Wound-Released Chemical Signals May Elicit Multiple Responses from an Agrobacterium tumefaciens Strain Containing an Octopine-Type Ti Plasmid

Agrobacterium tumefaciens has the unique ability to stably modify the genomes of host plants in a way that is beneficial to the bacterium (45). It does this by transferring approximately 15 genes from a 200-kb plasmid (the Ti plasmid) to plant nuclei, where they become covalently integrated into the host genome. This transfer process requires the products of approximately 20 known vir genes located on a nontransferred portion of the Ti plasmid (44), as well as a smaller number of chromosomally encoded proteins. Most transferred genes can be categorized into two distinct groups. The first group leads to the production of opines, compounds that are released by the plant and used by the bacterium as nutrient sources (8), while the second group of transferred genes mediates the overproduction of the phytohormones auxin and cytokinin, which interfere with the plant’s natural phytohormone balance and cause neoplastic growth, resulting in crown gall tumors (1). An early event leading to tumorigenesis is the transcriptional induction of the vir regulon in response to a variety of chemical signals that are released from plant wounds. The most important of these signals consist of a family of related phenolic compounds, including acetosyringone, that act in combination with extracellular acidity and particular monosaccharides (16). These signals are perceived by the chromosomally encoded sugar-binding protein ChvE and the Ti plasmid-encoded proteins VirA and VirG (2, 18, 40). VirA is a transmembrance sensory histidine kinase which phosphorylates the response regulator VirG (20, 21, 43). Phospho-VirG binds to binding sites designated vir boxes found upstream of each vir promoter and coordinately activates transcription of these promoters (15, 22). The previously characterized vir regulon includes eight operons (36, 39). Of these, the virA, virB (6, 23), virD, and virG operons are essential for tumorigenesis, while virC and virE are required for efficient tumorigenesis, and virF is required for tumorigenesis on certain plant hosts (17). virH of the octopine-type Ti plasmid was thought not to be required for tumorigenesis, although none of the available mutations disrupts both genes of the virH operon. The tzs gene of nopaline-type Ti plasmids, which directs the production of a cytokinin precursor, is also part of the vir regulon, but disruptions of this gene have not been described. In an attempt to identify the homologous regions between octopine-type and nopaline-type Ti plasmids, Engler et al. (11) used the electron microscope to determine which sequences of the octopine-type Ti plasmid pTiAch5 were able to form heteroduplexes with the nopaline-type Ti plasmid pTiC58. This study revealed that these plasmids share four major regions of homology, consisting of approximately 30% of their sequences. At that time, genetic analysis of these regions was extremely rudimentary. In hindsight, we now know that every conserved locus directs a process of fundamental importance to the biology of these plasmids. For example, each conserved gene in one cluster was later shown to encode a vir gene (Fig. ​(Fig.1),1), while most of the nonconserved sequences were later shown to encode insertion sequence (IS) elements. The Engler study identified two conserved genetic loci at the left end of the known vir region that were not subsequently studied. The reported positions of these conserved sequences suggested that they might flank the virH operon, although our analysis necessitates a reevaluation of these mapping data. Since all the genes in the vir region that are essential for tumorigenesis are conserved between the two plasmids, we decided to identify these conserved genes and evaluate their roles in pathogenesis. FIG. 1 Octopine- and nopaline-type Ti plasmid vir regions. Solid parallelograms represent the regions of homology described by Engler and colleagues (11). The dashed parallelogram indicates the region conserved between these plasmids. Restriction fragments newly ...