The right end of the vir region of an octopine-type Ti plasmid contains four new members of the vir regulon that are not essential for pathogenesis.

We sequenced the virD-virE, virE-virF, and virF-T-DNA intergenic regions of an octopine Ti plasmid. Four newly described genes were induced by the vir gene inducer acetosyringone, two of which are conserved in the nopaline-type Ti plasmid pTiC58. One gene resembles a family of phosphatase genes. Each of these genes is dispensable for tumorigenesis.

Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid.

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.

The octopine-type Ti plasmid pTiA6 of Agrobacterium tumefaciens contains a gene homologous to the chromosomal virulence gene acvB.

Although the majority of genes required for the transfer of T-DNA from Agrobacterium tumefaciens to plant nuclei are located on the Ti plasmid, some chromosomal genes, including the recently described acvB gene, are also required. We show that AcvB shows 50% identity with the product of an open reading frame, designated virJ, that is found between the virA and virB genes in the octopine-type Ti plasmid pTiA6. This reading frame is not found in the nopaline-type Ti plasmid pTiC58. acvB is required for tumorigenesis by a strain carrying a nopaline-type Ti plasmid, and virJ complements this nontumorigenic phenotype, indicating that the products of these genes have similar functions. A virJ-phoA fusion expressed enzymatically active alkaline phosphatase, indicating that VirJ is at least partially exported. virJ is induced in a VirA/VirG-dependent fashion by the vir gene inducer acetosyringone. Primer extension analysis and subcloning of the virJ-phoA fusion indicate that the acetosyringone-inducible promoter lies directly upstream of the virJ structural gene. Although the roles of the two homologous genes in tumorigenesis remain to be elucidated, strains lacking acvB and virJ (i) are proficient for induction of the vir regulon, (ii) are able to transfer their Ti plasmids by conjugation, and (iii) are resistant to plant wound extracts. Finally, mutations in these genes cannot be complemented extracellularly.