Cases of new phytopathogenic strains that reduce crop and tree yields are regularly reported. However, the molecular mechanisms underlying such emergence are still not well understood. The acquisition by non-pathogenic environmental strains of new virulence genes by horizontal gene transfer has been suggested as a factor driving the emergence of new pathogenic strains. In this study, we tested this hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four secreted type 3 associated proteins (T3SP) to the non-pathogenic Xanthomonas strains CFBP 7698 and CFBP 7700, which lack genes. encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped into Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. The non-pathogenic wild-type strains induced a necrosis similar to the hypersensitive response (HR), while the strains supplemented with T3SS and T3SP suppressed this response. Such suppression depends on a functional T3SS. Among the plasmid-encoded T3SPs, Hpa2, Hpa1, and, to a lesser extent, XopF1 are collectively involved in deletion. The monitoring of the plant population size showed that the sole acquisition of a functional T3SS by non-pathogenic strains affects the growth within the foliar tissues. These results provide functional evidence that acquisition through horizontal gene transfer of one T3SS and four T3SPs by non-pathogenic environmental strains is not sufficient to render the strains pathogenic. In the absence of a canonical effector, the only acquisition of a T3SS appears to be counter-selective, and further acquisition of type 3 effectors is probably required to allow the emergence of new pathogenic strains.