genetic
and biochemical analysis of Agrobacterium-plant cell interaction
Agrobacterium tumefaciens is a gram-negative soil bacterium
that has the unique capacity to transfer DNA from its resident
Ti (tumor-inducing) plasmid, and proteins encoded by this plasmid,
into plant cells. The transferred DNA (the T-DNA) is ultimately
moved into the nucleus, integrated into the chromosomal DNA
and expressed. T-DNA expression results in the production of
i) growth factors that cause uncontrolled cell proliferation
and ii) novel amino acid-sugar conjugates that cannot be metabolized
by the plant cell but will serve as a carbon and nitrogen source
for the inciting bacteria. Thus, Agrobacterium engineers
the plant cell so that it proliferates indefinitely while producing
nutrients that are dedicated to bacterial growth. This system
can also be manipulated so that rather than transferring the
wild type T-DNA the Agrobacterium will transfer any DNA
cloned into an appropriate vector, thus allowing for routine
genetic engineering of plants.
The research in our lab is focused on two major questions in
relation to the Agrobacterium-mediated transformation
process:
How are plant derived signals recognized and how does this
recognition activate the expression of the virulence genes?
How are the transported DNA and protein molecules moved
out of the bacterium and into the plant cell?
signal recognition and transduction
Initiation of plant transformation by Agrobacterium
occurs when the virulence (vir) genes of the Ti plasmid
are activated. This occurs as a result of the activities of
a classic two component regulatory system. The sensor kinase
(VirA), a membrane bound dimeric histidine kinase responds to
plant derived compounds (certain sugars, phenolics and low pH)
by phosphorylating the response regulator (VirG) which, in turn,
activates transcription of the vir genes. We are particularly
interested in how the different signals are recognized by and
activate the VirA sensor kinase. The sugars are known to bind
to a periplasmic protein that subsequently interacts with the
periplasmic domain of VirA, resulting in an increase in the
sensitivity of the system to phenols. We have recently shown
that there must be transfer of signaling information concerning
the sugars from one dimer VirA subunit to another. Less is known
about phenol or pH recognition. While earlier genetic studies
have provided evidence that VirA binds the phenols, there is
no physical evidence of this. Rather, chemical and biochemical
evidence indicates that the phenolics bind other proteins in
the bacterium, and these may then interact with VirA. We have
used a different genetic approach to address this question,
selecting for changes in the sensitivity to, or specificity
of, particular phenolics. Recently isolated mutant strains are
hypersensitive to some phenolics but not others, and do not
map to either VirA or the Ti plasmid, indicating that proteins
other than VirA may play a key role in the signal recognition
process. The gene(s) responsible for this change in specificity
are currently being isolated and analyzed.
the VirB complex, a Type IV transport system
The second major research focus concerns the structure and
function of the VirB complex. The movement of DNA from Agrobacterium
into plant cells depends upon the activities of the Ti plasmid
virulence (vir) genes. Several of the Vir proteins are required
for the production of a single-stranded DNA covalently attached
to VirD2 at its 5' terminus. At some time during the transfer
process the single-stranded DNA-binding protein VirE2 coats
the VirD2-T-strand. VirE2 and VirD2-T-strand, as well as the
broad host range plasmid RSF1010 can be independently transferred
to the plant cell via a membrane bound complex consisting of
the 11 VirB proteins and VirD4. This complex is the prototype
of the Type IV secretion system that many gram negative bacterial
pathogens (e.g. Bordetella pertussis, Brucella suis)
use for virulence factor transfer to host eukaryotic cells.
The VirB complex can also mediate the conjugal transfer of RSF1010
between agrobacteria, thus providing a useful genetic system
for monitoring VirB complex activity. Intriguingly, the presence
of the RSF1010 plasmid in wild type strains will inhibit the
transfer of the T-DNA or VirE2 to plant cells, evidently as
a result of competition between the conjugal intermediate of
RSF1010 and the normal substrates for the VirB complex. The
long term objectives are to understand how the VirB complex
assembles and functions.
Towards this end, a variety of genetic and biochemical methods
have been developed to study the interaction of VirB proteins
with themselves and with transported substrates. These include:
saturation mutagenesis of the virB genes (virB7-10) that
are proposed to form the transport channel between the inner
and outer membranes, characterization of high molecular weight
VirB complexes revealed by chemical cross-linking and immunoblot
analysis, test of VirB interactions with each other, or transported
substrates, using the yeast two-hybrid system. Our genetic analysis
of VirB-mediated conjugal transfer or RSF1010 between agrobacteria
revealed that the presence of the VirB complex in the recipient
strain increased the frequency of conjugal transfer by 3-4 orders
of magnitude. This has allowed us to conduct experiments demonstrating
that there are functional subsets of the VirB proteins. The
biochemical bases of their activities, either alone or in combination,
are being investigated.
Finally, we are examining the interaction of VirE2 with the
VirB transport complex. Our results have revealed a small portion
of the C-terminal region of VirE2 that is required for virulence
when expressed in the bacterium. Interestingly, if this same
mutant protein is expressed in a transgenic plant, that plant
will respond to virE2 mutants of Agrobacterium as if
they were fully virulent. This result suggests that the C-terminal
region of VirE2 is required for transport by the VirB complex.
Studies on the interaction of this transport signal and the
VirB complex are in progress.
selected
publications
Lynn, D. G. and A. N. Binns. 2007. Control of virulence gene
expression in Agrobacterium tumefaciens. In: Agrobacterium:
From Biology to Biotechnology. Eds: Tzvi Tzfira and Vitaly Citovsky.
Springer Press; In press.
Binns, A. N. 2007. A brief history of research
on Agrobacterium tumefaciens. In: Agrobacterium: From Biology
to Biotechnology.
Eds: Tzvi Tzfira and Vitaly Citovsky. Springer Press; In press.
McCullen, C. A. and A. N. Binns. 2006. Interactions
between Agrobacterium tumefaciens and plant cells required for
interkingdom macromolecular
transfer. Ann. Rev. Cell and Devel. Biol. 22:101-127.
Cascales, E., K. Atmakuri, Z. Liu, A. N. Binns and
P. J. Christie. 2005. Agrobacterium tumefaciens oncogenic suppressors
inhibit T-DNA
and VirE2 protein substrate binding to the VirD4 coupling protein.
Mol. Microbiol. 58:565-579.
Wise, A. A., L. Voinov, and A. N. Binns. 2005.
Intersubunit complementation of sugar signal transduction in VirA
heterodimers and post-translational
regulation of VirA activity in Agrobacterium tumefaciens. J.
Bacteriol. 187:213-223.
Höppner, C., Z. Liu, N. Domke, A. N. Binns and
C. Baron. 2004. VirB1 orthologs from Brucella suis and pKM101 complement
defects of the lytic transglycosylase required for efficient type
IV secretion form Agrobacterium tumefaciens. J. Bacteriol 186:1415-1422.
Nair, G., Z. Liu and A. N. Binns. 2003. Re-examining
the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium
tumefaciens strain C58. Plant Physiol. 133:989-999.
Liu, Z. and A. N. Binns. 2003. Functional subsets of the VirB
Type IV transport complex proteins of Agrobacterium tumefaciens.
J. Bacteriol. 185:3259-3269.
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