EARLY EVENTS IN THE AGROBACTERIUM TUMEFACIENS - PLANT INTERACTION

Tumor formation by Agrobacterium tumefaciens involves the transfer and integration of a defined segment (T-DNA) of tumor-inducing (Ti) plasmid DNA into the plant tumor genome. A set of plasmid genes outside the T-DNA, the vir genes, are thought to mediate the transfer process. We have performed a detailed molecular and genetic analysis of a limited host range Ti plasmid of Agrobacterium in order to compare it to the well characterized wide host range plasmids. These studies identified two regions of the limited host range plasmid, designated T(,A)-DNA and T(,B)-DNA, which become stably integrated into the plant tumor genome. These plasmid regions are separated by more than twenty five kilobase-pairs of not integrated plasmid DNA. Furthermore, whereas the wide host range oncogenes are localized immediately adjacent to one another within a single T-DNA region, homologous counterparts to these genes are divided among the two widely separated T-DNA regions on the limited host range plasmid. Factors both within the T-DNA and vir regions contribute to the narrow host range conferred by the limited host range plasmid. Within the T-DNA a defective cytokinin biosynthetic gene limits host range and introduction of the wide host range cytokinin gene into the limited host range T-DNA region expands host range. However, in order to fully restore a wide host range phenotype, the virA and virC loci from the wide host range plasmid must also be introduced into the limited host range strain. The wide host range virC operon was characterized and shown to consist of two protein products of 26 and 23 kilodaltons in size. The wide host range virD operon was shown to encode a site-specific endonuclease. This endonuclease cleaves at unique and identical locations within the 24 base-pair direct repeats which flank the T-DNA. Nucleotide sequence and protein data indicate that two proteins of 16 and 48 kilodaltons encode this endonuclease activity.