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

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.     

Tri1 Encodes the Cytochrome P450 Monooxygenase for C-8 Hydroxylation during Trichothecene Biosynthesis in Fusarium sporotrichioides and Resides Upstream of Another New Tri Gene

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.     

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

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.     

Fusarium Tri4 encodes a multifunctional oxygenase required for trichothecene biosynthesis

Fusarium graminearum and Fusarium sporotrichioides produce the trichothecene mycotoxins 15-acetyldeoxynivalenol and T-2 toxin, respectively. In both species, disruption of the P450 monooxygenase-encoding gene, Tri4, blocks production of the mycotoxins and leads to the accumulation of the trichothecene precursor trichodiene. To further characterize its function, the F. graminearum Tri4 (FgTri4) was heterologously expressed in the trichothecene-nonproducing species Fusarium verticillioides. Transgenic F. verticillioides carrying the FgTri4 converted exogenous trichodiene to the trichothecene biosynthetic intermediates isotrichodermin and trichothecene. Conversion of trichodiene to isotrichodermin requires seven biochemical steps. The fifth and sixth steps can occur nonenzymatically. Precursor feeding studies done in the current study indicate that wild-type F. verticillioides has the enzymatic activity necessary to carry out the seventh step, the C-3 acetylation of isotrichodermol to form isotrichodermin. Together, the results of this study indicate that the Tri4 protein catalyzes the remaining four steps and is therefore a multifunctional monooxygenase required for trichothecene biosynthesis.     

Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture

Fusarium head blight caused by Fusarium graminearum is a disease of cereal crops that not only reduces crop yield and quality but also results in contamination with trichothecenes such as nivalenol and deoxynivalenol (DON). To analyze the trichothecene induction mechanism, effects of 12 carbon sources on the production of DON and 3-acetyldexynivalenol (3ADON) were examined in liquid cultures incubated with nine strains of 3ADON-producing F. graminearum. Significantly high levels of trichothecene (DON and 3ADON) production by sucrose, 1-kestose and nystose were commonly observed among all of the strains tested. On the other hand, the levels of trichothecene biosynthesis induced by the other carbon sources were strain-specific. Tri4 and Tri5 expressions were up-regulated in the sucrose-containing medium but not in glucose. Trichothecene accumulation in the sucrose-containing medium was not repressed by the addition of glucose, indicating that trichothecene production was not regulated by carbon catabolite repression. These findings suggest that F. graminearum recognizes sucrose molecules, activates Tri gene expression and induces trichothecene biosynthesis.     

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.     

Tri16 Is Required for Esterification of Position C-8 during Trichothecene Mycotoxin Production by Fusarium sporotrichioides

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.     

Fusarium Tri8 encodes a trichothecene C-3 esterase

Mutant strains of Fusarium graminearum Z3639 produced by disruption of Tri8 were altered in their ability to biosynthesize 15-acetyldeoxynivalenol and instead accumulated 3,15-diacetyldeoxynivalenol, 7,8-dihydroxycalonectrin, and calonectrin. Fusarium sporotrichioides NRRL3299 Tri8 mutant strains accumulated 3-acetyl T-2 toxin, 3-acetyl neosolaniol, and 3,4,15-triacetoxyscirpenol rather than T-2 toxin, neosolaniol, and 4,15-diacetoxyscirpenol. The accumulation of these C-3-acetylated compounds suggests that Tri8 encodes an esterase responsible for deacetylation at C-3. This gene function was confirmed by cell-free enzyme assays and feeding experiments with yeast expressing Tri8. Previous studies have shown that Tri101 encodes a C-3 transacetylase that acts as a self-protection or resistance factor during biosynthesis and that the presence of a free C-3 hydroxyl group is a key component of Fusarium trichothecene phytotoxicity. Since Tri8 encodes the esterase that removes the C-3 protecting group, it may be considered a toxicity factor.     

The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes

We report for the first time the complete structure and sequence of the trichothecene biosynthesis gene cluster (i.e. Tri5-cluster) from Fusarium graminearum F15, a strain that produces 3-acetyldeoxynivalenol (3-ADON). A putative tyrosinase and polysaccharide deacetylase gene flank the Tri5-cluster: the number of pathway genes between them is less than half the total number of steps necessary for 3-ADON biosynthesis. In comparison with partial Tri5-cluster sequences of strains with 15-acetyldeoxynivalenol and 4-acetylnivalenol chemotypes, the Tri5-cluster from strain F15 contains three genes that are apparently unnecessary for the biosynthesis of 3-ADON (i.e. Tri8 and Tri3, which are expressed, and pseudo-Tri13, which is not expressed). In addition, the Tri7 gene was missing from the cluster. Recombinant TRI3 protein showed limited trichothecene C-15 acetylase activity. In contrast, recombinant TRI8 protein displayed no C-3 deacetylase activity, suggesting that the loss or alteration of function contribute directly to the chemotype difference.     

Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene in Fusarium sporotrichioides and Gibberella pulicaris.

Ancymidol, a plant growth regulator, inhibited biosynthesis of diacetoxyscirpenol by Gibberella pulicaris (Fusarium sambucinum) in a defined liquid medium. Ancymidol also inhibited biosynthesis of T-2 toxin by a wild-type strain of Fusarium sporotrichioides and biosynthesis of diacetoxyscirpenol, deacetylated calonectrin, and dideacetylated calonectrin by mutant strains of this species. Ancymidol-treated cultures accumulated the hydrocarbon trichodiene, a biosynthetic precursor of the trichothecenes. Ancymidol did not block trichodiene accumulation by a trichodiene-producing mutant strain of F. sporotrichioides. Ancymidol appears to block the trichothecene biosynthetic pathway after formation of trichodiene and before formation of trichothecenes containing four or more oxygen atoms.     

Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene in Fusarium sporotrichioides and Gibberella pulicaris

Ancymidol, a plant growth regulator, inhibited biosynthesis of diacetoxyscirpenol by Gibberella pulicaris (Fusarium sambucinum) in a defined liquid medium. Ancymidol also inhibited biosynthesis of T-2 toxin by a wild-type strain of Fusarium sporotrichioides and biosynthesis of diacetoxyscirpenol, deacetylated calonectrin, and dideacetylated calonectrin by mutant strains of this species. Ancymidol-treated cultures accumulated the hydrocarbon trichodiene, a biosynthetic precursor of the trichothecenes. Ancymidol did not block trichodiene accumulation by a trichodiene-producing mutant strain of F. sporotrichioides. Ancymidol appears to block the trichothecene biosynthetic pathway after formation of trichodiene and before formation of trichothecenes containing four or more oxygen atoms.     

Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum

Fusarium head blight is one of the most important diseases of wheat worldwide due to crop losses and the contamination of grains with trichothecene mycotoxins. The biosynthesis of trichothecenes by Fusarium spp. is highest during infection, but relatively low levels are produced from saprophytic growth in axenic culture. A strain of Fusarium graminearum was constructed where the promoter from the TRI5 trichothecene biosynthesis gene was fused to GFP. Using this strain in large-scale nutrient profiling, a variety of amines were identified that significantly induce TRI5 expression. Analysis of trichothecene levels in the culture filtrates revealed accumulation of the toxin to over 1000 ppm in response to these inducers, levels either greater than or equivalent to those observed during infection. From this work, we propose that products of the arginine-polyamine biosynthetic pathway in plants may play a role in the induction of trichothecene biosynthesis during infection.