Part:BBa_K4729503

virG TiBo542 N80D CDS

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 (N80D)

Based on the characteristics of parts BBa_K4729501 and BBa_K4729502, we designed a supervirulent vir G carrying a mutation that confers the constitutive phenotype to improve the transformation efficiency even further.

Design

In order to identify the correct site for the mutation in the supervrulent virG, its sequence was aligned with vir G (pRiA4b), where the mutation site was known to be at position 54. By analyzing the sequence alignment, the asparagine (N) of interest was identified at position 80 of virG (pTiBo542).

Figure 1: Global alignment of the protein sequence was done using the Needleman-Wunch algorithm.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 234
  Illegal BamHI site found at 410
  Illegal BamHI site found at 702
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 547
  
• 1000
  COMPATIBLE WITH RFC[1000]