A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins

Plant protoplasts constitute a versatile system for transient gene expression and have been widely used with several plant species for the functional characterization of genes and studies of diverse signaling pathways. However, such a system has not been developed for grapevine (Vitis vinifera L.) due to the challenges of large-scale isolation of viable grapevine protoplasts. Here, we report a simplified method for obtaining high yields and excellent viability of isolated protoplasts from young grapevine leaves. In addition, both the conditions for isolation and transfection of grapevine mesophyll protoplasts were modified, and the system was shown to be suitable for protein expression and studies of protein subcellular localization and protein-protein interactions. In addition, we heterologously and transiently expressed the Arabidopsis thaliana disease resistance protein RPW8.2, which has previously been reported to confer broad-spectrum resistance to several biotrophic pathogens in different plant families, as a fluorescent fusion protein in grapevine protoplasts. We observed that expression of the RPW8.2 fusion protein was induced in response to application of exogenous salicylic acid and following infection by the grapevine downy mildew pathogen, Plasmopara viticola. These results illustrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and investigation of the activity of disease resistance proteins in grapevine.