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Susan P. Mccormick - One of the best experts on this subject based on the ideXlab platform.
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structural and functional characterization of the tri101 trichothecene 3 o acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum kinetic insights to combating Fusarium head blight
Journal of Biological Chemistry, 2008Co-Authors: Graeme S. Garvey, Susan P. Mccormick, Ivan RaymentAbstract:Abstract Fusarium head blight (FHB) is a plant disease with serious economic and health impacts. It is caused by fungal species belonging to the genus Fusarium and the mycotoxins they produce. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by ∼100-fold but has demonstrated limited resistance to FHB in transgenic cereal. To understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes toward B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three-dimensional structures for the ternary complexes for both of these enzymes with coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein.
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Effects of xanthotoxin treatment on trichothecene production in Fusarium sporotrichioides.
Canadian journal of microbiology, 2008Co-Authors: Nancy J. Alexander, Susan P. Mccormick, Judith A. BlackburnAbstract:There are 4 P450 oxygenases involved in the biosynthesis of T-2 toxin in Fusarium sporotrichioides. Exactly how these enzymes react to antimicrobial plant defense compounds is unknown. Xanthotoxin (8-methoxypsoralen) is a phototoxic furanocoumarin that acts as a P450 oxygenase inhibitor. The current study shows that the addition of concentrations of 1.0 mmol/L or less of xanthotoxin to liquid cultures of F. sporotrichioides NRRL3299 can effectively block T-2 toxin production and cause an increase in accumulation of trichodiene, the hydrocarbon precursor of trichothecenes. The addition of xanthotoxin to liquid cultures of a trichodiene-accumulating F. sporotrichioides Tri4– mutant caused a 3- to 10-fold increase in trichodiene accumulation, suggesting that xanthotoxin not only blocks trichothecene oxygenation reactions, but may in some way also promote the synthesis of trichodiene. Feeding studies showed that 2 of the 4 P450 oxygenases, TRI4 and TRI1, were more sensitive to xanthotoxin, while oxygenases TR...
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Structural and functional characterization of the TRI101 trichothecene 3-O-acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum: Kinetic insights to combating Fusarium head blight
The Journal of biological chemistry, 2007Co-Authors: Graeme S. Garvey, Susan P. Mccormick, Ivan RaymentAbstract:Fusarium head blight (FHB) is a plant disease with serious economic and health impacts. It is caused by fungal species belonging to the genus Fusarium and the mycotoxins they produce. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by approximately 100-fold but has demonstrated limited resistance to FHB in transgenic cereal. To understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes toward B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three-dimensional structures for the ternary complexes for both of these enzymes with coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein.
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Expression of Tri15 in Fusarium sporotrichioides
Current Genetics, 2004Co-Authors: Nancy J. Alexander, Susan P. Mccormick, Troy M. Larson, James E. JurgensonAbstract:In the fungus Fusarium sporotrichioides , biosynthesis of trichothecene mycotoxins requires at least three genetic loci: a core 12-gene cluster, a smaller two-gene cluster, and a single-gene locus. Here, we describe the Tri15 gene, which represents a fourth locus involved in trichothecene biosynthesis. Tri15 is predicted to encode a Cys_2-His_2 zinc finger protein and is expressed in a manner similar to genes in the core trichothecene gene cluster. However, disruption of F. sporotrichioides Tri15 does not affect production of T-2 toxin, the major trichothecene produced by this fungus. This result suggests that Tri15 is not necessary for the production of toxin. Cultures with exogenously added T-2 toxin have high levels of Tri15 expression and no detectable expression of the trichothecene biosynthetic genes Tri5 and Tri6 . The expression analysis is consistent with Tri15 being a negative regulator of at least some of the trichothecene biosynthetic genes. In F. graminearum , Tri15 has been mapped to linkage group 2 and is therefore unlinked to the main trichothecene biosynthetic gene cluster.
