Identification of a carboxy-terminal glutamine-rich domain in agrobacterium tumefaciens coupling protein VirD4 required for recognition of T-Strand DNA and Not VirE2 as a substrate for transfer to plant cells

Agrobacterium tumefaciens transfers DNA and proteins to a plant cell inciting crown gall tumor disease on most plants. VirD4 targets the DNA and protein substrates to a type IV secretion (T4S) apparatus for translocation into the plant cell. Several bacteria with VirD4 homologs use T4S for intercellular export of microbial macromolecules to eukaryotic and prokaryotic hosts. How the VirD4 proteins recognize the diverse substrates is not well understood. To identify functional domains of A. tumefaciens pTiA6 VirD4, we introduced random 19-codon and targeted 10-codon insertions throughout the coding region. Analysis of 21 mutants showed that only the carboxy-terminal end of VirD4 is tolerant of an insertion. Sequence comparison of VirD4 proteins of Agrobacterium spp. and their close relative, Rhizobium etli, showed that these proteins contain a highly conserved C-terminal end, but the immediate upstream regions share no discernible sequence similarity. The conserved region sequence is rich in the amino acid glutamine (6/13 Q). Using site-specific and deletion mutagenesis, we demonstrated that the conserved Q-rich region is required for VirD4 function and for the specific recognition of VirD2-linked T-strand DNA as a substrate for translocation to plants. The Q-rich region is not required for the transfer of a second A. tumefaciens substrate, VirE2, to plants or a promiscuous Escherichia coli IncQ plasmid to another A. tumefaciens strain. We identified Q-rich sequences at or near the C terminus of several VirD4 homologs, including the E. coli F plasmid TraD. In F TraD, the Q-rich sequence maps to a region required specifically for the conjugative transfer of the F plasmid.