Agrobacterium gene transfer: progress on a "poor man's vector" for maize.

Sometime plant pathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, has captured the attention of plant biologists because of its natural ability to transfer genes to plant cells. It engages in such gene transfer in nature, splicing its genes to plant DNA without regard for the National Institutes of Health guidelines. This intriguing microbe over the last decade has been investigated, disarmed, and put to work in plant genetic engineering laboratories worldwide. The list of plants on which its talents have been exploited for gene transfer grows steadily. The desire to use Agrobacterium as a gene vector for maize, a plant outside its "normal host range," has driven the development of highly sensitive detection systems for gene transfer. It has been known for nearly a century that Agrobacterium tumefaciens can cause galls at wound sites on a wide variety of plants, including most dicots and many gymnosperms. About three decades ago, the biological motive for this activity came to light: the cells of the gall produce novel amino acid and sugar derivatives, generically called opines, that are specific growth substrates for the bacterium. Different strains of Agrobacterium produce (in galls) and consume different opine repertoires. Strains have became known accordingly as octopine, nopaline, or other types, depending on their diagnostic opine. The gall on the host plant thus serves as an ever-growing factory that converts normal plant metabolites into a form useful only to the inciting Agrobacterium. Nearly two decades ago, the mechanism of this opine-mediated subversion of the host plant cells was shown to be an elegant gene transfer process. The transferred genes are situated on plasmids, called tumor-inducing (Ti) plasmids, that also carry opine catabolic genes. A specific segment of Ti plasmid DNA, called T-DNA (transferred DNA), travels from bacterium to plant cell nucleus and becomes integrated into the plant's chromosomal DNA. The eukaryotic-style T-DNA genes are transcribed into active mRNAs. It is not known whether such T-DNA gene expression can precede integration. An elaborate mechanism for T-DNA transfer is encoded by a series of virulence (vir) genes on the Ti plasmid that map outside T-DNA. These vir genes are inducible by chemical signals released by wounded plant cells. Acetosyringone and related metabolites are the best-studied inducers. The mechanism of T-DNA transfer appears analogous in many ways to interbacterial plasmid conjugation. T-DNA is flanked by 25-base-pair direct repeats that serve as targets for cleavage by the virD2-encoded nuclease. The T-DNA intermediate is a single-stranded DNA, covalently joined to VirD2 protein at its 5' end. T-DNA transfer into the plant cell nucleus is apparently facilitated by two kinds of address labels on the package: there are nuclear localization sequences both in VirD2 protein and in VirE, a single-strand-binding protein that coats the T-strand. It is not known whether any Vir proteins facilitate T-strand duplication or integration. T-DNA gene products induce a gall by causing host plant cells to divide rapidly in an uncontrolled manner. Three T-DNA genes encode enzymes that catalyze synthesis of indoleacetic acid, a phytohormone causing cell expansion, and ribosyltrans-zeatin, a cell-division factor. Additional T-DNA genes encode enzymes that produce octopine, nopaline, or other opines. The function ofa number of other T-DNA genes is unknown. A decade ago two events signaled the beginning of the era of plant genetic engineering. First, Agrobacterium and its Ti plasmid were used as a gene vector system for producing transgenic plants. Tobacco cells were transformed by Agrobacterium carrying a "disarmed" Ti plasmid into which foreign genes had been inserted, with concomitant inactivation of the ribosyl-trans-zeatin synthesis gene. Transformed cells were regenerated to form healthy genetically engineered tobacco plants that transmitted disarmed T-DNA, including the foreign genes, to their progeny (1). Second, work in several laboratories showed that a non-plant gene (a bacterial gene conferring resistance to kanamycin) could be instructed to function efficiently in a plant cell by splicing a plant-active promoter-the T-DNA nopaline synthase promoter-to the coding region of the bacterial gene (2-4). This enabled accurate selection oftransformed plant cells. The future for Agrobacterium as a gene vector for crop improvement began to look bright. Indeed, Agrobacterium had transformed more than just tobacco cells and plants. In a real sense it had transformed our vision of what could be achieved by application of molecular biology to plant breeding and genetics. For the first time it seemed possible that novel desirable genes could cross genus, family, even kingdom boundaries and find use in crop improvement. The promise and eventual success of Agrobacterium as gene vector inspired the development of additional techniques for introducing DNA into plant cells. Under suitable conditions, naked DNA was shown to enter plant cell protoplasts and penetrate the nucleus, where it integrated into the chromosome (5). In addition, more violent physical means of introducing DNA through cell walls into intact plant cells were developed: bombardment with DNA-coated microprojectiles (6) and microinjection ofDNA solution (7). All of these newer procedures brought about insertion of the entire donor DNA molecule, in multiple copies, fragmented at unknown sites and sometimes mixed with carrierDNA, in contrast to the more clean and tidy insertion pattern for Agrobacterium T-DNA. Despite these new technologies which had no host range limitation, interest persisted in adapting Agrobacterium T-DNA transfer to a wider range of plant transformation projects. The reasons for this were several. First, it is a "poor man's vector system," requiring no hightech microinjection equipment or protoplast regeneration skills. Second, it is a "clean" system, introducing only the desired DNA package. Third, it is perceived to be a "natural" gene transfer system, more acceptable to those who feel that natural is better. And finally, based on the broad known host range of Agrobacterium, a universal host range, at least for the T-DNA transfer process, seemed plausible. That galls were not formed on some dicots and many monocots (8) could have any of several explanations. Perhaps such plants fail to produce a compound that induces the vir