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Plant Physiol, January 2001, Vol. 125, pp. 9-14 Agrobacterium. A MemoirSyngenta, P.O. Box 12257, Research Triangle Park, North Carolina 27709
This little memoir is not a review;
the reader is directed to current authoritative
Agrobacterium reviews with genetic (23) or cell
biology emphasis (24). Likewise, this is not an update on recent
advances in plant genetic engineering, which are the subject of a
recent book (13). Rather, I invite you to join me on a foray through
the story of Agrobacterium transformation of plant cells.
Our journey will take us back in time about 30 years, and we will note
early contributions from laboratories around the globe, including
Belgium, the Netherlands, France, Australia, and several in the United
States. The scientists in our story represented many disciplines, from
traditional ones such as plant pathology, microbiology, and chemistry
to younger fields such as molecular biology, plant tissue culture, and
plant metabolic chemistry. Many in the course of investigating
Agrobacterium found intellectual haven in the newly emerging
field of plant molecular biology. Beginning at a time when bulk DNA was
analyzed as a macromolecule, our story spans the birthing and growth of
recombinant DNA technology.
Lest the experiments we revisit seem simple when viewed from the 21st
century, our first stop will be a museum of molecular biology research
in the time about which I will write, circa 1970. The catalog of
restriction endonucleases was unrecognizably thin. What few enzymes
were available often were tainted. Kits were unknown. Procedures often
did not work. We sized DNA and determined its percentage of G and
C in the model E ultracentrifuge. We measured small volumes with
5-, 20-, 50-, or 100-µL glass capillaries. We cultured our plant
calli in jelly jars and fleakers. Instead of laminar flow hoods we
worked in still air hoods. A few years later when the plasmid came into
our lives, we taught ourselves how to do gel electrophoresis, and we
designed and built our own gel rigs. (The one with the agarose wicks
was known, of course, as the wicked gel.) We made combs from square
aluminum rod, using double-stick tape to mount teeth that were pieces
of glass cut with great difficulty from microscope slides. Each of us
hoarded his or her own collection of glass teeth, and it was not
uncommon to hear an anguished voice cry "Who took my teeth?"
Research in this period presented unique challenges. The first cloning
of DNA was out of sight, just over the horizon, and of course PCR was
not yet conceived.
With this setting in mind, then, let us turn our attention to the crown
gall problem and consider what was known at the beginning of the 1970s.
Dr. Armin Braun of the Rockefeller University (New York),
whom many regard as the godfather of the crown gall story, first demonstrated that tumor cells are transformed, i.e. they can be freed
from Agrobacteria and grown in vitro without the
supplemental auxin and cytokinin required by normal plant cells in
vitro (5). Braun kept tumor lines growing on hormone-free medium quite
literally for decades. He reasoned that Agrobacterium must
give these cells something, and he proposed that this gift must
replicate because it is never lost by dilution. He proposed for it the
term TIP (tumor inducing principle).
Georges Morel of the Institut National Recherche Agronomique on the
grounds of the Palais de Versailles in France discovered copious
amounts of new metabolites Rob Schilperoort at the State University of Leiden, the
Netherlands, as part of his Ph.D. research, prepared DNA filters with crown gall DNA and found that they bound radiolabeled
Agrobacterium DNA amazingly well. The thesis and other
publications of Schilperoort (see citations in 6) were an important
factor in the founding of our Seattle Crown Gall Group. Microbiologist
Gene Nester, plant viral RNA biochemist Milt Gordon, and I, an
organic-chemist-turned-DNA-hybridizer, all were intrigued by the idea
of gene transfer to plants. We realized that we three might
collaboratively do a much more definitive type of experiment to
identify bacterial DNA in tumors The first contribution of our Seattle Crown Gall Group to the problem
was a negative one that showed how large a challenge lay ahead. We
found that the DNA-filter results reported by Schilperoort were caused
by impurities (polysaccharides) in the DNA extracted from tumor cells,
and that this technique did not have the sensitivity to detect 1%
bacterial DNA in model mixtures (6). (One bacterial genome per plant
cell would constitute approximately 0.1%.) We next employed DNA
renaturation kinetic analysis, which tested whether a high
concentration of tumor DNA ("driver DNA") could make labeled
Agrobacterium DNA ("labeled probe") renature faster. We
showed that this method was sensitive enough to detect one copy of the
bacterial genome per three tumor cells, but tumor DNA did not drive our
labeled probe (6). It was a clear negative result. We recognized that
this method could only detect DNA corresponding to a significant
fraction of our labeled probe. The bacterial genome contains perhaps a
few thousand genes, so the acceleration of renaturation by even 10 specific bacterial genes in the tumor cells (a fraction of 1% of total
bacterial DNA probe) would be below the limit of detection.
