Agrobacterium tumefaciens-mediated Transformation

The genus Agrobacterium stands for saprophytic bacteria species and lives in the soil microflora1. Four of these Agrobacterium species cause neoplastic diseases in various plants. These are: “crown gall” caused by Agrobacterium tumefaciens, “hairy root” caused by Agrobacterium rhizogenes, and “cane gall” caused by A. rubi. The last one, which is called A. vitis and also a new species, was recently shown to cause tumors and necrotic lesions on a grape vine along with a few other plant species2. Several hundreds of different plant species are infected by virulent Agrobacterium species. Generally, these species are woody and herbaceous dicotyledons, but monocotyledons could be infected as well3. However, among them, A. rhizogenes has attracted more attention, and has been discovered that it can be used as a tool for genetic transformation4,5. The discovery of the transformation of Agrobacterium into its host to generate crown gall tumors is a defining moment in Agrobacterium research, and following discoveries finally developed this bacteria as a model system for horizontal gene transfer and, most importantly, as a tool for plant transformation. For this reason, among the four mentioned species, A. tumefaciens is by far the most important and best studied6.

Figure 1: Crown Gall growing on stem cells. From Clemson University – USDA Cooperative Extension Slide Series. Photo by Edward L. Barnard

Two elements are essential for the development of Agrobacterium gall disease (pathogenesis). These are transformation and tumorigenesis1. How this organism is a potential biological delivery system can be understood by investigating how it functions in its nature. In nature, Agrobacterium is able to, particularly, sense and recognize signal molecules like low molecular weight phenol compounds and sugar compounds secreted by wounded plant tissue cells, (acetosyringone and its derivatives) in a complex soil environment. It moves into wounded and susceptible plant tissue by chemotaxis, the place it enters and proliferates in the host intercellular spaces12,13. A. tumefaciens seems to transform plant cells right after exposure to fresh wounds14. Cell proliferation and the DNA replication machinery have been activated only in wounded plant tissue. Therefore this situation has been proved. Plant recombination processes and/or DNA repair enzyme activities improve the T-DNA integration14. The T-DNA carries two sets of oncogenic genes which encode enzymes involved in the synthesis of plant hormone auxin and cytokinin and in modulating the influences of phytohormones in the plant cells, respectively15. Their activity causes tumorigenesis. The second set of T-DNA genes encodes enzymes involved in opine synthesis at the same time. These compounds supply a selective advantage to Agrobacterium1,7. This alteration of the secondary metabolism of plants results in aberrant cell proliferation and synthesis of nutritive compounds. All these compounds are used by A. tumefaciens as carbon and nitrogen sources6.

As previously mentioned, Agrobacterium tumefaciens has been revealed to have a specific region called T-DNA, which is subsequently identified as tumor-inducing (Ti) plasmid, and the molecular basis for the genetic transformation of plant cells lies behind it. The mechanism comprises the transfer and stable integration of this plasmid into the plant nuclear genome2. The T-DNA region is mobile and takes a role in tumor formation and opine biosynthesis in plants. T-DNA has several regions to control the whole mechanism on its own. The vir region is one of these regions and includes approximately 35 virulence genes grouped in at least eight operons (virA, virB, virC, virD, virE, virG, virF, and virH). All of these encoded virulence proteins are responsible for multiple important roles in the bacteria and the host cell together, where they regulate T-DNA transfer and integration7. Another region, which is responsible for opine metabolism, functions in opine uptake and production8,9. The T-DNA region is defined and limited by highly homologous, directly repeated 25e28 bp T-DNA border sequences10,11. The crucial mechanism depends on these regions6.

Figure 2: A Ti plasmid.

The T-DNA structure and functions allow the foundation of the biotechnological use of Agrobacterium in genetic transformation.  The two bp direct repeat borders are necessary for T-DNA transfer to flank the transferred DNA. The original wild-type oncogenes (auxin and cytokinin) and opine synthase genes from the T-DNA can be substituted by genes of interest16. Hence, desired DNA placed between the borders can be transferred to the host cell. T-DNA has no capability of mediating its transfer so the vir genes are required for the T-DNA transfer and integration6. The ability of vir genes led to the development of binary and super-binary transformation vectors, as a major tool toward increasing the range of species and the efficiency that are suitable for Agrobacterium-mediated transformation17.

Figure 3: A binary vector re-engineered for transformation.
Figure 4: A helper Ti plasmid carrying the vir genes.

The agrobacterium-mediated transformation process is still complex, and yet not fully deciphered.  The transformation process can be examined in several steps. Firstly, wounded plant cells secrete signal molecules that are recognized by the bacteria VirA/VirG2-component signal transduction system. After an attachment to a healthy susceptible plant cell, activation of the vir genes enables the VirD1/VirD2 border-specific endonucleases to target the T-DNA border sequences. They extract the T-DNA from the Ti plasmid and release single-stranded T-DNA via a strand-replacement mechanism16. Subsequently, the VirD2 protein covalently attaches to the 50’ end of the T-strand. The VirD2/T-strand complex is then transferred to the plant cytoplasm via a type IV secretion system (T4SS) formed by 11 VirB proteins and VirD418. Other bacterial virulence proteins (VirE2, VirE3, VirF, and VirD5) also use the same route to pass into the host cell16. Although its existence has not yet been demonstrated in plants, the “mature T-complex” is believed to be assembled inside the host cell by associating the VirD2-conjugated T-strand complex with VirE22. It has been proposed that both VirD2 and VirE2 proteins protect the single-stranded T-strand from exonucleolytic attacks inside the plant cytoplasm by attaching to its 5’ end19. Both proteins include nuclear localization signals and serve as pilot proteins to direct the “mature T-complex” to the plant nucleus2. The accompanying proteins are released by targeted proteolysis in the nucleus and the uncoated single-strand T-DNA becomes a double-stranded molecule. Finally, the T-DNA is integrated into the host genome16. After the successful integration, the expression of T-DNA-encoded genes leads to the synthesis of bacterial proteins which promote tumor formation6.

