Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV secretion pathway

Agrobacterium tumefaciens uses a type TV secretion system to deliver oncoge nic nucleoprotein particles and effector proteins, such as the multifunctio nal VirE2 protein, to plant cells. In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export, A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abun dance. Deletion of virE1 did not affect transcription but decreased transla tion of virE2, as shown by analysis of lacZ transcriptional and translation al fusions, VirE2 was stable for a prolonged period, more than 6 h, when it was expressed in cis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans with virE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments. Vir E1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synt hesized in A, tumefaciens, VirE1 self-association was demonstrated by using bacteriophage lambda cI repressor fusion and pull-down assays, and evidenc e of VirE1 homomultimerization in vivo was obtained by native polyacrylamid e gel electrophoresis and gel filtration chromatography. A putative VirE1-V irE2 complex with a molecular mass of about 70 to 80 kDa was detected by ge l filtration chromatography of extracts from wild-type cells, whereas highe r-order VirE2 complexes or aggregates were detected in extracts from a virE 1 mutant, Taken together, our findings show that virE1 contributes in sever al ways to VirE2 export:(i) virE1 regulates efficient virE2 translation in the context of expression from the native P-virE promoter; (ii) the VirE1 s ecretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a pr edicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 comp lexes or aggregates.