In this article we will discuss about the process of T-DNA transfer.
Once plant exudates activating expression of vir genes and Agrobacterium attach to the plant cells, the T-DNA region in the Ti plasmid undergo excision process to transfer into the plant cells. Excision process involves active participation of vir genes such as virD, virC and virE.
Separation of single stranded T-DNA from Ti plasmid requires conserved cis-acting 25 bp imperfect direct repeat border sequence that delineates the ends of the T-DNA in both Ti and Ri plasmids. T-DNA transfer process will be unsuccessful if right border sequence is deleted. This indicates that right border is more crucial than left border suggesting the polarity of T-DNA transfer.
Processing of T-DNA begins with the attachment if virD2 protein. virD2 proteins are directly involved in processing of T-DNA from Ti plasmid.
Both virD1 and D2 are engaged in endonucleolytic cleavage of 25 bp T-DNA borders. virD1 functions as topoisomerase and convert Ti plasmid DNA from a supercoiled state to a relaxed form VirD2 protein acts as endonuclease, associates strongly with 5′ end of the DNA molecule through tyrosine amino acid.
Endonuclease introduces a site specific nick on bottom strand of DNA particularly at border regions. Binding of virC1 protein at conserved border sequence in overdrive regions stimulates virD endonuclease attachment and also facilitate nicking at the T-DNA borders.
DNA repair machinery of bacteria activates immediately to initiate damage control by the synthesis of a new DNA strand to fill remaining gap in the bottom strand at 3′-OH end of the border cleavage using upper DNA strand as a template.
Replacement of DNA strand synthesis is continuous until the left border cleavage site is reached. Bottom strand is then released as a linear single strand T-DNA molecule. Once T-DNA process completes as bottom strand is released, virD2 protein then remains permanently attached to 5′ end of the T-strand (Fig. 14.7). Generally expression of vir gene under in vitro condition produces one T-strand copy per bacteria cell.
Vir Proteins Escorting the T-DNA Strand:
Both virD2 and virE2 protein can escort single stranded T-DNA molecule and perform important functions in transferring out of bacterial cell and intergrating the T-DNA once the T- complex is inside the plant cell. The mobilized T-strand is thought to be packaged by virE2 into hollow, cylindrical filament.
Both virE2 and D2 proteins contains functional nuclear localization signal (NLS) sequence, virE2 has shown to contain two independently active NLSs located within the central region of the molecule while virD2 has been demonstrated to carry one functional NLS at its carboxyl terminus.
VirD2 protein plays a major role by acting as pilot function to guide the T-strand from bacteria into the plant cell. This protein is covalently attached to the 5′ end of the single stranded T-DNA. Similarly, virE2 is a single stranded DNA binding protein. VirE2 is the most abundant among different vir proteins accumulated in Agrobacterium induced by acetosyringone.
The virD2 and E2 proteins are co-operatively associated with the T-strand; often referred to as T- complex may safe-guard the T-DNA molecules from bacterial and plant cell nucleases. The virE operon consists of two open reading frames coding for proteins of 7 kD (virE1) and 60.5 kD (virE2) both of which are indispensable for virulence.
Recent findings have shown that Agrobacterium transfers virE2 proteins and T-DNA and virD2 complex separately. During T-DNA transfer, vir J protein (ACVB) plays an important role suggesting that ACVB (virJ) is a single stranded DNA binding protein which co-operatively interacts with the T-strand and facilitate its export from bacteria.
Another T-DNA accompanying protein is virF. It is also exported likely to the plant. Wild type octopine type Agrobacterium strains that contain virF genes triggers tumours on a wide range of dicotyledonous plants. It is strongly believed that virF performs an important function in plant rather than in bacteria. It can stimulate plant cells to divide and become more susceptible to transformation.
Some of the earlier studies on T-DNA movement across the cell boundaries were heavily focused based on bacterial conjugal systems, including the role played by F– pilus in mobilizing conjugal DNA into the recipient cell. However, later extended work provides more clarity of information after discovering the role of virB operon responsible in the formation of membrane associated T-DNA transport apparatus.
At least six virB proteins have been restricted to A. tumeifaciens’ inner membrane and at least three virB genes (virBa, vir10 and B1) are essential for virulence. Recent findings on T-DNA movement across cell boundaries highlighted involvement of type IV secretion system. This system constitutes upto twelve proteins that form two functional components.
One is filamentous virulence pilus and the other one is transporter complex. In Agrobacterium, the type IV transport comprises proteins encoded by the virD4 gene and by eleven open reading frames of the virB operons. Some of the virB (virB1, virB2 and virB5) proteins are likely to interact with the host cell reporters.
