Agrobacterium-mediated transient expression in citrus leaves: a rapid tool for gene expression and functional gene assay

In this study, we present a method for transient expression of the type III effector AvrGf1 from Xanthomonas citri subsp. citri strain A(w) in grapefruit leaves (Citrus paradisi) via Agrobacterium tumefaciens. The coding sequence of avrGf1 was placed under the control of the constitutive CaMV 35S promoter in the binary vectors pGWB2 and pGWB5. Infiltration of grapefruit leaves with A. tumefaciens carrying these constructs triggered a hypersensitive response (HR) in grapefruit 4 days after inoculation. When transiently expressed in grapefruit leaves, two mutants, AvrGf1 Delta N116 and AvrGf1 Delta C83, failed to induce an HR. Moreover, using bioinformatics tools, a chloroplast transit signal was predicted at the N terminus of AvrGf1. We demonstrated chloroplast localization by using an AvrGf1::GFP fusion protein, where confocal images revealed that GFP fluorescence was accumulating in the stomatal cells that are abundant in chloroplasts. Transient expression in citrus has the potential for aiding in the development of new disease defense strategies in citrus.