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Plant Physiology 133:948-955 (2003) © 2003 American Society of Plant Biologists Agrobacterium tumefaciens and the Plant: The David and Goliath of Modern Genetics1Friedriech Miescher Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
Since the first reports on GM crops in the 1980s (Bevan, 1984  ), the amount of debate surrounding the generation of transgenic plants and the political and economical ramifications of their development have seldom been far from the public eye. It could be said that attention on the use of transgenically modified crop plants is substantially greater than on any other area of plant science. To date, the majority of commercially produced GM crops have been created by biolistically delivering transgenes into embryonic tissue (Padgette et al., 1995; James, 2002). However, as our understanding of the mechanisms of T-DNA integration grows and further information is reported on the proteins involved, the biolistic approach may well be superseded by a more sophisticated and "natural" solution to the problem.
Agrobacterium tumefaciens is a soil-borne bacterium that, in nature, is capable of inserting a defined fragment of its DNA into the genome of dicotyledonous plants (for review, see Tzfira and Citovsky, 2002
The aim of this review is to discuss recent advances and future prospects in the field of A. tumefaciens-mediated plant transformation (for more detailed review, see Tzfira and Citovsky, 2002
Agrobacterium is a soil-borne bacterium that, in the presence of a wounded plant, moves toward it, attaches itself to the wound site, and proceeds to transform the cell. The sugars and phenolic compounds exuded by the wounded plant not only signal the pathogenic opportunity to the bacterium but also induce transcription of the virulence genes. These virulence genes are located on a specific plasmid known as the tumor-inducing (Ti) plasmid, which also contains the transferred DNA (T-DNA). Virulence proteins have roles ranging from transcriptional activation to T-DNA processing and export, with certain proteins also having a function in the host (Fig. 1). Agrobacterium has evolved to transfer the T-DNA, which codes for: (a) plant hormone producing enzymes that stimulate growth of a tumor, and (b) metabolic enzymes responsible for producing opines, metabolizable only by Agrobacterium. The resultant crown gall is a microcosm where the bacteria can thrive.
Virulence proteins VirD1 and VirD2 act together in the processing of the single-stranded T-DNA from the Ti-plasmid, during which the VirD2 protein becomes covalently bound to the 5' terminus (Fig. 2). This covalent association is thought to remain until the last stages of T-DNA integration into the host genome because localization experiments have shown the accumulation of VirD2 in the nucleus. One of the other vital virulence proteins is VirE2. This virulence protein was originally shown to be a single-stranded DNA-binding protein that binds in a sequence-nonspecific manner. Many more roles have been put forward for this versatile protein from involvement in cytoplasmic trafficking and nuclear import to formation of a transmembrane pore (Dumas et al., 2001
Other virulence proteins play a role in constituting the membrane channel, representative of the bacterial type IV secretion system, through which the T-DNA and certain virulence proteins are secreted. The type IV secretion system is thought to be related to the ancient conjugation system, which allows the secretion of DNA-protein complexes (for review, see Christie, 2001
Many features of T-DNA and the nature of its delivery and integration have made it an invaluable tool in plant biotechnology. Not least of these features is the fact that the actual transfer and integration do not rely on genes encoded by the T-DNA itself. In fact, the only necessary sequences on the T-DNA are the border sequences (right and left) that delineate it. Between the border sequences, genes of scientific interest can be cloned, allowing for great flexibility in what is transferred to the host cells.