Indirect genetic evidence that Agrobacterium
might carry a virus or plasmid with tumor-inducing genes emerged from
two kinds of experiments published in 1971. Hamilton and Fall at the
University of Pennsylvania (Philadelphia) discovered that strain C58,
when grown at 37° (28° is optimal), lost virulence irreversibly.
They proposed that tumor induction must be a plasmid- or virus-born trait because of its susceptibility to "curing" (12). At the same time, plant pathologist Allen Kerr at the Waite Institute
in Adelaide, South Australia, was attempting to develop a
biocontrol microbe to protect plants against crown gall disease. He
co-inoculated avirulent and virulent Agrobacteria into the
same sunflower plant. When he re-isolated the "avirulent" strain
from the gall, it had become virulent! This transfer of virulence
suggested to Kerr the existence of an extrachromosomal element as
vector for tumor induction (16).
Back in Seattle, Gene Nester read these reports and became convinced
that there must be a plasmid in Agrobacterium. He and Alice
Montoya reproduced the transfer of virulence with our own strains.
Bruce Watson, a student in Milt Gordon's lab, reproduced the C58
curing experiment also. (The reproduction of published claims was
clearly an important activity for our group, cast as we found ourselves
in the role of iconoclasts. It was essential to know what could be
believed.) Nevertheless, Bruce Watson repeatedly had no luck when he
looked for plasmids in Agrobacterium using established
methods (i.e. methods that were established for small plasmids).
In 1974, Ivo Zaenen (Fig. 1) at the
University of Ghent (Belgium) cracked the crown gall problem
wide open for everyone. Working in the laboratory of Jeff Schell
and Marc van Montagu, Ivo Zaenen was the first to lay eyes on the
megaplasmids of Agrobacterium. I asked him recently
how he had succeeded where others had failed. He replied that at first
he did not recognize what he had found. He was using alkaline Suc
gradients to look for something else: a replicating form of an
Agrobacterium phage called PS8 (whose DNA was once claimed
to be in tumor DNA). He eventually found plasmids ranging from 96 × 106 to 156 × 106
Mr in 11 virulent strains and not in eight
avirulent strains (22). His publication in the prestigious
Journal of Molecular Biology is a landmark.
When news of this discovery came to us in Seattle, it set off a flurry of experiments and launched a vigorous competition between the Seattle and Ghent groups. We quickly isolated plasmid DNA from several Agrobacterium strains by Zaenen's method. Both groups found that strain C58 lost a mega-plasmid when grown at 37°. Transfer of virulence was mediated by transfer of a plasmid. It quickly emerged that the genes for catabolism of octopine and nopaline were located on their respective giant plasmids, which the Ghent group christened Ti (tumor-inducing) plasmid.
At last with the Ti plasmid of our Agrobacterium strain in hand, we felt confident that we had the right probe to look for TIP in crown gall tumors. But when we performed renaturation kinetic analysis with the whole plasmid as probe, we got the by now too familiar result: It was not there. Our experiment ruled out the presence of the entire plasmid, but just as before, we recognized that a few genes could be there without our noticing any kinetic change. In order to settle the issue, we decided to cut the Ti plasmid into specific fragments and test each piece by renaturation kinetic analysis. It was a brute force experiment involving everyone in the lab (Fig.
2). In order to label our probe to
maximum specific activity, Martin Drummond seized the fresh
32P-dCTP the moment we received it from New
England Nuclear and labeled our plasmid DNA by nick translation.
