<i>Nicotiana benthamiana VASCULAR-RELATED NAC-DOMAIN7-2</i> (<i>NbVND7-2</i>) has a role in xylem formation during interfamily grafting

VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a transcription factor gene that plays a critical role in xylem differentiation. The ectopic expression of VND7 induces the formation of secondary cell walls with spiral patterns in multiple plant cell types. In the present study, we identified four homologs of VND7 in Nicotiana benthamiana and assigned them the names NbVND7-1 to NbVND7-4. In particular, NbVND7-1 and NbVND7-2 were highly expressed during N. benthamiana and Arabidopsis thaliana (Nb/At) interfamily grafting. Analysis of the promoter β-glucuronidase (GUS) reporter lines of NbVND7 genes elucidated the expression of NbVND7-1 and NbVND7-2 in xylem tissues of intact and grafted plants, and those of NbVND7-3 and NbVND7-4 in internal phloem tissues. Gene network analysis revealed the downstream genes of each NbVND7 homolog and highlighted the association of NbVND7-1 and NbVND7-2 with xylem formation. A β-estradiol-inducible system for NbVND7-2 demonstrated that NbVND7-2 promotes ectopic xylem vessel differentiation in N. benthamiana seedlings and in the stem tissues at graft junctions. Induction of NbVND7-2 at graft junctions enhanced ectopic xylem formation in the callus tissues proliferated at the graft boundary, accelerated the initiation of water transport from stock to scion, and enhanced scion stem growth after grafting. This study reveals a role for NbVND7 genes in xylem formation that can enhance Nicotiana interfamily grafting.