Agrobacterium delivers VirE2 protein into host cells via clathrin-mediated endocytosis

can cause crown gall tumors on a wide range of host plants. As a natural genetic engineer, the bacterium can transfer both single-stranded DNA (ssDNA) [transferred DNA (T-DNA)] molecules and bacterial virulence proteins into various recipient cells. Among -delivered proteins, VirE2 is an ssDNA binding protein that is involved in various steps of the transformation process. However, it is not clear how plant cells receive the T-DNA or protein molecules. Using a split-green fluorescent protein approach, we monitored the VirE2 delivery process inside plant cells in real time. We observed that delivered VirE2 from the bacterial lateral sides that were in close contact with plant membranes. VirE2 initially accumulated on plant cytoplasmic membranes at the entry points. VirE2-containing membranes were internalized through clathrin-mediated endocytosis to form endomembrane compartments. VirE2 colocalized with the early endosome marker SYP61 but not with the late endosome marker ARA6, suggesting that VirE2 escaped from early endosomes for subsequent trafficking inside the cells. Dual endocytic motifs at the carboxyl-terminal tail of VirE2 were involved in VirE2 internalization and could interact with the μ subunit of the plant clathrin-associated adaptor AP2 complex (AP2M). Both the VirE2 cargo motifs and AP2M were important for the transformation process. Because AP2-mediated endocytosis is well conserved, our data suggest that the pathogen hijacks conserved endocytic pathways to facilitate the delivery of virulence factors. This might be important for to achieve both a wide host range and a high transformation efficiency.