The bases of crown gall tumorigenesis.

The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120). Most of the genes required for tumorigenesis are found on large extrachromosomal elements called Ti plasmids. Indeed, transfer of Ti plasmids into certain nonpathogenic bacteria converts them into tumorigenic pathogens (43). Ti plasmids are generally referred to by the types of opines whose catabolism they direct (see below). However, this nomenclature is becoming less satisfactory as we discover that all known Ti plasmids direct the catabolism of more than one opine and that opine catabolic genes are found in a variety of combinations in different plasmids. The Ti plasmids pTiA6NC, pTi15955, pTiAch5, pTiR10, and pTiB6S3, which are widely considered to be functionally identical, are generally referred to as octopine-type Ti plasmids (or, less frequently, octopine, mannityl opine-type Ti plasmids). The DNA sequencing of these plasmids was initiated almost 20 years ago (21) and was recently completed in our three laboratories. The resulting 194,140nucleotide sequence is a composite assembly of sequences from all of the plasmids listed above. The close similarity of these plasmids is exemplified by the sequence of a 42-kb segment of the vir regions of pTiA6NC and pTi15955. These sequences differ at only one base, and this polymorphism is silent at the amino acid level. We have no evidence for polymorphisms elsewhere except for a large deletion that is unique to pTiA6NC (Fig. 1). The restriction map deduced from this sequence agrees almost perfectly with the published restriction map of pTiAch5 (25). All known and suspected genes are depicted in Fig. 1, and their demonstrated or putative functions are described in Table 1. The DNA sequence of this Ti plasmid provides a useful framework to review the roles of this plasmid in the biology of plant infection and colonization. This Ti plasmid contains 155 open reading frames (ORFs), almost all of which are likely to encode functional proteins (Fig. 1 and Table 1). The overall G1C composition of this plasmid is 55%, although a few segments are considerably richer in A’s and T’s, particularly in the T region (see below). Overall, the Ti plasmid exhibits a modular structure with genes of similar function or purpose grouped together. Thus, we can define five components: (i) the T region, which codes for sequences that are transferred to the plant host; (ii) the vir region, which directs the processing and transfer of the TDNA; (iii) the rep region, which is required for replication of the Ti plasmid; (iv) the tra and trb loci, which direct the conjugal transfer of the Ti plasmid; and (v) genes that direct uptake and catabolism of opines. An exception to this clustering is the tra and trb loci, the two gene sets required for conjugal transfer, which are separated from each other by 60 kb.