In the functional clarity of these virB proteins, the secreted virB1 from Agrobacterium cell is known to establish the cell-to-cell contact between Agrobacterium and the host plant cell wall, whereas virB2, virB5 and virB7, the main components of the Agrobacterium pilus.
T-DNA inside the Plant Cell:
Once the single stranded T-DNA complex enters plant cells, its final destiny is to reach the nucleus and consequently integrate into the plant DNA. The probable size of the T-complex is around 13 nm in diameter and presence of nuclear-localizing activities in both virD2 and E2 facilitate transport to nucleus.
Several in vitro experiment have confirmed NLS activities, for example, when NLS containing protein is fused to β-glucoronidase reporter gene, it can be easily targeted to nucleus of plant cell, as virD2 is covalently attached to T-DNA. Therefore, virD2 and E2 likely to-operate with cellular factors to mediate T-complex nuclear entry and integration into the host genome (Table 14.1).
Involvement of plant proteins in T-complex nuclear import and integration into the plant DNA has been given special attention and heavily focused. The plant encoded proteins identified as Arabidopsis importin α (α-karyopherin) or AtKAPα bind specifically to the nuclear targeting sequence of D2 and E2. Recently, another identified plant protein known as cyclophilin has many a positive role in T-DNA transport.
VirD2 protein might act as a chaperone to hold virD2 in order to maintain its conformation during T-DNA trafficking through the plant cells. Three members of Arabidopsis cyclophilin chaperone family—RocA, Roc4 and CypA, have been identified, which are involved in the maintenance of conformation stability.
Extended investigation presented some other plant proteins called vip1 and vip2, primarily involved in facilitation of virE2 nuclear localization signal (NLS) recognized by importin α and promote T-DNA integration. In the importin-dependent pathway importin α binds NLS containing proteins and subsequently associates with nuclear pore complex (NPC).
Once T-complex is inside the nucleus, GTP binding protein Ran mediates release of T-DNA accompanied proteins. VirE2 gets favourable treatment inside the plant cytoplasm after binding with vip1 protein. Vip1 is indispensable for successful transport of virE inside the nucleus. It however, does not interact with virD2.
Vip1 protein has a certain homology to basic zipper proteins, localized to cell nucleus and presumed to be involved as plant transcription factor. Another virE2-interacting protein vip2 might target the T-strand into high profile transcription sites of the plant chromosomes and promote integration (Fig. 14.8).
T-DNA Integration and its Genes Expression:
T-DNA integration into the host cell genome is the final step leading to tumour formation. Integration involves illegitimate recombination. Most of the T-DNA entry into the plant nucleus does not stably integrate. T-DNA enters the nucleus as a single strand and integrates plant DNA either by strand invasion of the locally denatured plant DNA.
Although the T-strand is known to be converted into double-stranded DNA in the host cell nucleus, it is however, not clear whether the T-DNA integrates as a double-stranded or single-stranded molecule. Several evidences have shown that majority of the invading T-DNA molecules do not integrate into the host genome.
VirD2 protein has some role to play in T-DNA integration process as it covalently linked and persist in front of the T-strand. Participation of virD2 in T-DNA integration in plants is based on two lines of evidences. The integration of the 5′ end of the T-strand into plant DNA is precise, usually contains a few 5′ nucleotides, which are deleted during integration into plant genome. This may happen only to safeguard T-DNA from exonucleus during integration. VirE2 may play only in indirect role in T-DNA integration. Functions of various vir genes are summarised in the Table 14.1.
Several mutant studies in Arabidopsis revealed that there are possible roles of other plant proteins such as RAT5, histone H2A protein interact with virD2 protein and also interact directly with the incoming T-strand. Integration is very precise and proceeds at active transcription sites. Successful T-DNA integration is followed by the specific set of oncogenes gene expression. Their encoded proteins are involved in the synthesis of plant hormones. Over production of these ultimately result in tumourogenesis or hairy root formation.
In an extended work it was documented that new proteins called vip1 and vip2 primarily involved in facilitating virE2 nuclear localization signal (NLS) recognized by importin α and promote the integration of T-strand. In the importin-dependent patyway, importin α binds to NLS containing proteins and subsequently associates with importin β.
Further, participation of importin β in interaction with the nuclear pore complex (NPC) takes place immediately. Once T-complex is inside the nucleus, GTP-binding protein, Ran mediates release of the proteins accompanied T-strand. VirE2 gets favourable treatment inside the plant cytoplasm after binding with vip1 protein.
Vip protein is indispensable for successful transport of virE2 inside the nucleus (Fig. 14.9). It however, does not interact with virD2. vip1 has a certain homology to basic zipper proteins that are restricted or localized to cell nucleus and likely to be involved in plant transcription factors. Another virE2 interacting protein vip2 might target T-strand into high profile transcriptionally active sites in the plant chromosomes and also promote integration.
