www.checkbiotech.org ; www.raupp.info ; www.czu.cz

Researchers at the Virginia Bioinformatics Institute (VBI) at Virginia Tech,
Colorado State University and Duke University Medical Center have developed
a new method to determine gene function on a genome-wide scale in the fungal
pathogen Alternaria brassicicola, February 2006.

A. brassicicola, which causes black spot disease in cultivated Brassica,
is a destructive fungus that may lead to considerable leaf loss in
economically important crops including canola, cabbage and broccoli.

Genomic methods that allow the disruption of several thousand genes are
needed because they allow high-throughput identification of genes and gene
function. Such procedures are widely applicable and would be extremely
useful in allowing scientists to investigate the key events that occur when
a host interacts with a pathogen.

Associate Professor Christopher Lawrence of VBI, the director of the project
and one of the authors of the study, remarked: ?The development of this
protocol is timely as the genome sequence of A. brassicicola is scheduled
for completion in 2006. We now have in our hands a versatile method that
will allow us to dissect the pathogen?s nucleotide sequence information and
establish the function of many of the individual genes in this filamentous
fungus.?

He added: ?A. brassicicola has consistently been used in studies with the
weedy mustard plant Arabidopsis. The genome sequence of Arabidopsis was
determined in 2001 and many methods are available to ascertain gene function
in this plant. We now have a means to identify key fungal and plant genes
that interact and ultimately lead to disease development or resistance. This
is an extremely powerful research tool.?

The generation of gene disruption mutants has been a limiting step for the
analysis of gene function in most filamentous fungi. The new method takes
advantage of a novel linear DNA construct that greatly improves the
efficiency of targeted gene disruption. The DNA construct includes an
antibiotic-resistance marker gene, which allows for easy selection of the
new mutants, as well as a short partial target gene that integrates and
disrupts genes in the pathogen?s genome.

Richard Oliver, director of the Australian Centre for Necrotrophic Fungal
Pathogens and professor of Molecular Plant Pathology at Murdoch University,
Perth, commented: ?The new disruption method looks highly promising as a
tool for functional genomic studies. The authors looked at over 20 genes and
were able to produce transformants and inactivated genes or knock-outs in
each experiment. In most cases, the efficiency of gene disruption was 100%,
which represents a considerable improvement over previously reported methods
and makes large-scale functional analysis of individual genes feasible.?

Dr. Yangrae Cho of VBI, lead scientist and author of the paper, remarked:
?The high throughput system described in this study should allow for the
systematic analysis of large sets of candidate genes in A. brassicicola such
as those encoding cell-wall-degrading enzymes and other genes of interest in
pathogen-plant interactions.?

The new gene disruption method may also find applications in the study of
fungal pathogens that directly impact humans and human health. In addition
to causing numerous plant diseases, Alternaria are involved in the
development of chronic airway diseases such as asthma, allergy and chronic
rhinosinusitis. Gene disruption methods could help in identifying molecules
from the fungus that trigger inflammatory and other types of immune
responses in humans. By understanding how fungi modulate immune responses in
humans, new ways of developing therapeutics for these conditions could be
identified.

The work was funded by the National Science Foundation under grant number
0443991.

The research appears in vol.19, no.1, 2006, of the journal Molecular
Plant-Microbe Interactions, in the article "A high throughput targeted gene
disruption method for Alternaria brassicicola functional genomics using
linear minimal element (LME) constructs.?

Virginia Bioinformatics Institute (VBI) at Virginia Tech has a research
platform centered on understanding the ?disease triangle? of
host-pathogen-environment interactions in plants, humans and other animals.
By successfully channeling innovation into transdisciplinary approaches that
combine information technology and biology, researchers at VBI are
addressing some of today?s key challenges in the biomedical, environmental
and plant sciences.

[www.vbi.vt.edu]

------------------------------------------
Posted to Phorum via PhorumMail