The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens

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Abstract
The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T-DNA processing and undefined virulence functions. Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root-inducing plasmid pRiA4b and to that of the octopine-type plasmid pTiA6NC. However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC. T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively. virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids. Non-polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4. An independently regulated fifth ORF (orf5) is located immediately downstream of 3' end of virD4 and encodes a polypeptide of 97.4 kDa. The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A. tumefaciens. Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence. In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non-polar mutants that were tested for virulence. virD3 and orf5 non-polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants. These data suggest that virD3 and orf5 are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.