AGROBACTERIUM VIRE2 PROTEIN MEDIATES NUCLEAR UPTAKE OF SINGLE-STRANDED-DNA IN PLANT-CELLS

Agrobacterium genetically transforms plant cells by transferring a sin gle-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2, VirE2 binds single-stranded nucleic acid coope ratively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone co uld mediate nuclear localization of ssDNA, In this study, fluorescentl y labeled ssDNA accumulated in the plant cell nucleus specifically whe n microinjected as a complex with VirE2, Microinjected ssDNA alone rem ained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2- ssDNA complex import in the presence of wheat germ agglutinin or a non hydrolyzable GTP analog, both known inhibitors of protein nuclear impo rt, and (ii) the retardation of import when complexes were prepared fr om a VirE2 mutant impaired in ssDNA binding and nuclear import.