Transient gene expression system in zoysiagrass leaf mesophyll protoplasts

Transient expression of genes in protoplasts is a versatile technique for rapid functional characterization of genes by assessing protein localization or effector–reporter responses. In addition, protoplasts have been widely used for generating transgenic or gene-edited plants in model or crop plants. Zoysiagrass (Zoysia japonica Steud.) is one of the most economically important turf plants used in many living places or natural fields, but its management is labor-intensive. Therefore, genetic manipulation using valuable genes is highly demanded in zoysiagrass. Although transient expression systems in zoysiagrass facilitates the identification of valuable zoysiagrass genes, transient expression use in the zoysiagrass protoplasts is yet to be reported. Here we describe the methodology and feasibility for transient expression of genes in the zoysiagrass protoplasts isolated from green leaves. We obtained more than 70% of transfection efficiency in the zoysiagrass protoplasts using polyethylene glycol-mediated transfection. Additionally, we showed the feasibility of cellular, biochemical, and molecular approaches using the zoysiagrass protoplasts transiently expressing genes of interest through fluorescent microscopic observation, immunoblot analysis, and luciferase-based promoter activity assay. Along with the genome draft of the Zoysia family, transient expression will be a valuable tool to rapidly investigate gene functions. An initial assessment of gene function using protoplasts facilitate the identification of valuable genes that can improve the economic value of the Z. japonica or its closely related species by gene manipulation.