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

Abstract

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 dispensible for tumorigenesis.Infection and colonization of plant or animal hosts often require a multifaceted attack against the host. For example, many pathogenic bacteria, including Bordetella pertussis, Yersinia spp., and Pseudomonas syringae, release multiple toxins and other virulence factors, any one of which may be dispensable for pathogenesis (5,7,8). It seems plausible that the plant pathogen Agrobacterium tumefaciens might similarly use more than one approach to invade and colonize host plants. This bacterium is well-known to transfer fragments of plasmid-encoded DNA (T-DNA) by a conjugation-like mechanism to the nuclei of host plants, where they are integrated into the host genome (23). This T-DNA directs the production of phytohormones (leading to formation of crown gall tumors) and of compounds called opines, which serve the colonizing bacteria as sources of nutrients. While it is not clear whether A. tumefaciens uses additional strategies to attack host plants, another species of Agrobacterium (A. vitus) releases a root macerating pectinase, as well as transferring T-DNA (22). This pectinase is not required for detectable tumorigenesis, while T-DNA transfer is not required for root maceration, indicating that this organism uses at least two independent strategies for pathogenesis.One way to search for undescribed strategies for host infection and colonization is to identify genes that are induced during infection and not required for T-DNA transfer. Approximately 25 genes, called vir genes, are required for T-DNA transfer (23,25). All vir genes are coordinately induced during infection by plant-released chemical signals, including phenolic compounds such as acetosyringone (11). This induction requires the sensory histidine protein kinase VirA, the response regulator VirG, and the periplasmic sugar-binding protein ChvE. Importantly, several of the genes in this regulon, including virH, virK, virL, and virM, are not required for efficient tumorigenesis (15). While it is possible that some or all of these genes play ancillary, dispensable roles in T-DNA transfer, at least some of these genes may direct other processes. We have recently demonstrated that the VirH2 protein catalyzes the O demethylation of several phenolic compounds, converting them to forms that are inactive as vir gene inducers (16). The functions of virK, virL, and virM are not understood, although virK strongly resembles a gene found on a symbiotic megaplasmid of Rhizobium sp. strain NGR234 and is thus unlikely to have any direct role in T-DNA transfer.As part of an ongoing effort to identify new genes that are part of the vir regulon and to study their roles in pathogenesis, we have sequenced approximately 15 kb of DN...

The Bases of Crown Gall Tumorigenesis

“…These include virD5, -E3, -F, -H, -J, -K, -L, -M, P, and -R (50,52). However, the lack of an apparent role in tumorigenicity could be a consequence of functional redundancy.…”

VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals

“…Phospho-VirG activates expression of at least 14 vir operons, containing a total of 30 genes (3,5,6) on the octopine-type Ti plasmid, whereas far fewer VirG-inducible genes have been identified on the nopaline-type Ti plasmid (7). The products of some vir genes play direct roles in T-DNA transfer, whereas the products of other vir genes are not required for tumorigenesis and probably play other roles during the initial stages of infection (8).…”

Indoleacetic acid, a product of transferred DNA, inhibits vir gene expression and growth of Agrobacterium tumefaciens C58

“…The response regulator, VirG, is phosphorylated by VirA (Asp-52) and then directly activates a regulon of Ϸ30 genes on the tumor-inducing (Ti) plasmid (9). Interestingly, only Ϸ20 of the 30 genes are required for plant cell transformation under laboratory conditions (10).…”