Figure 5: A simplified model of the Agrobacterium-mediated transformation process.

Conclusion

Agrobacterium tumefaciens is the most successful plant genetic engineer in nature. This bacterium has evolved the ability to deliver DNA into plant cells. The expression of the T-DNA not only results in the proliferation of the cell carrying it but also causes such transformed cells to produce nutrients that serve as carbon and nitrogen sources to the infecting bacterium. Plant and agricultural scientists have been able to harness the DNA transfer activity of Agrobacterium so that it is now possible to genetically engineer a wide variety of plants. In addition to this remarkable technical advance, the study of Agrobacterium continues to supply novel insights into the general mechanisms in which both plant and animal pathogens transfer macromolecules into host cells, resulting in disease states in the host organisms20.

References:

  1. Escobar, M. A., & Dandekar, A. M. (2003). Agrobacterium tumefaciens as an agent of disease. Trends in plant science8(8), 380-386.
  2. Gelvin, S. B. (2003). Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiology and molecular biology reviews67(1), 16-37.
  3. Otten, L., Burr, T., & Szegedi, E. (2008). Agrobacterium: a disease-causing bacterium. In Agrobacterium: from biology to biotechnology(pp. 1-46). Springer, New York, NY.
  4. Nilsson, O., & Olsson, O. (1997). Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiologia plantarum100(3), 463-473.
  5. Taylor, C. G., Fuchs, B., Collier, R., & Kevin Lutke, W. (2006). Generation of composite plants using Agrobacterium rhizogenes. Agrobacterium protocols, 155-168.
  6. Păcurar, D. I., Thordal-Christensen, H., Păcurar, M. L., Pamfil, D., Botez, C., & Bellini, C. (2011). Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology76(2), 76-81.
  7. Valentine, L. (2003). Agrobacterium tumefaciens and the plant: the David and Goliath of modern genetics. Plant Physiology133(3), 948-955.
  8. Zambryski, P. C. (1992). Chronicles from the Agrobacterium-plant cell DNA transfer story. Annual review of plant biology43(1), 465-490.
  9. Nester, E. W., Gordon, M. P., Amasino, R. M., & Yanofsky, M. F. (1984). Crown gall: a molecular and physiological analysis. Annual Review of Plant Physiology35(1), 387-413.
  10. Yadav, N. S., Vanderleyden, J., Bennett, D. R., Barnes, W. M., & Chilton, M. D. (1982). Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proceedings of the National Academy of Sciences79(20), 6322-6326.
  11. Wang, K., Herrera-Estrella, L., Van Montagu, M., & Zambryski, P. (1984). Right 25 by terminus sequence of the nopaline t-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell38(2), 455-462.
  12. Shaw, C. H. (1991). Swimming against the tide: chemotaxis in Agrobacterium. BioEssays13(1), 25-29.
  13. Zambryski, P. C. (1992). Chronicles from the Agrobacterium-plant cell DNA transfer story. Annual review of plant biology43(1), 465-490.
  14. BRAUN, A. C. (1982). A history of the crown gall problem. In Molecular biology of plant tumors(pp. 155-210). Academic Press.
  15. . Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis.
  16. Lacroix, B., Li, J., Tzfira, T., & Citovsky, V. (2006). Will you let me use your nucleus? How Agrobacterium gets its T-DNA expressed in the host plant cell. Canadian journal of physiology and pharmacology84(3-4), 333-345.
  17. Lee, L. Y., Fang, M. J., Kuang, L. Y., & Gelvin, S. B. (2008). Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells. Plant methods4(1), 1-11.
  18. Christie, P. J. (2004). Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research1694(1-3), 219-234.
  19. Dürrenberger, F., Crameri, A., Hohn, B., & Koukolíková-Nicola, Z. (1989). Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proceedings of the National Academy of Sciences86(23), 9154-9158.
  20. Binns, A., & Campbell, A. (2001). Agrobacterium tumefaciens‐mediated Transformation of Plant Cells. e LS.

Figure References:

  1. https://www.forestryimages.org/browse/detail.cfm?imgnum=5224023 (Date of Access: 17.08.2022)
  2. Păcurar, D. I., Thordal-Christensen, H., Păcurar, M. L., Pamfil, D., Botez, C., & Bellini, C. (2011). Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology76(2), 76-81.
  3. Păcurar, D. I., Thordal-Christensen, H., Păcurar, M. L., Pamfil, D., Botez, C., & Bellini, C. (2011). Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology76(2), 76-81.
  4. Păcurar, D. I., Thordal-Christensen, H., Păcurar, M. L., Pamfil, D., Botez, C., & Bellini, C. (2011). Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology76(2), 76-81.
  5. Păcurar, D. I., Thordal-Christensen, H., Păcurar, M. L., Pamfil, D., Botez, C., & Bellini, C. (2011). Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology76(2), 76-81.

Inspector: Furkan EKER

Yorum bırakın

E-posta adresiniz yayınlanmayacak. Gerekli alanlar * ile işaretlenmişlerdir