Daniela Sciaky digested the labeled DNA with SmaI (purified
by Alice Montoya
A reviewer of the manuscript describing our findings required that we separate the doublet 3AB and determine which fragment was in the tumor. Although initial cloning experiments were just beginning in our group, we had no idea how to clone these blunt-ended SmaI fragments, and we found no enzyme that would cut one member of the doublet and spare the other. In desperation I finally managed to separate fragment 3A from 3B by a heroic serial electrophoresis of 4 d duration. We found that 3B was the fragment in the plant cells, and the paper was accepted (7). Resolution of the band 10 triplet showed us that 10C was the member in T-DNA, and when we subsequently determined the fragment map of our Ti plasmid, fragments 3B and 10C were contiguous, showing that T-DNA was a single segment of the Ti plasmid.
By this time, genomic Southern blots had been developed and were clean enough to show T-DNA bands; renaturation kinetic analysis was a dying art that nobody mourned. The Southern blots showed recognizable intact Ti plasmid fragments and in addition "border fragments" that were different in different tumor lines, suggesting attachment of T-DNA to plant genomic DNA. By analysis of Southerns of nuclear DNA, chloroplast DNA and mitochondrial DNA, the T-DNA of several tumor lines was proven to be located in the nuclear fraction (8, 21). In 1979, I moved from the University of Washington to Washington University in St. Louis, and focussed on nopaline Ti plasmids, while the founding group in Seattle continued with the octopine strain. My new group at Washington University, the Seattle group, and Patti Zambryski in the Ghent group (Fig. 3) all succeeded in cloning T-DNA fragments from tumor DNA. When we sequenced through the junctions of T-DNA and plant DNA, comparing plasmid DNA with T-DNA, we found a 25-bp imperfect direct repeat on the Ti plasmid at the edges of what is incorporated into the plant genome. These border sequences define T-DNA on the plasmid but not in the plant: they are not transferred intact to the plant cell (reviewed in 4).
vir Genes Transposon mutagenesis of the Ti plasmid in Leiden, in Seattle, and in Ghent showed that all mutations affecting tumor induction mapped to a sector of approximately 42 kb, separate from T-DNA, called the virulence (vir) region. The vir genes constitute a regulon inducible by acetosyringone and other phenolics that are found in plant wound juice (19). These compounds, directly or indirectly, affect the "antenna" protein VirA, which autophosphorylates, then phosphorylates VirG, a transcriptional activator for all of the vir genes. T-DNA is excised from the Ti plasmid by endonuclease VirD2, with facilitation by VirD1 and VirC1. VirD2 nicks the bottom strand of the right border sequence after the third base and attaches to the 5' end at the nick, forming the "leading" end of the T-strand to be delivered to the plant. The details of left border scission are not clear, but VirD2 produces a similar nick there. The vir E2 gene encodes a single-strand binding protein essential for tumor induction, that can alternatively be expressed in the plant with equal effect. The VirB operon consists of 11 open reading frames, which encode the T-DNA conduit from bacterium to plant. The structural and functional similarity of many of these to proteins involved in plasmid transfer to other bacteria has led to the view that T-DNA transfer has evolved from plasmid conjugation (reviewed in 23 and 24). T-DNA Genes Transposon hits in T-DNA were found to eliminate opine production or to alter tumor morphology or to have no recognizable effect at all. The morphology mutations were eventually shown to eliminate cytokinin autonomy ("rooty" tumors) or auxin autonomy ("shooty" tumors). T-DNA genes were shown to encode a two-step pathway to the plant auxin indoleacetic acid and an enzyme producing the cytokinin isopentenyladenosine 5'monophosphate (reviewed in 4). Most importantly, no mutation in T-DNA blocked T-DNA transfer. All of the genes affecting the process of T-DNA export to the plant cell mapped in the vir region. This fact would greatly simplify the disarming of T-DNA and construction of vir region-containing helper plasmids lacking any T-DNA.
