of miR396 and its target genes GRF1 and GRF3 in the syncytium is required for proper formation. To gain a better understanding of this coordinated regulation, we used quantitative real-time PCR to assess the abundance of primary (pri)-miRNA396a, pri-miRNA396b and mature miRNA396 in transgenic Arabidopsis plants overexpressing either wild-type variants of the GRF1 or GRF3 coding sequences or miR396-resistant variants. We also included a grf1/grf2/grf3 triple mutant in these analyses. We observed significant decreases in the abundance of pri-miRNA396a, pri-miRNA396b and mature miR396 in the transgenic plants overexpressing GRF1 or GRF3, particularly with the miRNA396-resistant variants. In contrast, the primary transcripts and mature miRNA396 abundance were significantly increased in the grf1/grf2/grf3 triple knockout mutant. These results demonstrate that homeostasis between miR396 and the target genes GRF1 and GRF3 is established through reciprocal feedback regulation, in which GRF1/GRF3 and miR396 negatively regulate each other's expression. In addition, we found that constitutive expression of GRF1 or GRF3 decreases the mRNA abundance of other GRFs, even those that are not targeted by miR396, as well as their own endogenous transcripts, which documents further regulatory facets of this equilibrium.