Professor of Microbiology & Plant Pathology
My lab is interested in the biology and the molecular biology of the plant pathogen, Agrobacterium tumefaciens. This organism causes tumors, called crown galls, on susceptible plants. Tumor induction results from the transfer of a small piece of DNA, called T-DNA, from the bacterium to the plant cell during infection. The T-DNA becomes integrated into plant cell nuclear DNA and expression of genes on this segment causes the normal plant cell to differentiate into a tumor cell. Expression of additional T-DNA genes causes the plant tumor cells to produce and secrete novel small carbon compounds called opines. In turn, Agrobacterium cells can utilize opines as sole carbon and energy sources. In the bacterium, the T-DNA and the genes for opine catabolism reside on a large, extrachromosomal virulence element called the Ti plasmid. This plasmid itself is transmissible from the bacterium to recipient bacteria by a mating mechanism called conjugation.
Plant signalling and Ti plasmid conjugation. T-DNA transfer is initiated when the bacterium receives a proper chemical signal from the wounded plant. Similarly, Ti plasmid conjugal transfer requires a specific plant signal, in this case, an opine secreted by the tumor. These signalling events are crucial to the biology of the interaction between Agrobacterium and its host plant, and also between this bacterium and other related bacteria in the plant rhizosphere (Figure 1).
We are investigating the nature and the mechanism of the signalling events that lead to Ti plasmid conjugal transfer. Using approaches ranging from microbial physiology through genetics to molecular biology, we have dissected the communication mechanism operative in telling bacterial donor cells to initiate Ti plasmid conjugal transfer. We have identified, mapped, and are sequencing the loci of the Ti plasmid required for conjugal transfer and opine utilization. We have cloned, sequenced and analyzed a gene, called accR, which acts as the central negative regulator for controlling expression of conjugal transfer and opine catabolism operons. It is this gene product that senses the presence of the opine signal molecules produced by the transformed plant cells. We are examining the interaction of this repressor protein with operator sequences present in the promoters regulated by the signal opine.
We have identified a second level of regulation governing expression of Ti plasmid conjugal transfer functions. Donor bacteria induced for conjugation by the signal opines themselves secrete a second messenger that is required for maximum induction of the conjugal transfer operons within the donor population. This soluble effector molecule, called Agrobacterium auto inducer (AAI) functions in conjunction with the product of a second regulatory gene called traR. The product of this gene, TraR, is a transcriptional activator which itself requires AAI in order to induce expression of the traR operons. We have cloned and sequenced the traR gene and identified it as a homologue of luxR, a gene activator in Vibrio fischeri required for activation of the lux bioluminescence genes. Like TraR, LuxR requires a soluble co-inducer called VAI. Chemically, VAI and AAI are related, and are cross functional. We have identified, cloned and sequenced the Ti plasmid gene encoding AAI production. This gene, called traI is the first gene in a large operon encoding other Tra functions. Furthermore, traI requires AAI and TraR for expression. traI is a homologue of luxI, the V. fischeri gene responsible for AAI synthesis. Recently we have identified and sequenced a gene which acts to suppress the TraR:AAI system. Our analysis indicates that the product of this gene , TraM, acts as an antiactivator by binding to TraR protein.
Finally, the expression of traR is regulated by AccR. This completes the circle by putting the TraR-AAI regulatory circuit under control of the opine-responsive regulon (Figure 2). Thus, a model is emerging whereby opines regulate catabolism and expression of traR by repression, and conjugal transfer is secondarily regulated by activation mediated by TraR-AAI. We currently are examining the regulatory sequences recognized by the TraR-AAI system as well as studying the structure of TraR and determining the mechanisms by which AAI transforms this protein into an active transcriptional regulator and by which TraM modulates this interaction.