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A Genetic and Biochemical Approach to Study Trichothecene Diversity in Fusarium sporotrichioides and Fusarium graminearum
Fungal genetics and biology : FG & B, 2001Co-Authors: Daren W. Brown, Nancy J. Alexander, Susan P. Mccormick, Robert H. Proctor, Anne E. DesjardinsAbstract:The trichothecenes T-2 toxin and deoxynivalenol (DON) are natural fungal products that are toxic to both animals and plants. Their importance in the pathogenicity of Fusarium spp. on crop plants has inspired efforts to understand the genetic and biochemical mechanisms leading to trichothecene synthesis. In order to better understand T-2 toxin biosynthesis by Fusarium sporotrichioides and DON biosynthesis by F. graminearum, we compared the nucleotide sequence of the 23-kb core trichothecene gene cluster from each organism. This comparative genetic analysis allowed us to predict proteins encoded by two trichothecene genes, TRI9 and TRI10, that had not previously been described from either Fusarium species. Differences in gene structure also were correlated with differences in the types of trichothecenes that the two species produce. Gene disruption experiments showed that F. sporotrichioides TRI7 (FsTRI7) is required for acetylation of the oxygen on C-4 of T-2 toxin. Sequence analysis indicated that F. graminearum TRI7 (FgTRI7) is nonfunctional. This is consistent with the fact that the FgTRI7 product is not required for DON synthesis in F. graminearum because C-4 is not oxygenated.
Thomas M. Hohn - One of the best experts on this subject based on the ideXlab platform.
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Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides.
Applied and environmental microbiology, 1999Co-Authors: Susan P. Mccormick, Nancy J. Alexander, Susan E. Trapp, Thomas M. HohnAbstract:We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.
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The TRI11 Gene of Fusarium sporotrichioides Encodes a Cytochrome P-450 Monooxygenase Required for C-15 Hydroxylation in Trichothecene Biosynthesis
Applied and environmental microbiology, 1998Co-Authors: Nancy J. Alexander, Thomas M. Hohn, Susan P. MccormickAbstract:Several genes in the trichothecene biosynthetic pathway of Fusarium sporotrichioides have been shown to reside in a gene cluster. Sequence analysis of a cloned DNA fragment located 3.8 kb downstream from TRI5 has led to the identification of the TRI11 gene. The nucleotide sequence of TRI11 predicts a polypeptide of 492 residues (Mr = 55,579) with significant similarity to members of the cytochrome P-450 superfamily. TRI11 is most similar to several fungal cytochromes P-450 (23 to 27% identity) but is sufficiently distinct to define a new cytochrome P-450 gene family, designated CYP65A1. Disruption of TRI11 results in an altered trichothecene production phenotype characterized by the accumulation of isotrichodermin, a trichothecene pathway intermediate. The evidence suggests that TRI11 encodes a C-15 hydroxylase involved in trichothecene biosynthesis.
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Accumulation of Trichothecenes in Liquid Cultures of a Fusarium sporotrichioides Mutant Lacking a Functional Trichothecene C-15 Hydroxylase
Applied and environmental microbiology, 1997Co-Authors: Susan P. Mccormick, Thomas M. HohnAbstract:A mutant strain of Fusarium sporotrichioides NRRL 3299 produced by disruption of Tri11, a gene encoding a cytochrome P-450 monooxygenase, was shown to be altered in its ability to biosynthesize T-2 toxin. This mutant strain produced four trichothecenes that were not observed in cultures of the parent strain. The compounds were identified as isotrichodermin, 8-hydroxyisotrichodermin, 8-hydroxyisotrichodermol, and 3,4,8-trihydroxytricothecene on the basis of their nuclear magnetic resonance and mass spectra. This is the first report of these 8-hydroxytrichothecenes as metabolites of F. sporotrichioides. The accumulation of isotrichodermin and the results of whole-cell feeding experiments with a Tri11(sup-) strain confirm that oxygenation of C-15 is blocked.
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Structural characterization of 15-hydroxytrichodiene, a sesquiterpenoid produced by transformed tobacco cell suspension cultures expressing a trichodiene synthase gene from Fusarium sporotrichioides.
