Identifying the Downstream Effectors of miRNA 160 During Soybean Root Nodule Development

Soybean is an excellent candidate for sustainable agriculture due to its production of nutritious, versatile beans and the ability to form symbiotic organs called root nodules that perform nitrogen fixation. As demand for both yield and sustainable agriculture continue to increase, root nodules offer an attractive alternative to expensive and environmentally harmful nitrogen fertilizers. Understanding root nodule formation may open genetic engineering avenues for optimizing nitrogen fixation performance and transferring the nodule-formation ability to other plants. A major candidate in soybean root nodule formation is microRNA 160 (miR160), which mediates auxin sensitivity by targeting repressor auxin response factor (ARF) transcription factors during nodule development. However, the specific downstream effectors of this interaction remain unknown. This project seeks to resolve these effectors by evaluating the cellular, spatiotemporal, and DNA-binding activity of ARF 16-2 (a key target of miR160), by confocal microscopy, DamID-Seq, and protein-DNA interaction studies. So far, 16-2promoter:GUS or tdT fusions reveal ARF 16-2 is transcriptionally active in dividing and differentiating tissues such as primary root tips, lateral root primordium, and emerging nodules. In addition, 16-2 promoter:GUS or tdT fusions show that this gene is transcriptionally active in the parenchyma and root stele of mature nodules. Interestingly, 16-2 promoter:16-2 CDS:tdT translational fusions show a reduction of tdT signal in mature nodule tissues, underscoring post-transcriptional regulation of 16-2, likely by miR160 during nodule maturation. When the genomic binding profile of 16-2 is identified and evaluated, we expect to identify key downstream genes affected by its activity during nodule development.