Scientists at the Virginia Bioinformatics Institute and Duke University Medical Center have pinpointed a fungal gene that appears to play an important role in the development and virulence of Alternaria brassicicola. A. brassicicola, a destructive fungal pathogen that causes black spot disease on most cultivated Brassica crops worldwide, results in considerable leaf loss in many economically important crops including canola, cabbage and broccoli. Sensitivity to spores of Alternaria species is also clinically associated with human respiratory disorders such as allergy, asthma, and chronic sinusitis.
Spores, which are often termed conidia in some fungi, are an essential part of the developmental cycle of A. brassicicola and arise from the branching filaments or hyphae that make up the fungus. In the study, the investigators show that disruption of the AbNPS2 gene drastically impacts the integrity of the cell wall of fungal spores produced in the reproductive phase of A. brassicicola's life cycle. The AbNPS2 gene most likely directs the synthesis of a molecule that plays an essential role in maintaining the structure of the cell wall of the conidia.
Associate Professor Christopher Lawrence of the Virginia Bioinformatics Institute and the Department of Biological Sciences at Virginia Tech, director of the study, remarked: "Typical A. brassicicola spores are hydrophobic. Water droplets placed on a lawn of fungal hyphae bearing normal spores are repelled and easily roll off the surface. When the AbNPS2 gene is disrupted, the linkage of the outermost layer of the fungal spore cell wall to the middle layer appears to be disturbed, destroying the cell wall's regular architecture and making the spores permeable to water. This has drastic effects on the viability of the spores."
Kwang-Hyung Kim, doctoral student and the lead author on the paper, stated: "What we have been able to show is that mutation of the AbNPS2 gene is accompanied by structural changes that occur in the cell wall of the spores, a decrease in spore germination rate, lower survival rates for the spores under adverse environmental conditions, and a reduced ability of the fungus to damage the host plant. These observations may open up a route to develop new and innovative research strategies aimed at understanding the host-pathogen interaction for this destructive plant pathogen."
The investigators used a wide range of experimental approaches to look in detail at the link between the function of the gene and its impact on the structure of the spore cell wall. A recently developed gene disruption method was used to generate the fungal mutants. Bioinformatic and gene prediction tools were applied to probe the structure and organization of the AbNPS2 gene and the surrounding region in the recently sequenced A. brassicicola genome, a collaborative project nearing completion with Washington University Genome Sequencing Center in St. Louis. Electron microscopy revealed some of the dramatic structural changes in the cell wall arising from disruption of the gene.
Dr. Nancy Keller, Professor in the Department of Plant Pathology at the University of Wisconsin, Madison and international expert in fungal secondary metabolism, commented: "The finding that a non-ribosomal peptide synthetase is integral to conidial morphology further illustrates the versatile and essential role of these secondary metabolites in fungal biology. For years long ignored, the function of natural products is rapidly becoming one of the hot topics in fungal biology; the findings reported in this study by Dr. Lawrence's research group further underline their importance."
The AbNPS2 gene encodes a large protein known as a non-ribosomal peptide synthetase. This protein directs the synthesis of secondary metabolites known as non-ribosomal peptides. However, the functions of many of these proteins and the subsequent synthesized metabolites are largely unknown. Dr. Lawrence added: "To the best of our knowledge, this is the first report that a fungal non-ribosomal peptide synthetase is associated with cell wall construction in fungal spores. The putative secondary metabolite produced by the protein encoded by this gene could serve as a physical bridge in the layers of the cell wall or function as a regulator of cell wall biosynthesis. Future work will focus on identifying the role of the product of the AbNPS2 gene which should allow us to get an important handle on the precise series of molecular events that give rise to these drastic effects on spore viability."
The work was funded by the National Science Foundation and the United States Department of Agriculture.
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