Phytochemistry, 1996Co-Authors: Michael Zook, Thomas M. Hohn, Kermit M. Johnson, Raymond HammerschmidtAbstract:Tobacco (Nicotiana tabaccum) cell suspension cultures transformed with a gene encoding trichodiene synthase, a sesquiterpene synthase from the fungus Fusarium sporotrichioides, produced a novel sesquiterpenoid derived from the in vivo production of trichodiene. Mass and nuclear magnetic resonance spectroscopic analyses identified the new compound as 15-hydroxytrichodiene. The in vivo hydroxylation of trichodiene by transformant tobacco cell suspension cultures demonstrates that the introduction of a foreign sesquiterpene synthase gene can result in the production of novel sesquiterpenoid metabolites.
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Isolation and characterization of Tri3, a gene encoding 15-O-acetyltransferase from Fusarium sporotrichioides.
Applied and environmental microbiology, 1996Co-Authors: Susan P. Mccormick, Thomas M. Hohn, Andanne E. DesjardinsAbstract:An acetyltransferase gene (Tri3) was isolated from Fusarium sporotrichioides by complementation of a previously identified Tri3- mutant and shown to be closely linked to three other trichothecene biosynthetic pathway genes. Comparison of the Tri3 sequence with its cDNA revealed the presence of four introns. The Tri3 cDNA contains a 1,539-bp open reading frame that encodes a protein with a molecular mass of 57,418 Da. Regulation of Tri3 transcription in liquid cultures appeared identical to that of other trichothecene pathway genes. Disruption of the Tri3 gene resulted in the accumulation of deacetylated calonectrins rather than T-2 toxin. The results of whole-cell feeding experiments with Tri3- strains suggested that 15-O-acetylation is blocked. Cell-free feeding experiments confirmed that Tri3- strains are able to acetylate a trichothecene C-3 hydroxyl group but are unable to acetylate a trichothecene C-15 hydroxyl group. Our results show that Tri3 encodes an acetyltransferase that converts 15-decalonectrin to calonectrin.
Nancy J. Alexander - One of the best experts on this subject based on the ideXlab platform.
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Effects of xanthotoxin treatment on trichothecene production in Fusarium sporotrichioides.
Canadian journal of microbiology, 2008Co-Authors: Nancy J. Alexander, Susan P. Mccormick, Judith A. BlackburnAbstract:There are 4 P450 oxygenases involved in the biosynthesis of T-2 toxin in Fusarium sporotrichioides. Exactly how these enzymes react to antimicrobial plant defense compounds is unknown. Xanthotoxin (8-methoxypsoralen) is a phototoxic furanocoumarin that acts as a P450 oxygenase inhibitor. The current study shows that the addition of concentrations of 1.0 mmol/L or less of xanthotoxin to liquid cultures of F. sporotrichioides NRRL3299 can effectively block T-2 toxin production and cause an increase in accumulation of trichodiene, the hydrocarbon precursor of trichothecenes. The addition of xanthotoxin to liquid cultures of a trichodiene-accumulating F. sporotrichioides Tri4– mutant caused a 3- to 10-fold increase in trichodiene accumulation, suggesting that xanthotoxin not only blocks trichothecene oxygenation reactions, but may in some way also promote the synthesis of trichodiene. Feeding studies showed that 2 of the 4 P450 oxygenases, TRI4 and TRI1, were more sensitive to xanthotoxin, while oxygenases TR...
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Expression of Tri15 in Fusarium sporotrichioides
Current Genetics, 2004Co-Authors: Nancy J. Alexander, Susan P. Mccormick, Troy M. Larson, James E. JurgensonAbstract:In the fungus Fusarium sporotrichioides , biosynthesis of trichothecene mycotoxins requires at least three genetic loci: a core 12-gene cluster, a smaller two-gene cluster, and a single-gene locus. Here, we describe the Tri15 gene, which represents a fourth locus involved in trichothecene biosynthesis. Tri15 is predicted to encode a Cys_2-His_2 zinc finger protein and is expressed in a manner similar to genes in the core trichothecene gene cluster. However, disruption of F. sporotrichioides Tri15 does not affect production of T-2 toxin, the major trichothecene produced by this fungus. This result suggests that Tri15 is not necessary for the production of toxin. Cultures with exogenously added T-2 toxin have high levels of Tri15 expression and no detectable expression of the trichothecene biosynthetic genes Tri5 and Tri6 . The expression analysis is consistent with Tri15 being a negative regulator of at least some of the trichothecene biosynthetic genes. In F. graminearum , Tri15 has been mapped to linkage group 2 and is therefore unlinked to the main trichothecene biosynthetic gene cluster.
