In this article we will discuss about the organization of Ti Plasmid.
Agrobacterium tumeifaciens harbours a large circular DNA known as Ti plasmid, which exists as an independent extra chromosomal entity of 200 kb size. The most spectacular regions of Ti plasmid are the presence of T (transfer) DNA and vir regions.
Other components are origin of replication and regions for opine catabolism. There are two types of Ti plasmids based on the synthesis of opine class of aminoacids, one is nopaline Ti plasmid and another octopine Ti plasmid.
In octopine Ti, plasmid there are two T-regions, one is 13 kb and other is 8 kb located at very close proximity (Fig. 14.1). Presence of OS regions on Ti plasmid controls the synthesis of opine class of amino acids. Opine synthesis is controlled by enzyme octopine synthase and nopaline synthase at loci called OCS and NOS on octopine and nopaline Ti plasmids respectively.
Transfer DNA (T-DNA):
Although both species of Agrobacterium contains unique T-DNA in their plasmids, the nature of genes and T-DNA numbers vary considerably. This has been evidenced in octopine and nopaline type of Ti plasmids in which, octopine has two T-DNA regions of 13 kb on left hand and 8 kb right hand regions.
In contrast, nopaline type consists of single 23 kb T-DNA in their Ti plasmid. T-DNA region is organized into two sets of oncogenes. One of the sets of three genes is capable of inducing tumour in transformed plants. Three genes are iaam (tms 1), iaaH (tms 2) and ipt (tmr). These encode enzymes invovled in the synthesis of phytohormones (Fig. 14.2).
The iaam and iaaH genes encode trptophan monoxygenase and indole acetamine hydrolase, primarily responsible for auxin synthesis namely, indole acetic acid. The ipt gene encodes enzyme isopentanyl transferase, which uses isopentanyl pyrophosphate and adenosine monophosphate to synthesize cytokinin-isopentanyl adenosine. These three genes are expressed as a part of T-DNA in plant host chromosomes after successful integration.
Second set of T-DNA genes are involved in the synthesis and transport of unusual amino acids or sugar conjugates termed as opines. Plant cells are incapable of metabolizing the opines. However, Agrobacterium carries catabolism loci that enable it to use opine as a sole nitrogen and carbon source.
On either side of the T-DNA regions are flanked by conserved 25 bp czs-acting imperfect direct repeats known as border sequence. These conserved border sequences are present at both the ends of T-DNA in all Ti and Ri plasmids (Fig. 14.3). Both left and right border sequences are recognized and cut by specific endonuclease, which is one of the gene products of vir gene and initiates transfer of T-DNA.
Right border sequence, situated in front of the single-stranded T- DNA facilitates T-DNA transport in polar fashion. Any damage or deletion of this border sequence will severely hamper T-DNA transfer process. The role of right border is more crucial than left border in the transfer of T-DNA from bacteria to plant cell.
Although a number of genes are present within T-DNA border sequence, they do not, however, play any role in the transport of T-DNA. Thus, any DNA sequence placed between these two borders sequences will be easily mobilized into the plant cell. All the above factors suggest that border signals of T-DNA can only direct processing and transport of T-DNA rather than integration into plant DNA.
Virulence genes are unique regions and important components of both Ti and Ri plasmids. This region consists of at least ten organized operons of 35 kb. Most of these vir operons are involved in T-DNA transfer, but their physical attachment with T-DNA is not required essentially.
At least twenty one vir genes are involved in virulence functions and organized into ten operons such as vir A, vir G, vir B, vir C, vir D1, D2, D4, vir E2, Vir F1, vir H and vir J/ACVB. The poly-cistronic nature of almost all vir genes, with the exception of virA and D are organized into operons.
Plant Signaling and vir Genes Induction:
Regulation of vir genes in bacteria after attachment to plant cell is potentially a fascinating process. Generally, their expression is extremely at a low profile in Agrobacterium under normal conditions. High profile expression of certain genes requires signal molecules.
The activated vir genes involved in the synthesis and mobilization of T-DNA into plant cell is well regulated and depends on the ability of bacteria to sense phenolics and sugars synthesized during plant wound response. Induction of vir genes is a low process and takes 8—16 h to reach maximum level of expression.
Transcriptional activation of Agrobacterium virulence machinery is a ‘two component’ signal-transduction system. Two of the vir operons, virA and virG, have been shown to participate in signal induction process. Genetic evidence on direct sensing of phenolic compounds by virA protein has been documented.
