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in-cites,
November 2005
http://www.in-cites.com/papers/AndrewBent.html
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An
interview with:
Dr. Andrew Bent |
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the interview below, in-cites talks with Dr. Andrew Bent about
the highly cited paper he and colleague Dr. Steve Clough
published in 1998, "Floral dip: a simplified method for Agrobacterium-mediated transformation of
Arabidopsis
thaliana," (Plant J. 16[6]: 735-43, December
1998). This paper is currently ranked at #1 in the field of
Plant & Animal Sciences in the ISI
Essential Science Indicators
Web product. Dr. Bent’s record in this field includes 14
papers cited a total of 1,867 times to date. He is an
Associate Professor in the Department of Plant Pathology at
the University of Wisconsin, Madison.
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Why, in your view, is your
paper highly cited?
Clearly, it is a "methods" paper. It describes how to
transform Arabidopsis with exogenous DNA. Transgenic plants are now
a common part of many more people’s research, in part because
Arabidopsis is now so easy to transform. It is "just" a
method, and we can’t take even half of the credit for developing
the method, but it is interesting to see how the method really has
revolutionized plant biology.
What are the circumstances that led you to your work?
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“Transgenic plants are a common part of many more people's research, in part because
Arabidopsis is now so easy to transform.”
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While a postdoctoral scientist in Brian Staskawicz’s
laboratory, working to clone a disease resistance gene in the early
days of map-based cloning, we confronted the need to test many
overlapping genome segments to identify the region that complemented
our mutant phenotype. Tissue culture-based transformation was
available and we did this, but it was quite laborious. Ken Feldmann
and David Marks had recently had success directly treating
Arabidopsis seeds with Agrobacterium. That method was very
tough to reproduce, but it showed that superficial application of Agrobacterium,
without tissue culture, could generate transformants, transforming
the plant after divergence of the male and female germ lines. Doug
Dahlbeck and I tried a "clip and squirt" method pioneered
by Hong-Gil Nam, applying Agrobacterium to the base of plant
rosettes where secondary inflorescences were emerging. It worked for
us but it wasn’t ideal—still not very reproducible. In the
meantime, we had also worked out ways of applying bacteria to
Arabidopsis via vacuum infiltration, as part of our development of
the Arabidopsis-Pseudomonas pathosystem. We adapted vacuum
infiltration to these little plants by growing them through a screen
placed tightly on the soil, to keep most of the soil in place while
dunking the plants in liquid. We also brought Silwet L-77 to
Arabidopsis, to help bacteria penetrate into the plant interior
through stomates and other small openings. This was a suggestion
from Steve Lindow (many other surfactants don’t work well with
plants). I also made a few unsuccessful attempts at vacuum
infiltration of Agrobacterium into Arabidopsis. These were
small bits of the puzzle.
Then came the big result from across the ocean. Nicole Bechtold
and Jeff Ellis, working with Georges Pelletier, were also trying to
transform Arabidopsis without tissue culture. Astutely, they were
focused on getting Agrobacterium to persist on the plant as
it flowered. They had heard talks about how our labs applied Pseudomonas
bacteria to Arabidopsis by vacuum infiltration, and they tried that
approach. It worked! They, along with Ken Feldmann, really are the
ones who showed everyone the way forward. I saw their poster, which
they shared prior to publication at the international Arabidopsis
meeting at Ohio State University, and immediately became quite
excited. We tweaked their method, figured out that we could leave
plants intact rather than uprooting and re-planting them, sent an
e-mail to the community about it, and published it as a method in
our Science paper on the first cloning of an NB-LRR
resistance gene (AF Bent, et al., "RPS2 of Arabidopsis
thaliana: a leucine-rich repeat class of plant disease
resistance genes," Science 265:1856-60, 1994).
Would you summarize the paper briefly and describe its significance
for your field?
The above information was all out there and under revision by
many in the Arabidopsis community, prior to the Clough and Bent
work. There were stories flying around, including anecdotes and tall
tales, about how to get Arabidopsis transformation to work better.
Steve and I noted that very few people were approaching it
scientifically, with controls and replications and quantitative
data, to dissect out what mattered. We simply applied the scientific
method, and slogged through many experiments to test many variables
in the method. I might add that our work was funded by soybean
farmers of the central U.S., through the North Central Soybean
Research Program, with the goal of learning how to apply the method
to soybean and other crops. We found that for Arabidopsis, the means
of Agrobacterium culture preparation wasn’t very important,
and that one could dispense with the uprooted plants, the tissue
culture media, the hormones, and even the vacuum infiltration, but
that the sucrose mattered a great deal. Silwet L-77 surfactant made
the whole thing reproducible if dipping rather than using vacuum.
There were other details that were very briefly examined, for
example about Agrobacterium strain and Arabidopsis ecotype
(some were better than others). Other labs were on to similar ideas,
and partly similar protocols were available through the grapevine
(and via the new "world-wide web"). The main contribution
of our paper was that we provided a rational, peer-reviewed
dissection and a simplified method that people could use.
The significance of the method (everyone’s collective method,
not just our version) has been truly revolutionary. It has
facilitated routine testing of any DNA construct in an intact,
uniformly and stably transformed plant. It has facilitated
positional cloning of many genes. But most significantly, it has
allowed insertional mutagenesis of Arabidopsis. The forward genetic
uses (including variations such as promoter trapping) are very
valuable, but the arrival of reverse genetics has been the biggest
revolution. The sequence-indexed knockout collections, presently
over 300,000 independent plant lines from various sources, represent
a crucial and very widely used resource that is not available for
any other plant species. So floral dip has been one of those methods
that is not "just a method."
Where has this research gone since the publication of your paper? Is
the floral dip method widely used?
Bechtold and Pelletier, as well as Christine Desfeux, Steve
Clough, myself, and a few others, have learned more about where and
when Agrobacterium transforms Arabidopsis in this method.
Female gametes are apparently the primary target for productive
transformation. We’ve discovered that Agrobacterium has to
reach the interior of the floral gynoecium prior to locule closure.
This is an issue that may prevent transformation of other species,
this requirement for access to female gametophytes. The defensive
responses of other plant species against Agrobacterium also
appear to be an issue.
But in Arabidopsis, the floral dip method is very widely used.
Inflorescences are sometimes sprayed with Agrobacterium
rather than dipped.
Where do you see this research going 10 years from now?
For Arabidopsis, we may have all that we need in terms of a
stable transformation method. But transient transformation of
Arabidopsis could be better. Gene replacement at sites of homology
remains a desirable goal for all plants, so that is another area for
future focus. Perhaps most important, we and other labs have spent
significant amounts of time trying to get similarly simple
transformation methods working for other plant species, especially
the economically significant crop species. That has not yet worked,
other than for a few Arabidopsis relatives in the Brassicaceae.
It is just an amazing coincidence that Arabidopsis thaliana
was chosen as the model genetic organism for plants due primarily to
its small size, rapid generation time, and small genome, and it
subsequently turned out to be the species that is amenable to this
incredibly simple transformation method. But there is now increased
understanding of where and why the transformation method works. Stan
Gelvin, Vitaly Citovsky, and others are identifying molecular
mechanisms of T-DNA handling within the plant. Hopefully the
remaining barriers to similar transformation of other plant species
can be surmounted in the future.
Andrew Bent
Department of Plant Pathology
University of Wisconsin - Madison
Madison, WI, USA
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in-cites, November 2005
http://www.in-cites.com/papers/AndrewBent.html
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