Development of Agrobacterium tumefaciens infiltration of infectious viral vectors of grapevine geminivirus A and defective viral genomes directly into greenhouse-grown grapevine and Nicotiana benthamiana plants

Grapevine virus infectious clones are important tools for fundamental studies, but also because of their potential for translational applications for grapevine improvement.  Although several grapevine virus infectious clones have been developed, there has been difficulty in directly infecting mature grapevine plants, and many of the viruses used still cause disease symptoms in grapevine plants making them less likely candidates for biotechnological applications in grapes. Here, we developed an improved Agrobacterium tumefaciensinfiltration method that can be used to deliver DNA plasmids and viral infectious clones directly into ~20-40 cm-high (above soil) greenhouse-grown grapevine plants. We developed infectious clones for two isolates of grapevine geminivirus A (GGVA):  isolate Longyan – China (GenBank accession number: KX570611; GGVA-76) and Super Hamburg - Japan (GenBank: KX570610; GGVA-93). We also constructed the defective forms of both isolates based on the defective viral genome of the Nagano Purple isolate (GenBank: KX570608.1). Neither virus caused any obvious symptoms when inoculated to plants of grapevine varieties Colombard, Salt Creek, Cabernet Sauvignon, and Vaccarèse. However, the two GGVA isolates, and the co-infection of the wild-type and defective viral genomes induced different symptom severity and viral titer in Nicotiana benthamiana plants. The two GGVA isolates used here were found to accumulate to different titers in different parts/branches of the infected grapevine plants. The GGVA infectious clones and the improved grapevine infiltration technique developed here provide new, valuable tools that can be applied to grapevine plants, possibly even for translational applications such as disease management and desired trait improvements.