The virulence (vir) genes of A. tumeifaciens are induced by low molecular weight phenolic compounds and carbohydrates like certain monosaccharides through two-component regulatory system consisting of virA and virG proteins. Both virA and virG are constitutively expressed in Agrobacterium cell.
The presence of plant cells that are a potential target for the T-DNA transfer is sharply sensed by virA proteins. There are some contrasting similarities between virA protein and other bacterial sensory proteins such as chevA, NtrB etc., which firmly participate in chemotaxis.
The virA sensory proteins span the inner membrane with its C-terminal region located in the cytoplasm and its N-terminal domain in the periplasm. The C-terminal region can be divided into three domains: a linker, a protein kinase, and a phosphoryl including N-terminal region of the virA protein including the periplasmic domain is not essential for vir gene induction.
The plant phenolic signal chemical such as acetosyringone related compounds are perceived by virA sensory protein. The virA is an autophosphorylation protein and it recognizes three types of signals. Most of the plant signal molecules are low molecular weight phenols carrying an orthomethoxy group. Some of these signal molecules acts as strong or weak inducer of vir genes.
Strong inducers are acetosyringone and weak inducers are ferulic acid, syringic acid and acetovanillone. The strong inducing potential is due to their para substituents that facilitate the dissociation of the proton from the hydroxyl group. Several other phenolic compounds induce expression of the A. tumeifaciens vir loci.
Doltonei (1986) identified at least seven plant phenolics, which can induce transcription of vir loci, when co-cultivation with plant cells under in vitro condition. The data from screening assays indicated that seven simple plant phenolic compounds such as p-hydroxynenzoic acid, B-resoryllic acid, protocatechonic acid, pyrogallic acid, gallic acid and vanillin induce virE locus. These seven phenolics are known to be constituents of wounded plants, widely distributed among angiosperms (Fig. 14.4).
In addition, certain pyranose sugars like arabinose and sugar derivative (galacturonic acid) strongly enhance the inducing activity of phenolics. Certain sugar binding proteins encoded by chromosomal ChvE are recognized by these sugars.
This sugar-protein complex can cause change in conformation of virA after interaction with its periplasmic domain that strongly enhances sensitive virA towards phenolics signaling. The same protein is also known to have involved in response to acidic pH. However, information on pH of the environment received by virA is not known.
VirG protein exhibits some of the amino acid sequences indicating the presence of several domains, which are the characterization of DNA binding proteins. There is reason to believe that one of the domains present in the N-terminal half of this protein is presumed to interact with transcription unit of cell, i.e., sigma subunit of alfa RNA polymerase.
Similarly, C-terminal domains contain a helix-turn helix DNA binding motif and amino acid residue in the region of this protein interact with specific bases on the DNA. There are three vir boxes located upstream from virG transcription start site.
Only phosphorylated virG protein acts as a transcription activator and induces expression of other vir genes. Transcription occurs from two promoters on activation by phosphorylated G bound to vir boxes upstream from the promoter.
Consolidating the overall process, Agrobacterium, when it comes in contact with plant wounded site containing signal compounds or inducers, its membrane spanning virA protein directly sense the phenolic compounds. The sugar binding protein (encoded by chvE) interacts with virA causing conformation change and increased its sensitivity towards phenolics.
Autophosphorylation of virA proteins follows immediately and subsequently interact with virG. The interaction is at the level of phosphorylating conserved aspartic acid. The phosphorylated G protein then becomes transcription activator and able to induce expression of remaining vir genes situated on operon vir sequence of the Ti plasmid (Fig. 14.5).
The direct sensing of signal molecules results in the auto regulation of vir regulatory genes. Binding of phosphorylated virG to two promoters favours transcription. Promoter I involves increasing transcription rate by sensing inducer molecules and transcription from promoter II is increased due to environmental influence.
Chromosomal Genes Involved in Tumourogenesis:
Virulence nature of Agrobacterium is due to the participation of both virulence genes and chromosomal genes. Bacterial chromosomes contain several gene loci like chA, chB, chE and all of which are involved in virulence nature Agrobacterium (Fig. 14.6).
At least one gene (chvE) is involved in recognizing plant cells coupled for transformation. There is strong evidence that virA and virG controls expression of five virulence related proteins and two of which are encoded by Ti plasmid and remaining by bacterial chromosomes.