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Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides.

ABSTRACT: 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.

SUBMITTER: McCormick SP

PROVIDER: S-EPMC91713 | biostudies-literature | 1999 Dec

REPOSITORIES: biostudies-literature

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Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides.

McCormick S P SP Alexander N J NJ Trapp S E SE Hohn T M TM

Applied and environmental microbiology 19991201 12

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 alte ...[more]

PMID: 10583973

Similar Datasets

Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides.

Project description:Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the "in vivo" characterization of Deltatri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.

| S-EPMC2762587 | biostudies-literature

Isolation and characterization of Tri3, a gene encoding 15-O-acetyltransferase from Fusarium sporotrichioides.

Project description: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.

| S-EPMC167806 | biostudies-other

The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis.

Project description: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.

| S-EPMC124697 | biostudies-literature

Tri16 is required for esterification of position C-8 during trichothecene mycotoxin production by Fusarium sporotrichioides.

Project description: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.

| S-EPMC201190 | biostudies-literature

Tri6 encodes an unusual zinc finger protein involved in regulation of trichothecene biosynthesis in Fusarium sporotrichioides.

Project description:In Fusarium sporotrichioides, several genes required for biosynthesis of the trichothecene mycotoxin T-2 toxin are closely linked. Further characterization of this gene cluster has revealed a gene, Tri6, that specifies a 217-amino-acid protein with regions similar to Cys2His2 zinc finger proteins. Temporal expression of Tri6 is similar to that of trichothecene biosynthetic pathway genes. Analysis of Tri6 transcripts indicated that transcription is initiated in two regions and that within each region there may be at least four initiation sites. Disruption of Tri6 resulted in a mutant that did not produce trichothecenes but that did accumulate low levels of the trichothecene precursor trichodiene. The Tri6 mutant was unable to convert six trichothecene biosynthetic intermediates to T-2 toxin, and transcription of two biosynthetic genes, Tri4 and Tri5, was greatly reduced in the mutant relative to the wild type. In addition, the product of Tri6 functioned as a transcriptional activator in Saccharomyces cerevisiae when fused to the DNA binding region of GAL4. These results indicate that Tri6 encodes a protein involved in the transcriptional regulation of trichothecene biosynthetic genes in F. sporotrichioides.

| S-EPMC167455 | biostudies-other

Tri1 encodes the cytochrome P450 monooxygenase for C-8 hydroxylation during trichothecene biosynthesis in Fusarium sporotrichioides and resides upstream of another new Tri gene.

Project description: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.

| S-EPMC150100 | biostudies-literature

Detoxification and Excretion of Trichothecenes in Transgenic Arabidopsisthaliana Expressing Fusarium graminearum Trichothecene 3-O-acetyltransferase.

Project description:Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces trichothecenes including deoxynivalenol (DON), nivalenol (NIV), and 3,7,15-trihydroxy-12,13-epoxytrichothec-9-ene (NX-3). These toxins contaminate grains and cause profound health problems in humans and animals. To explore exploiting a fungal self-protection mechanism in plants, we examined the ability of F. graminearum trichothecene 3-O-acetyltransferase (FgTri101) to detoxify several key trichothecenes produced by F. graminearum: DON, 15-ADON, NX-3, and NIV. FgTri101 was cloned from F. graminearum and expressed in Arabidopsis plants. We compared the phytotoxic effects of purified DON, NIV, and NX-3 on the root growth of transgenic Arabidopsis expressing FgTri101. Compared to wild type and GUS controls, FgTri101 transgenic Arabidopsis plants displayed significantly longer root length on media containing DON and NX-3. Furthermore, we confirmed that the FgTri101 transgenic plants acetylated DON to 3-ADON, 15-ADON to 3,15-diADON, and NX-3 to NX-2, but did not acetylate NIV. Approximately 90% of the converted toxins were excreted into the media. Our study indicates that transgenic Arabidopsis expressing FgTri101 can provide plant protection by detoxifying trichothecenes and excreting the acetylated toxins out of plant cells. Characterization of plant transporters involved in trichothecene efflux will provide novel targets to reduce FHB and mycotoxin contamination in economically important plant crops.

| S-EPMC8145220 | biostudies-literature

Fusarium sporotrichioides

Project description:EST sequencing

| PRJNA137 | ENA

Fusarium sporotrichioides

Project description:Fungus that produces toxic trichothecenes, such as T-2 toxin

| PRJNA9527 | ENA

The defense response in Arabidopsis thaliana against Fusarium sporotrichioides.

Project description:UNLABELLED: BACKGROUND:Certain graminaceous plants such as Zea mays and Triticum aestivum serve as hosts for Fusarium sporotrichioides; however, molecular interactions between the host plants and F. sporotrichioides remain unknown. It is also not known whether any interaction between Arabidopsis thaliana and F. sporotrichioides can occur. To understand these interactions, we performed proteomic analysis. RESULTS:Arabidopsis leaves and flowers were inoculated with F. sporotrichioides. Accumulation of PLANT DEFENSIN1.2 (PDF1.2) and PATHOGENESIS RELATED1 (PR1) mRNA in Arabidopsis were increased by inoculation of F. sporotrichioides. Furthermore, mitogen-activated protein kinase 3 (MPK3) and mitogen-activated protein kinase 6 (MPK6), which represent MAP kinases in Arabidopsis, were activated by inoculation of F. sporotrichioides. Proteomic analysis revealed that some defense-related proteins were upregulated, while the expression of photosynthesis- and metabolism-related proteins was down regulated, by inoculation with F. sporotrichioides. We carried out the proteomic analysis about upregulated proteins by inoculation with Fusarium graminearum. The glutathione S-transferases (GSTs), such as GSTF4 and GSTF7 were upregulated, by inoculation with F. graminearum-infected Arabidopsis leaves. On the other hand, GSTF3 and GSTF9 were uniquely upregulated, by inoculation with F. sporotrichioides. CONCLUSIONS:These results indicate that Arabidopsis is a host plant for F. sporotrichioides. We revealed that defense response of Arabidopsis is initiated by infection with F. sporotrichioides.

| S-EPMC3507649 | biostudies-literature

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