An important role for both VirD2 and VirE2 has been demonstrated by analyzing the virulence of Agrobacterium with either of these genes deleted (Otten et al., 1984
Further research has established a role for VirD2 in the nuclear import of the T-DNA, confirming the functionality of the putative nuclear localization signals (Ballas and Citovsky, 1997
One of the major contributions of Agrobacterium research to plant research has been the use of T-DNA as a mutagen. To date, it has not been possible to target T-DNA to any particular locus in the genome with any great efficiency. This has the advantage that, in a library containing a large number of independently transformed seeds, the plant genome would be saturated with individual T-DNA integrations. In such a library, there is a high likelihood that there are T-DNA insertions in every open reading frame, and the resultant mutant plants can then be screened by phenotype or using reverse genetics to identify specific mutant line of interest. The first large-scale T-DNA-tagged library was made available by Krysan et al. (1999 ). It contained over 60,000 independent mutants and marked a change in the direction of Arabidopsis genetic research from chemical mutagenesis followed by chromosome walking and mapping to reverse genetics. Using T-DNA with a known sequence offers a myriad of possibilities for researchers such as using enhancer traps or genetic complementation. The sequence-independent nature of T-DNA integration means that identification of the sequences adjacent to the T-DNA became possible by cloning a bacterial vector between the border sequences and cloning the genome sequences by plasmid rescue. There is now a >75% chance of finding a plant with a T-DNA located in a gene of interest, with the obvious exception of insertions, which create a dominant lethal phenotype.
As more and more T-DNA insertion sites have been sequenced, a wealth of information has been generated that, upon analysis, can provide us with insights into the subtle preferences for T-DNA integration. Most recently, Alonso et al. (2003
These observations are consistent with a study that looked specifically at the conservation of the T-DNA borders and the genomic sequences after integration (Brunaud et al., 2002
To identify plant factors that are involved in the transformation process, T-DNA-tagged mutant Arabidopsis libraries were screened to identify plants which were resistant to Agrobacterium transformation (RAT; Nam et al., 1999
All of the genes identified could feasibly have an impact on transformation of recalcitrant species, but one key target area is the stable integration of the T-DNA into the host genome. Table I summarizes the Arabidopsis mutants that have been identified either through the screen or by reverse genetics to be specifically involved in the integration step. The rat5 mutant is the best characterized to date and was shown to be mutated in one of the histone genes (H2A-1; Mysore et al., 2000
The observation of the random, as opposed to targeted, nature of T-DNA integration has led to the suggestion that the likely mechanism is through non-homologous end joining (NHEJ). NHEJ is the DNA repair mechanism that joins double-stranded breaks (DSBs) irrespective of sequence. This is the process involved in well-characterized cases such as variable-diversity-junction (VDJ) joining in the mammalian immune system (Pastink et al., 2001 ). NHEJ is an unfaithful repair mechanism that often incorporates filler DNA, such as T-DNA, into the repair site.
Further insights into mechanisms of T-DNA integration have been gained by using yeast as a model system. In contrast to the situation in plants, integration of T-DNA in yeast usually occurs by homologous recombination (HR), but, in the absence of any homology, T-DNA can also integrate using NHEJ. The use of yeast mutants has been very valuable in understanding the balance between HR and NHEJ. It has been shown that in a Yku70 mutant, which is deficient in NHEJ, T-DNA integrates solely by HR (van Attikum et al., 2001
Ligase IV is the specific ligase responsible for NHEJ and has therefore been the focus of a great deal of recent research in plants. Two independent Arabidopsis ligase IV mutants have been characterized with respect to T-DNA integration and sensitivity to gamma radiation (DSB) and methyl methane sulfonate. Both mutants showed a loss in ability to repair DSBs, as indicated by the gamma plantlet phenotype. Less clear, however, was the role ligase IV might play in T-DNA integration. Friesner and Britt (2003
Recent research, using VirD2 as bait in a yeast two-hybrid assay, allows new insights into the alternative integration mechanism. VirD2 was shown to interact with Arabidopsis TATA-binding protein and CAK2M, a nuclear kinase (Bako et al., 2003
With rapid advances being made in Agrobacterium and transgenic plant research, the possibilities for crop improvement are numerous. One recent advance epitomizes the current situation: coffee (Coffea canephorea) plants that have been genetically modified to contain less caffeine (Ogita et al., 2003 ). The caffeine biosynthesis gene, coding for obromine synthase, was targeted for down-regulation using the concept of RNA interference. The transgenic coffee plantlets obtained after Agrobacterium-mediated transformation had a 50% to 70% reduction in caffeine. These decaffeinated coffee plants will, in theory, bring an end to the expensive, industrial decaffeination that also results in a loss of taste. One problem remains. Even though the traditional breeding time of 25 years has been reduced to one year, the plants that were transformed were C. canephorea and not Coffea arabica. The difficulties in transforming C. arabica, which is the commercially valuable species, could be addressed by understanding the mechanics of Agrobacterium-mediated transformation and using the knowledge to improve the transformation rate.