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A Genetic and Biochemical Approach to Study Trichothecene Diversity in Fusarium sporotrichioides and Fusarium graminearum
Fungal genetics and biology : FG & B, 2001Co-Authors: Daren W. Brown, Nancy J. Alexander, Susan P. Mccormick, Robert H. Proctor, Anne E. DesjardinsAbstract:The trichothecenes T-2 toxin and deoxynivalenol (DON) are natural fungal products that are toxic to both animals and plants. Their importance in the pathogenicity of Fusarium spp. on crop plants has inspired efforts to understand the genetic and biochemical mechanisms leading to trichothecene synthesis. In order to better understand T-2 toxin biosynthesis by Fusarium sporotrichioides and DON biosynthesis by F. graminearum, we compared the nucleotide sequence of the 23-kb core trichothecene gene cluster from each organism. This comparative genetic analysis allowed us to predict proteins encoded by two trichothecene genes, TRI9 and TRI10, that had not previously been described from either Fusarium species. Differences in gene structure also were correlated with differences in the types of trichothecenes that the two species produce. Gene disruption experiments showed that F. sporotrichioides TRI7 (FsTRI7) is required for acetylation of the oxygen on C-4 of T-2 toxin. Sequence analysis indicated that F. graminearum TRI7 (FgTRI7) is nonfunctional. This is consistent with the fact that the FgTRI7 product is not required for DON synthesis in F. graminearum because C-4 is not oxygenated.
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Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides.
Applied and environmental microbiology, 1999Co-Authors: Susan P. Mccormick, Nancy J. Alexander, Susan E. Trapp, Thomas M. HohnAbstract:We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.
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TRI12, a trichothecene efflux pump from Fusarium sporotrichioides : gene isolation and expression in yeast
Molecular & general genetics : MGG, 1999Co-Authors: Nancy J. Alexander, Susan P. Mccormick, T. M. HohnAbstract:Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides are present within a gene cluster. Here we report the complete sequence for TRI12, a gene encoding a trichothecene efflux pump that is located within the trichothecene gene cluster of F. sporotrichioides. TRI12 encodes a putative polypeptide of 598 residues with sequence similarities to members of the major facilitator superfamily (MFS) and is predicted to contain 14 transmembrane-spanning segments. Disruption of TRI12 results in both reduced growth on complex media and reduced levels of trichothecene production. Growth of tri12 mutants on trichothecene-containing media is inhibited, suggesting that TRI12 may play a role in F. sporotrichioides self-protection against trichothecenes. Functional analysis of TRI12 was performed by expressing it in yeast strains that were co-transformed with a gene (TRI3) encoding a trichothecene 15-O-acetyltransferase. In the presence of the TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TRI3 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TRI3 alone. PDR5, a transporter of the ABC superfamily, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TRI3 strains. These results confirm the involvement of TRI12 in the trichothecene efflux associated with toxin biosynthesis, and demonstrate the usefulness of yeast as a host system for studies of MFS-type transporters.
Chris M Maragos - One of the best experts on this subject based on the ideXlab platform.
