Myrothecium roridum Tri4 encodes a multifunctional oxygenase required for three oxygenation steps

The biosyntheses of both macrocyclic trichothecenes in Myrothecium roridum and simple trichothecenes in Fusarium species begin with the cyclization of farnesyl pyrophosphate to form the sesquiterpene hydrocarbon trichodiene. A previous study showed that Myrothecium has a cluster of 3 genes that are homologous with Fusarium trichothecene genes: Tri4, a P450 oxygenase; Tri5, the sesquiterpene cyclase; and Tri6, a zinc-finger regulatory gene. Fusarium graminearum Tri4 (FgTri4) and M. roridum MrTri4 (MrTri4) have 66.9% identity. In this study, MrTri4 was expressed in Fusarium verticillioides. Liquid cultures of transformant strains expressing MrTri4 converted exogenous trichodiene to isotrichodiol, indicating that MrTri4 controls 3 oxygenation steps and that the product of MrTRI4 is isotrichodiol.     

Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis

Fusarium Tri4 encodes a cytochrome P450 monooxygenase (CYP) for hydroxylation at C-2 of the first committed intermediate trichodiene (TDN) in the biosynthesis of trichothecenes. To examine whether this CYP further participates in subsequent oxygenation steps leading to isotrichotriol (4), we engineered Saccharomyces cerevisiae for de novo production of the early intermediates by introducing cDNAs of Fusarium graminearum Tri5 (FgTri5 encoding TDN synthase) and Tri4 (FgTri4). From a culture of the engineered yeast grown on induction medium (final pH 2.7), we identified two intermediates, 2alpha-hydroxytrichodiene (1) and 12,13-epoxy-9,10-trichoene-2alpha-ol (2), and a small amount of non-Fusarium trichothecene 12,13-epoxytrichothec-9-ene (EPT). Other intermediates isotrichodiol (3) and 4 were identified in the transgenic yeasts grown on phosphate-buffered induction medium (final pH 5.5-6.0). When Trichothecium roseum Tri4 (TrTri4) was used in place of FgTri4, 4 was not detected in the culture. The three intermediates, 1, 2, and 3, were converted to 4,15-diacetylnivalenol (4,15-diANIV) when fed to a toxin-deficient mutant of F. graminearum with the FgTri4+ genetic background (viz., by introducing a FgTri5- mutation), but were not metabolized by an FgTri4- mutant. These results provide unambiguous evidence that FgTri4 encodes a multifunctional CYP for epoxidation at C-12,13, hydroxylation at C-11, and hydroxylation at C-3 in addition to hydroxylation at C-2.     

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.     

Tri1 in Fusarium graminearum encodes a P450 oxygenase

Gibberella zeae (asexual state Fusarium graminearum) is a major causal agent of wheat head blight and maize ear rot in North America and is responsible for contamination of grain with deoxynivalenol and related trichothecene mycotoxins. To identify additional trichothecene biosynthetic genes, cDNA libraries were prepared from fungal cultures under trichothecene-inducing conditions in culture and in planta. A gene designated LH1 that was highly expressed under these conditions exhibited only moderate (59%) similarity to known trichothecene biosynthetic cytochrome P450s. To determine the function of LH1, gene disruptants were produced and assessed for trichothecene production. Gene disruptants no longer produced 15-acetyldeoxynivalenol, which is oxygenated at carbon 7 (C-7) and C-8, but rather accumulated calonectrin and 3-deacetylcalonectrin, which are not oxygenated at either C-7 or C-8. These results indicate that gene LH1 encodes a cytochrome P450 responsible for oxygenation at one or both of these positions. Despite the relatively low level of DNA and amino acid sequence similarity between the two genes, LH1 from G. zeae is the probable homologue of Tri1, which encodes a cytochrome P450 required for C-8 oxygenation in F. sporotrichioides.     

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.     

Screening of putative oxygenase genes in the Fusarium graminearum genome sequence database for their role in trichothecene biosynthesis

In the biosynthesis of type B trichothecenes, four oxygenation steps remain to have genes functionally assigned to them. On the basis of the complete genome sequence of Fusarium graminearum, expression patterns of all oxygenase genes were investigated in Fusarium asiaticum (F. graminearum lineage 6). As a result, we identified five cytochrome P450 monooxygenase (CYP) genes that are specifically expressed under trichothecene-producing conditions and are unique to the toxin-producing strains. The entire coding regions of four of these genes were identified in F. asiaticum. When expressed in Saccharomyces cerevisiae, none of the oxygenases were able to transform trichodiene-11-one to expected products. However, one of the oxygenases catalyzed the 2beta-hydroxylation rather than the expected 2alpha-hydroxylation. Targeted disruption of the five CYP genes did not alter the trichothecene profiles of F. asiaticum. The results are discussed in relation to the presence of as-yet-unidentified oxygenation genes that are necessary for the biosynthesis of trichothecenes.     

4-O-acetylation and 3-O-acetylation of trichothecenes by trichothecene 15-O-acetyltransferase encoded by Fusarium Tri3

In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50 microM), we observed 3-O-acetylation activity against isotrichodermol that was more than 10(5) times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15-diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.     

Fusarium graminearum Tri12p influences virulence to wheat and trichothecene accumulation

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.     

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.