DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) also interacts with WOX and KNOX proteins associated with wood formation in Populus trichocarpa.

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) from snapdragon (Antirrhinum majus) is a MYB/SANT protein that interacts with related MYB/SANT proteins, RADIALIS and DIVARICATA, through its N-terminal MYB/SANT domain. In addition to the MYB/SANT domain, DRIF contains a C-terminal domain of unknown function (DUF3755). Here we describe novel protein-protein interactions involving a poplar (Populus trichocarpa) homolog of DRIF, PtrDRIF1. In addition to interacting with poplar homologs of RADIALIS (PtrRAD1) and DIVARICATA (PtrDIV4), PtrDRIF1 interacted with members of other families within the homeodomain-like superfamily, including PtrWOX13c, a WUSCHEL-RELATED HOMEOBOX protein, and PtrKNAT7, a KNOTTED1-LIKE HOMEOBOX protein. PtrRAD1 and PtrDIV4 interacted with the MYB/SANT-containing N-terminal portion of PtrDRIF1, whereas DUF3755 was both necessary and sufficient for interactions with PtrWOX13c and PtrKNAT7. Of the two MYB/SANT domains present in PtrDIV4, only the N-terminal MYB/SANT domain interacted with PtrDRIF1. GFP-PtrDRIF1 expressed alone or with PtrRAD1 localized to the cytoplasm, whereas co-expression of GFP-PtrDRIF1 with PtrDIV4, PtrWOX13c or PtrKNAT7 resulted in nuclear localization of GFP-PtrDRIF1. Modified yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments using PtrDRIF1 as a bridge protein revealed that PtrDRIF1 simultaneously interacted with PtrRAD1 and PtrWOX13c, but could not form a heterotrimeric complex when PtrDIV4 was substituted for PtrRAD1. Moreover, a Y2H competition assay indicated that PtrKNAT7 inhibits the interaction between PtrRAD1 and PtrDRIF1. The discovery of an additional protein-protein interaction domain in DRIF proteins, DUF3755, and its ability to form heterodimers and heterotrimers involving MYB/SANT and wood-associated homeodomain proteins, implicates DRIF proteins as mediators of a broader array of processes than previously reported.