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observation of t 2 toxin and ht 2 toxin glucosides from Fusarium sporotrichioides by liquid chromatography coupled to tandem mass spectrometry lc ms ms
Toxins, 2011Co-Authors: M Busman, Stephen M Poling, Chris M MaragosAbstract:The trichothecenes produced by solid and liquid cultures of Fusarium sporotrichioides were evaluated with high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Along with the expected T-2 toxin HT-2 toxin and neosolaniol, two additional compounds were detected, which had ions 162 m/z higher than those in the mass spectra of T-2 toxin or HT-2 toxin. Fragmentation behavior of these two compounds was similar to that of T-2 toxin and HT-2 toxin. Based on LC-MS/MS behavior, it is proposed that the two compounds are T-2 toxin 3-O-glucoside and HT-2 toxin 3-O-glucoside. Production of the two glucosides was measured in kernels from wheat and oat inoculated with F. sporotrichiodes, as well as in cultures grown in liquid media and on cracked corn or rice. Production of glucosides in wheat and oats suggest that they may also be present in naturally contaminated cereals.
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Observation of T-2 toxin and HT-2 toxin glucosides from Fusarium sporotrichioides by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
Toxins, 2011Co-Authors: M Busman, Stephen M Poling, Chris M MaragosAbstract:The trichothecenes produced by solid and liquid cultures of Fusarium sporotrichioides were evaluated with high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Along with the expected T-2 toxin HT-2 toxin and neosolaniol, two additional compounds were detected, which had ions 162 m/z higher than those in the mass spectra of T-2 toxin or HT-2 toxin. Fragmentation behavior of these two compounds was similar to that of T-2 toxin and HT-2 toxin. Based on LC-MS/MS behavior, it is proposed that the two compounds are T-2 toxin 3-O-glucoside and HT-2 toxin 3-O-glucoside. Production of the two glucosides was measured in kernels from wheat and oat inoculated with F. sporotrichiodes, as well as in cultures grown in liquid media and on cracked corn or rice. Production of glucosides in wheat and oats suggest that they may also be present in naturally contaminated cereals.
Marian N. Beremand - One of the best experts on this subject based on the ideXlab platform.
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Tri16 is required for esterification of position C-8 during trichothecene mycotoxin production by Fusarium sporotrichioides.
Applied and Environmental Microbiology, 2003Co-Authors: Andrew W. Peplow, Isaac B. Meek, Melinda C. Wiles, Timothy D. Phillips, Marian N. BeremandAbstract:We previously characterized Tri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis in Fusarium sporotrichioides NRRL 3299. Sequence analysis of the region surrounding Tri1 revealed a gene, named Tri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption of Tri16 were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate that Tri16 encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of a Tri16 ortholog in Gibberella pulicaris R-6380 that is likely linked to a presumably inactive ortholog for Tri1.
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Tri1 Encodes the Cytochrome P450 Monooxygenase for C-8 Hydroxylation during Trichothecene Biosynthesis in Fusarium sporotrichioides and Resides Upstream of Another New Tri Gene
Applied and environmental microbiology, 2003Co-Authors: Isaac B. Meek, Andrew W. Peplow, Timothy D. Phillips, C. L. Ake, Marian N. BeremandAbstract:Many Fusarium species produce one or more agriculturally important trichothecene mycotoxins, and the relative level of toxicity of these compounds is determined by the pattern of oxygenations and acetylations or esterifications on the core trichothecene structure. Previous studies with UV-induced Fusarium sporotrichioides NRRL 3299 trichothecene mutants defined the Tri1 gene and demonstrated that it was required for addition of the oxygen at the C-8 position during trichothecene biosynthesis. We have cloned and characterized the Tri1 gene from NRRL 3299 and found that it encodes a cytochrome P450 monooxygenase. The disruption of Tri1 blocks production of C-8-oxygenated trichothecenes and leads to the accumulation of 4,15-diacetoxyscirpenol, the same phenotype observed in the tri1 UV-induced mutants MB1716 and MB1370. The Tri1 disruptants and the tri1 UV-induced mutants do not complement one another when coinoculated, and the Tri1 gene sequence restores T-2 toxin production in both MB1716 and MB1370. The DNA sequence flanking Tri1 contains another new Tri gene. Thus, Tri1 encodes a C-8 hydroxylase and is located either in a new distal portion of the trichothecene gene cluster or in a second separate trichothecene gene cluster.
