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

Abstract

Downy mildew of grapevine (<i>Vitis vinifera</i> L.), caused by the oomycete pathogen <i>Plasmopara viticola</i>, 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 <i>P. viticola</i>. In this study, we engineered loss-of-function mutations in the <i>VvPR4b</i> 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 <i>VvPR4b</i> knockout lines had increased susceptibility to <i>P. viticola</i>. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of <i>P. viticola</i> in <i>VvPR4b</i> knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that <i>VvPR4b</i> plays an active role in the defense of grapevine against downy mildew.