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Title:   Author  item
                                            KHATIBI, PIYUM - Virginia Polytechnic Institution & State Universityitem
                                            NEWMISTER, SEAN - University Of Wisconsinitem
                                            RAYMENT, IVAN - University Of Wisconsinitem
                                            McCormick, Susanitem
                                            Alexander, Nancyitem
                                            SCHMALE, DAVID - Virginia Polytechnic Institution & State University
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 12/4/2010
                                    Publication  Date: 2/1/2011
                                    Citation: Khatibi, P.A., Newmister, S.A., Rayment, I., McCormick, S.P., Alexander, N.J., Schmale, III, D.G. 2011. Bioprospecting for trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes with different abilities to modify the mycotoxin deoxynivalenol. Applied and Environmental Microbiology. 77(4):1162-1170.Interpretive Summary: In this research, we looked at different forms of a gene that may improve resistance to wheat head blight by more effectively locking up associated toxins. Fusarium Head Blight is a disease of cereal crops, caused by a fungus that produces toxins called trichothecenes. The disease has serious economic and health impacts, and these toxins are factors in the severity of the disease. One strategy that has been used to combat the disease is to introduce a Fusarium sporotrichioides gene, Tri101, which protects this fungus from its own toxins, into cereals such as wheat and barley. We found that the enzyme product of the Tri101 gene in other Fusarium species is up to 70 times better than the Fusarium sporotrichioides gene at modifying the toxin most commonly associated with U.S. outbreaks of wheat head blight. Wheat breeders and seed companies may be able to improve resistance to wheat head blight by utilizing this gene product in wheat.
                                Technical Abstract: The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activity of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activity of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species (11 strains) of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyldeoxynivalenol (3ADON) ranged from 63% to 99%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the slowest rate. Steady-state kinetic assays using seven recombinant, purified, orthologs produced catalytic efficiencies for DON acetylation ranging from 6.8 x 10(4) M-1-s-1 to 4.7 x 10(6) M-1-s-1. Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from over 30 species of Fusarium (including known trichothecene producers and non-producers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study.


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