In order to use the Ti plasmid as a vector, we needed a method of putting genes into T-DNA (and knocking some out, as well). In Ghent and in St. Louis, methods were developed for inserting DNA into any specific part of the Ti plasmid. The DNA to be inserted was cloned between pieces of T-DNA on a plasmid, introduced into the bacterium by conjugation or by transformation, and subjected to "forced recombination" (17, 20). A simpler approach to engineering T-DNA was to make a small separate T-DNA plasmid that could be manipulated directly. Although Agrobacterium, in nature, keeps vir genes and T-DNA on the same replicon, there is no requirement for this arrangement. If you place T-DNA on a separate replicon in Agrobacterium (a binary vector, as it is now called), the process of T-DNA transfer to the plant cell still occurs with good efficiency (9, 15). Thus, the T-DNA of a binary vector could be engineered directly in Escherichia coli and then transformed into Agrobacterim. Another problem for the genetic engineer was plant regeneration. All efforts to regenerate a plant from transformed cells were initially rewarded with only rare deletion mutants that had lost practically all of their T-DNA, a strong indication that at least part of T-DNA was inimical to plant regeneration. We discovered the critical part almost serendipitously. Tony Matzke and Ken Barton, post-docs in my group, introduced a yeast gene into T-DNA in a position that we thought might hit an oncogene (17). It turned out indeed to inactivate the cytokinin production gene. In collaboration with Andrew Binns at the University of Pennsylvania, we discovered that this single insertion event produced an engineered T-DNA that was completely disarmed. It produced transformants that synthesized nopaline but that could not grow autonomously without hormones. Binns identified the transformed plant cells by screening for nopaline production. In contrast to crown gall tumor cells, the tobacco cells transformed by multiple copies of this T-DNA were able to regenerate into normal plants that passed the T-DNA copies to progeny plants as Mendelian traits (1). By 1982 we had the first evidence that foreign DNA engineered between T-DNA borders and transformed into the plant nuclear DNA could be stably maintained in the plant genome and passed intact to progeny. Starting in about 1980, a formidable new group was assembled by Ernie Jaworski at Monsanto (our neighbor in St. Louis) to harness the T-DNA transfer technology for crop improvement. At this time Michael Bevan in my laboratory found himself in a race with Patti Zambryski's team in an effort to sequence the nopaline synthase (nos) gene and map its promoter and terminator by S1 nuclease protection (2, 10). Then a second race ensued amongst Bevan, Zambryski and her Ghent collaborators, and the Monsanto group to isolate the nos gene promoter and splice it to a kanamycin resistance coding region in order to create a selectable marker that might work in plant cells. If this scheme worked, then one would no longer have to screen for nopaline production to find transformed plant cells: one could select the cells with T-DNA inserts on kanamycin agar. The symbolic coming of age of genetic engineering occurred at the Miami Winter Symposium, January 18, 1983. During one session, Jeff Schell, Rob Horsch from Monsanto, and I all gave talks about Agrobacterium and its adaptation as a gene vector for plants. All three of us reported success with chimeric kanamycin resistance genes as a selectable marker for plant cells (3, 11, 14). I described initial success in transforming tobacco cells with binary vectors (which we called MiniTi at that time). In addition, I described our tobacco plants engineered with a disarmed Ti plasmid, and Southern blots proving that they passed their T-DNA insert to progeny intact. It was clear from the progress in all three groups that crop improvement by genetic engineering would become a reality. Reflections from 2000 Finding that T-DNA can integrate into a plant genome without benefit of homology was a real intellectual shock to me. The bacterial transformation studies I had made as a student and again as postdoc taught me the absolute need for good homology in those systems. Now illegitimate recombination seems the rule not only for T-DNA but also for foreign DNA integration in animal cells and indeed naked DNA delivery to plant protoplasts or bombardment of plant cells with DNA-coated microprojectiles. Incorporation of foreign DNA is clearly a process that cells carry out efficiently, perhaps in the course of repairing genomic damage. It is not a tradesecret of Agrobacterium, although we may yet discover some secret details of the process. Agrobacterium acted as an inspiration to others who have developed various means of DNA delivery. It gave us our first selectable marker (tumor induction). It gave us the promoters and terminators for the next generation of selectable markers (octopine/nopaline synthase promoters and nos terminator, bounded as they were by T-DNA borders and neighboring genes in this highly compact T-DNA). The wide host range of Agrobacterium (even wider now that monocot transformation is facile) inspired the idea that DNA incorporation may be a universal phenomenon. But perhaps the most important legacy from Agrobacterium has been its inspiration of confidence that foreign gene integration, even though DNA is sometimes delivered artificially, is a perfectly natural process. My most fervent wish is that well-meaning environmental proponents will come to recognize this and embrace the technology based on it.
* E-mail Marydell.chilton{at}syngenta.com; fax 919-541-8585.
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INTRODUCTION
An Idea Born Before...
octopine and nopaline
