New method enables gene disruption in destructive fungal pathogen

Blacksburg, Va. 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. This destructive fungus causes black spot disease, leading to considerable leaf loss in such economically important crops as 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.

"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," said Christopher Lawrence, associate professor at VBI, director of the project, and one of the authors of the study.

"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," Lawrence said. "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 percent, which represents a considerable improvement over previously reported methods and makes large-scale functional analysis of individual genes feasible."

Yangrae Cho of VBI, lead scientist and author of the paper, said, "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 such chronic airway diseases 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.

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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.


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