Mechanisms of Infection
Contents
Introduction
Vir Genes
Bacterial Cell Step
Plant Cell Step
Sensing Cell Signals
Agrobacterium Tumefaciens, and its related species A. rhizogenes, & A. vitris are the only known bacterial pathogens that invade plans by transferring their DNA to the plant, hence they have evolved as a major tool for plant genetic engineering. There are many different biological processes involved in this inter-kingdom DNA transfer, such as intercellular signalling, cell-to-cell DNA transport and DNA integration into the host cell nucleus (Tzfira & Citovsky, 2000).
The genetic modification of the plant occurs as a result of the integration to the plant genome of a specific fragment of DNA, termed the T-DNA, or Transfer DNA, which originates from the bacterial Ti (tumor inducing) plasmid. Expression of these genes leads to the formation of Opines; specific oligosaccharides used purely by A. tumefaciens as a source of carbon. From then on, expression of several oncogenic (onc) genes leads to the formation of tumours (Gaudin et al., 1994). The entire process is regulated and controlled by a set of genes known as Vir genes, which are activated by the detection of wounded plant phenolic compounds. The transfer process can be divided into 2 steps; the bacterial cell step and the plant cell step (Tinland, 1996).
Vir Gene |
Function |
| Vir A, Vir G | Sense phenolic compounds from wounded plant cells and induce expression of other virulence genes |
| VirD2 | Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis |
| Vir D1 | Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp border sequence |
| Vir D2 | Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA Complex. Also guides the T-DNA complex through the nuclear pores |
| Vir C | Binds to the 'overdrive' region to promote high efficiency T-strand Synthesis |
| Vir E2 | Binds to T-strand protecting it from nuclease attack, and intercalates with lipids to form channels in the plant membranes through which the T-complex passes |
| Vir E1 | Acts as a chaperone which stabilises Vir E2 in the Agrobacterium |
| Vir B & Vir D4 | Assemble into a secretion system which spans the inner and outer bacterial membranes. Required for Export of the T-complex and Vir E2 into the plant cell |
Click here to see how these vir genes interact to initiate infection.
Attachment & Penetration
The initial pre-penetration event in the soil rhizosphere is the conjugal transfer of the Ti plasmid, therefore increasing the number of pathogenic isolates in the soil. Quorum sensing proteins TraI and TraR induce the expression of genes required for bacterial cell mating and mobilisation of the plasmid. This response is also affected by opines produced by infected plants, which either suppress or activate a repressor of the TraR gene. The response is determined by the strain of A. tumefaciens present, and the opines being produced.
A. tumefaciens posess swimming motility which is mediates by flagella. The precise mechanism controlling chemotaxis is so far unknown, but it is thought that migration occurs towards sugars and amino acids which accumulate around plant roots in the rhizosphere. Some strains may also be attracted to specific plant compounds released from wounded plants such as acetosyringone, and also to opines. Mutants which are non motile are still virulent (disease causing), but are unable to infect a plant unless directly innoculated, which suggests that motility is an important part of the infection process.
Attachment to the plant is a two stage process, firstly involving a weak initial adhesion, then the bacteria synthesise cellulose fibrils which anchor them to the wounded plant cell surface. Some of the bacterial genes required for this process have been identified, namely chvA, chvB, pscA and att, as a mutation in any of these genes leaves the bacterium unable to attach to the plant. There are also molecules within the plant which are thought to be involved in the attachment process. One such molecule is vitronectin; an adhesive glycoprotein which is a component of the plant extracellular matrix (ECM). Vitronectin is more commonly associated with the cohesion of plant cells, thus having a role in plant structure and rigidity. The diagram below opens in a new window, and shows the mechanism by which the bacterium enters the plant and proleferates to form a gall.