Perhaps the most difficult hurdle facing the advance of Agrobacterium and transgenic plant research is not a technical but a political one. Decisions that will shape the future of plant research are about to be made as countries all over the world are preparing to draft new legislation to control the development of transgenic crops. There are many different parties contributing to the debate, each with their own concerns and goals. Many have obvious merit, but concessions will have to be made to find solutions and ensure the future of plant research. At a naïve level, there are the large corporations on one side and environmental groups on the other. Scientists are largely placed on the side of big business, and the farmers are portrayed as sharing concerns with the environmentalists. These boundaries are blurred, however, as is apparent with the farmers in India who crossed transgenic pest-resistant cotton (Gossypium hirsutum) with another variety to increase their yields, an act that would appear to be in violation of the patent on the resistance gene held by Monsanto (Jayaraman, 2001
Another major driving force for the progression toward the use of GM crops is the pressure imposed by the world's growing population. It is estimated that in the next 50 years, the world's population will grow from 6 to 9 billion, where it will hopefully stabilize (Evans, 1998 Tackling the problem of world hunger requires a multifaceted approach involving not only a country's transport infrastructure, its political transparency, and external industrial motives but also incorporation of biotechnological advances.
It is certainly true to say that industrial companies will always have to make a profit and environmentalists might claim that feeding the world may not be their first priority, but this does not change the fact that feeding 9 billion people remains our future. It should also be noted that in the case of "Golden Rice," rice (Oryza sativa) enriched for the vitamin A precursor B-carotene, the farmers need only pay proceeds to the industrial producers when annual profits exceed $10,000 (Thomson, 2002
To understand the implications of GM crops on the agricultural and economical future of the world and, in particular, developing countries, the International Service for the Acquisition of Agri-Biotech Applications (ISAAA) report by James (2002 ) provides valuable insight to the current situation and trends. In the 6-year period since the first commercial GM crop was introduced, growth in the amount of global coverage has been observed. A sustained growth rate of over 10% per annum has resulted in 145 million acres of land growing GM crops by the end of 2002. As can be seen in Figure 4A, the United States has by far the greatest impact by growing 66% of the world total. Perhaps surprisingly, Argentina is the second biggest grower of GM crops with 23%. This is a developing nation with a recent history of economic hardship that has enthusiastically embraced the biotechnological advances and will continue to be a model for developing nations with respect to the benefits or problems associated with these crops.
It is also of interest to investigate which countries have increased their GM output compared with the previous year. Figure 4B shows that in 2002, South Africa and China were the two countries whose output increased the most compared with the previous output figures. Is this an indication of the need in developing countries for this type of technology? It is certainly an indication that developing countries are likely to play a major role in the future of GM crops worldwide.
