Part:BBa_K4729501

virG TiBo542

General explanation of virulence

The mechanism for virulence and plant transformation is mostly conserved between A. tumefaciens and A. rhizogenes, with high similarity in the sequences of the virulence genes and their regulation (Moriguchi et al., 2001; Zhu et al., 2000). Therefore, most of the knowledge already available for A. tumefaciens can be extrapolated when working with rhizogenes strains. In fact, the swapping of Ti-plasmids in tumefaciens strains with Ri-plasmids has created some of the most commonly used Agrobacterium rhizogenes strains, including one of the strains used by our team, Arqua1.

The figure illustrates the key steps of Agrobacterium-mediated insertion of a target DNA (T-DNA) into the genome of a host plant. Originally, T-DNA and virulence genes were both harboured on the same plasmid, the Ti plasmid in A. tumefaciens or the Ri plasmid in A. rhizogenes. The picture shows transformation using a binary plasmid, meaning that the vir region of the Ti plasmid is separated on a helper plasmid. Virulence is induced if either phenolic compounds are secreted by the wounded plant (dicots, 1a) or have to be added manually (monocots, 1b). After diffusing through the outer membrane, these phenolic compounds are sensed by the membrane-bound sensor kinase VirA (2). VirA in turn autophosphorylates and activates VirG (3). VirG is the master regulator of the vir operon and binds as a transcription factor to the promoters of the virulence genes. These genes are involved in the transfer of the T-DNA into the host plant's genome (step 4-5). Agrobacterium-mediated transformation can be used to insert any gene region of interest into a plant's genome.

The Virulence Mechanism

Agrobacterium strains can transfer large DNA sequences to plant cells and integrate them into the plants' genome. Naturally, all the components for infection are present in a single, non-essential, 250 kbp plasmid (Ti-plasmid in A. tumefaciens or Ri-plasmid in A. rhizogenes).

The genes that code for the mechanism of plant infection and transformation are clustered in the vir (virulence) region, a ~30 kbp region of the Ri-plasmid. There are ca. 35 CDSs distributed in 11 operons in the vir region, which code for - among others - the type IV secretion system (vir B operon), proteins that excise and integrate the T-DNA in the hosts genome (C,D and E operons), and the two-component system that regulates the activation of the whole system (A and G operons). This two-component system can be understood as a “master switch” for the virulence genes.

Vir A is a trans-membrane sensor kinase that reacts to an acidic pH and phenolic compounds secreted by wounded plant tissue, causing it to phosphorylate the response regulator vir G. Among those phenolic compounds are acetosyringone, catechol and vanillin (Bolton et al., 1986). Once phosphorylated, vir G binds to the vir box region (TGAAAT) present in the promoters of virulence operons and upregulates their expression (Aoyama et al., 1989).

pTiBo542

There is a multitude of Agrobacterium strains, with differing characteristics and virulence strengths. The strain A281 in particular, which are able to transform a broader range of plant species and at a higher efficiency, is considered “supervirulent”. Introducing copies of its vir G and vir B operons in regular strains has been shown to recreate the “supervirulent” phenotype. In fact, the virG variant within the pTiBo542 plasmid has been observed to induce a more robust expression of vir genes compared to its virG counterpart on the pTiA6 plasmid. This heightened activity is primarily attributed to the existence of V7I and I106T mutations in the coding sequence of the variant. (Chen et al., 1991).

In planta experiments

Figure 2: Evaluation of the transformation efficiency in A. thaliana using 35S:RUBY:KanR + Ptac_TiBo542[VirG]_pSRK.

A. thaliana transformation via ARqua1 containing Ptac_TiBo542[VirG]_pSRK was the first construct tested. After 3 days we saw a total amount of 32% of plants which expressed RUBY. Seven days later, the number of RUBY-positive plants had increased to 57%. Thus, transformation efficiency after 3 days is 32% lower compared to the baseline with ARqua1 35S:RUBY:KanR, increasing by 24% after day 10.

Bibliography

Aoyama, T., Takanami, M., & Oka, A. (1989). Signal structure for transcriptional activation in the upstream regions of virulence genes on the hairy-root-inducing plasmid A4. Nucleic Acids Research, 17(21), 8711–8725.

Bolton, G. W., Nester, E. W., & Gordon, M. P. (1986). Plant Phenolic Compounds Induce Expression of the Agrobacterium tumefaciens Loci Needed for Virulence. Science, 232(4753), 983–985. https://doi.org/10.1126/science.3085219

Chen, C.-Y., Wang, L., & Winans, S. C. (1991). Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542. Molecular and General Genetics MGG, 230(1), 302–309. https://doi.org/10.1007/BF00290681

Jin, S., Song, Y., Pan, S. Q., & Nester, E. W. (1993). Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium. Molecular Microbiology, 7(4), 555–562. https://doi.org/10.1111/j.1365-2958.1993.tb01146.x

Moriguchi, K., Maeda, Y., Satou, M., Hardayani, N. S. N., Kataoka, M., Tanaka, N., & Yoshida, K. (2001). The complete nucleotide sequence of a plant root-inducing (Ri) plasmid indicates its chimeric structure and evolutionary relationship between tumor-inducing (Ti) and symbiotic (Sym) plasmids in rhizobiaceae11Edited by N.-H. Chua. Journal of Molecular Biology, 307(3), 771–784. https://doi.org/10.1006/jmbi.2001.4488

Zhu, J., Oger, P. M., Schrammeijer, B., Hooykaas, P. J. J., Farrand, S. K., & Winans, S. C. (2000). The Bases of Crown Gall Tumorigenesis. Journal of Bacteriology, 182(14), 3885–3895.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 702
  Illegal XhoI site found at 349
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]