Fig. 2: GmMYB176-interacting transcription factors and their DNA binding activity.

a GmMYB176- transcription factors obtained by co-IP assay GO annotation “Biological process” was retrieved and compared with the list of transcription factors obtained from in silico analysis of 30 bp GmCHS8 promoter fragment (Supplementary Table 2) using a Venn diagram. b Protein–protein interactions of GmMYB176 and GmMYB176S29A in planta with candidate transcription factors obtained by Co-IP. The interaction between the proteins was assayed by co-expression of translational fusions of candidate proteins with N-terminal (YN) and C-terminal (YC) halves of YFP. The proximity of the two fragments results in a functional fluorophore. The fluorescence indicates the presence and location of the interaction between GmMYB176 or GmMYB176S29A with the candidate transcription factors. Fluorescent intensity parameters were kept constant in all images. Scale bars = 50 μm. c GmCHS8 promoter (30 bp fragment) binding activity of GmMYB176 interacting candidates. Yeast cells carrying 30 bp GmCHS8 tandem repeats (30 bpTR) as a bait, were transformed with prey constructs fused to a GAL4 activation domain. Growth on SD lacking leucine and in the presence of Aureobasidin A (SD/-Leu/AbA) shows the activation of the reporter and indicates DNA binding activity. As a negative control, pGADT7 vector only was used. d Tissue-specific expression pattern of GmMYB176, GmbZIP4, and GmbZIP5 in soybean. RNA-seq data across different tissues were extracted from soybean whole genome database in Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Gmax) and a heatmap was constructed. Numbers to the right indicate the maximum value of fragments per kilobase of million mapped reads (FPKM). The color scale indicates expression values, green indicating low transcript abundance, and red indicating high levels of transcript abundance.