Bacterial Cell Step
The bacterial step involves all the processes leading to the production and export of the T-DNA complex, containing the information necessary to cause infection. The T-complex is made up of nucleoprotein; a single stranded T-DNA coated by VirE2 proteins, with a VirD2 protein attached at the 5I end (Howard & Citovsky, 1990). The VirD2 acts as a site specific endonuclease which recognises and cuts the left and right T-DNA borders with the VirD1. The VirE2 acts as a coat protein which envelopes the T-DNA, thus protectecting it from nuclease attack when it enters the plant cell (Tinland, 1996). These VirD2 and VirE2 proteins are also believed to have a role in targeting the T-DNA to the cell nucleus, once it has entered the plant cell. Once the T-DNA has been enveloped to prevent it from being degraded once outside the bacterial cell, it is referred to as the T-complex.
The T-complex requires a specific export system to deliver it across both the
bacterial envelope and the plant cell membrane and into the plant cell cytoplasm.
T-complex export occurs via a type IV secretion mechanism (right), comprising of a filamentous
pilus and a transporter complex that translocates substances through the cell membranes
(Salmond, 1994). In A. tumefaciens, the type IV secretion system is assembled
from proteins encoded by the virD4 gene and the virB operon (Tzfira and Citovsky, 2000).
Eleven VirB proteins are encoded by the VirB operon, all of which have a role to
play in the transport of the T-complex across the membrane. VirB1 initiates the
assembly, and VirB2 is the main structural protein in the pillus. The pilus was
proposed by Zupan et al. (1998) to sense contact with a plant cell and relay this
information back to the transporter complex and initiate the export of the T-complex.
Plant Cell Step
It is believed that the T-DNA complex passes into the plant cell nucleus by active nuclear uptake, as the size of the T-DNA complex (12.6nm diameter) (Citovsky et al. 1997) exceeds that of the diameter of the nuclear pores (9nm) (Forbes, 1992), although the size of the nuclear pore can increase to 23nm during nuclear uptake (Forbes, 1992). Unlike other mobile genetic elements such as retroviruses, T-DNA does not encode functions for transport and integration, therefore the DNA sequence is non-specific. It is this property of the T-DNA which makes it so useful; any DNA sequence inserted between the T-DNA borders will be transferred into the plant genome, enabling the efficient production of transgenic plants.
Integration into the plant cell genome occurs essentially at random, a process which is believed to be controlled by the host factors. As a result of research into Arabidopsis mutants, a VIP1 basic zipper transcription factor has been identified which appears to facilitate the entry of the T-complex into the cell nucleus. A second protein, VIP2, acts to target the T-complex to transcriptionally active DNA. Once integrated into the plant genome, the auxin and cytokinin biosynthetic genes are expressed, resulting in uncontrolled proliferation and growth of the gall. Opine biosynthetic genes are also expressed, and these opines are used only by the gall as its sole carbon source, making it almost independent of the plant.
Sensing Plant Signals
As A. tumefaciens is so ubiquitous in the soil, it needs a specific mechanism to enable it to assess whether a particular plant is suitable for infection or not. A. tumefaciens has adopted a two component regulatory system which is common in many prokaryotes, and involves the VirA and VirG proteins (Tzfira & Citovsky, 2000). VirA acts as a membrane sensor protein, whereas VirG regulates the cytoplasmic response to wounded plant cell phenolic compounds and promotes activation of all the Vir genes. VirG specifically interacts with the vir box; a conserved 12 base pair sequence located in the promotor sequence of all the vir genes. VirG is also able to induce its own expression, as it is produced from mRNA both in the presence and absence of plant phenolic compounds (Stachel & Zambryski, 1986). Compounds known to induce Vir gene expression include lignin, flavanoid precursors and acetosyringone (Stachel et al. 1985).
The signalling pathway is initiated when wounded plant phenolics interact with VirA, which can either be through direct or indirect interaction, depending on the nature of the signal intercepted (Tzfira & Citovsky, 2000). Through mutational analysis of VirA, Tzfira and Citovsky (2000) concluded that during signal transduction, VirA functions both as a protein kinase and a phosphotransferase.
Plant phenolics are known to be bacteriostatic at higher concentrations, a well known plant defense mechanism. To overcome this, a VirH protein is expressed as a result of VirG, which is believed to detoxify these harmful compounds. However, VirH expression will only occur after VirG has played its role in signalling during the infection process (Tzfira & Citovsky, 2000).