One final aspect of the current situation that is likely to eventually differ within developing countries is the type of GM crop grown. At present, there are four main types of GM crops currently grown worldwide (soybean [Glycine max], maize [Zea mays], cotton, and canola [Brassica napus]; Fig. 4C). It is also interesting to note that there are two main genetic modifications within the crop species being grown currently: crops that produce the Bacillus thuringiensis protein, which is toxic to insect pests; and herbicide-resistant plants, which allow selective growth of the crop at lower levels of herbicide usage. The impact on yield has been reported to be a 10% increase with the soybean in Argentina and 514,000-metric ton increase in cotton in China (James, 2002
With political and economical issues to one side, the most pertinent issue remaining is that of human health. It is obviously in nobody's interest to introduce a GM crop that will lead to illness. It was of concern that early GM products still carried the antibiotic-resistance gene that had been used in the initial selection for transformation. The alleged problem lies in the possibility that the resistance gene may be transferred to intestinal bacteria, then to pathogenic bacteria, which would then not respond to medically prescribed antibiotics, the resistance gene, therefore, imposing a negative effect on human health. Steps have since been taken to ensure minimal foreign DNA content in the final GM plants. One of the major obstacles in this area of the debate is the need to define acceptable risk. GM food is discussed in the realms of risk free, but it would be more constructive to compare it with food consumption risks in general. As much as 1 g of bacterial, viral, animal, and plant DNA is consumed every day, most of which is destroyed by stomach acid or by enzymes within the digestive system (Doerfler and Schubbert, 1997). Adding a small percentage (<0.001%) of GM DNA to a diet should pose no greater risk to health than non-GM DNA. It should also be considered that many countries worldwide have rigorous safety trials followed by field trials before general release into the environment is allowed. To date, no GM crops have been shown to be detrimental to mammalian health, although conscientious tests in the future remain of central importance.
The ethical debate is centered around the acceptability of taking genes from one species and moving them into another species, with species boundaries (ability to produce fertile offspring) being used to define "normal" and "natural." The question of "naturalness" is discussed in a recent review (Verhoog, 2003 ) where the differences between intrinsic (for example, playing God) and extrinsic (such as health risks) moral concerns are defined. Verhoog postulates that one of the biggest divides in the ethical debate is what an individual understands "nature" to be. If "nature" is considered to be neutral, then GM technology is viewed as neutral, in essence something that can be used for either good or evil. On the other hand, if "nature" is viewed as good, then doing anything to change it is seen as evil. Obviously, the ability of Agrobacterium for interkingdom DNA transfer opens the debate as to what is "natural." This is an organism that has evolved a mechanism of securing its own environment by genetically modifying another organism. Would using Agrobacterium to produce GM crops be morally acceptable?
At present, the field of Agrobacterium research is increasing our understanding of the bacterium itself; in particular, the mechanism by which the T-DNA is translocated into the plant cell and exactly which bacterial virulence proteins accompany it. This understanding is not only vital for the biotechnological application of Agrobacterium, but it also has implications in the understanding of human pathogens with the type IV secretion system.
There is a rapidly increasing wealth of knowledge available (http://www.bio.purdue.edu/about/faculty/gelvin/gelvinweb/main.html) about the plant factors used by Agrobacterium to ensure transport across the plant wall, membrane, and cytoplasm, nuclear import, and finally integration of the T-DNA. The application of this knowledge to improve transformation rates will bring gene technology to species that, at present, are recalcitrant to Agrobacterium and not transformation competent using a biolistic approach. The improvement of transformation protocols is a practical advance, but the really exciting advances will be in the types of modification and the application therein. Current research foci include biodegradable plastics in plants (Mittendorf et al., 1998 With so much promise, Agrobacterium could be the key to future agricultural progress. It can only be hoped that regular, constructive debate can lead to legislative solutions for the ethical, health, and political issues that are likely to play such an influential role in the development of our society.
The author would like to acknowledge the intellectual input from Prof. Stanton B. Gelvin. I would like to thank Prof. Barbara Hohn for interesting and informative discussion and her thorough reading of the manuscript, not to mention years of intellectual stimulation. Special thanks to Billy Valentine for technical advice and endless support. Received August 25, 2003; returned for revision August 29, 2003; accepted September 11, 2003.
http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.032243.
1 This work was supported by the National Science Foundation. * E-mail lisav{at}fmi.ch; fax 0041–61–6973976.
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