Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa.

Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)-DNA and TF-TF interactions. Functions of TF-TF interactions in wood formation are still in the early stages of identification. PtrMYB074 is a woody dicot-specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two-hybrid and bimolecular fluorescence complementation, we conducted a genome-wide screening for PtrMYB074 interactors and identified 54 PtrMYB074-TF pairs. Of these pairs, 53 are novel. We focused on the PtrMYB074-PtrWRKY19 pair, the most highly expressed and xylem-specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem-specific one of the pair's only two direct TF target genes. Using transient and CRISPR-mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074-PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought-tolerant phenotypes. Knowledge of the PtrMYB074-PtrWRKY19-PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.