CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine(Vitis vinifera L.)

Downy mildew of grapevine(Vitis vinifera L.),caused by the oomycete pathogen Plasmopara viticola,is one of the most serious concerns for grape production worldwide.It has been widely reported that the pathogenesis-related 4(PR4)protein plays important roles in plant resistance to diseases.However,little is known about the role of PR4 in the defense of grapevine against P.viticola.In this study,we engineered loss-of-function mutations in the VvPR4b gene from the cultivar“Thompson Seedless”using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance.Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred.Infection assays using leaf discs showed that,compared to wild-type plants,the VvPR4b knockout lines had increased susceptibility to P.viticola.This was accompanied by reduced accumulation of reactive oxygen species around stomata.Measurement of the relative genomic abundance of P.viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen.Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.