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.     

Isolation and characterization of mutants blocked in T-2 toxin biosynthesis

Mutants of Fusarium sporotrichioides NRRL 3299 that were blocked or altered in the biosynthesis of the trichothecene T-2 toxin were generated by UV treatment and identified by a rapid screen in which monoclonal antibodies to T-2 were used. Three stable mutants were isolated and chemically characterized. Two mutants accumulated diacetoxyscirpenol, which suggests that they were defective in the step required for the addition of a hydroxyl group to the C-8 position in the trichothecene core structure. The third mutant appeared to be partially blocked at an early step or regulatory point in the pathway. This represents the first isolation of mutants in a trichothecene biosynthetic pathway.     

Isolation and characterization of mutants blocked in T-2 toxin biosynthesis.

Mutants of Fusarium sporotrichioides NRRL 3299 that were blocked or altered in the biosynthesis of the trichothecene T-2 toxin were generated by UV treatment and identified by a rapid screen in which monoclonal antibodies to T-2 were used. Three stable mutants were isolated and chemically characterized. Two mutants accumulated diacetoxyscirpenol, which suggests that they were defective in the step required for the addition of a hydroxyl group to the C-8 position in the trichothecene core structure. The third mutant appeared to be partially blocked at an early step or regulatory point in the pathway. This represents the first isolation of mutants in a trichothecene biosynthetic pathway.     

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.     

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

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.     

Bioconversion of possible T-2 toxin precursors by a mutant strain of Fusarium sporotrichioides NRRL 3299.

Liquid cultures of a mutant strain of Fusarium sporotrichioides NRRL 3299 that accumulates trichodiene rather than T-2 toxin converted tricho-9-ene-2 alpha,3 alpha,11 alpha-triol, trichotriol (tricho-10-ene-2 alpha,3 alpha,9 alpha-triol), tricho-10-ene-2 alpha,3 alpha,9 beta-triol, 3 alpha-hydroxytrichothecene, and 3 alpha-acetoxytrichothecene to T-2 toxin. Other possible oxygenated precursors of T-2 toxin, including trichodiol (tricho-10-ene-2 alpha,9 alpha-diol), trichothecene, 4 alpha-hydroxytrichothecene, and 15-hydroxytrichothecene, were not metabolized. The results indicate that in the biosynthesis of T-2 toxin by F. sporotrichioides, (i) oxygenation at C-3 occurs prior to the second cyclization, (ii) this second cyclization involves two steps that may be nonenzymatic, and (iii) oxidation at C-3 precedes that at C-4 or C-15.     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil.

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Bioconversion of possible T-2 toxin precursors by a mutant strain of Fusarium sporotrichioides NRRL 3299

Liquid cultures of a mutant strain of Fusarium sporotrichioides NRRL 3299 that accumulates trichodiene rather than T-2 toxin converted tricho-9-ene-2 alpha,3 alpha,11 alpha-triol, trichotriol (tricho-10-ene-2 alpha,3 alpha,9 alpha-triol), tricho-10-ene-2 alpha,3 alpha,9 beta-triol, 3 alpha-hydroxytrichothecene, and 3 alpha-acetoxytrichothecene to T-2 toxin. Other possible oxygenated precursors of T-2 toxin, including trichodiol (tricho-10-ene-2 alpha,9 alpha-diol), trichothecene, 4 alpha-hydroxytrichothecene, and 15-hydroxytrichothecene, were not metabolized. The results indicate that in the biosynthesis of T-2 toxin by F. sporotrichioides, (i) oxygenation at C-3 occurs prior to the second cyclization, (ii) this second cyclization involves two steps that may be nonenzymatic, and (iii) oxidation at C-3 precedes that at C-4 or C-15.     

Accumulation of Trichothecenes in Liquid Cultures of a Fusarium sporotrichioides Mutant Lacking a Functional Trichothecene C-15 Hydroxylase

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.     

Heterologous expression of two trichothecene P450 genes in Fusarium verticillioides

Fusarium graminearum Z-3639 and F. sporotrichioides NRRL3299 produce the trichothecene mycotoxins 15-acetyldeoxynivalenol and T-2 toxin, respectively. These toxins differ in oxygenation at C-4, C-7, and C-8. In F. sporotrichioides, Tri1 (FsTri1) controls C-8 hydroxylation. To determine the function of an apparent F. graminearum Tri1 (FgTri1) homolog, both FsTri1 and FgTri1 genes were heterologously expressed in the trichothecene-nonproducing species F. verticillioides by fusing the Tri1 coding regions to the promoter of the fumonisin biosynthetic gene FUM8. FsTri1 and FgTri1 have been partially characterized by disruption analysis, and the results from these analyses suggest that FsTri1 most likely has a single function but that FgTri1 may have two functions. Transgenic F. verticillioides carrying the FsTri1 (FvF8FsTri1) converted exogenous isotrichodermin and calonectrin to 8-hydroxyisotrichodermin and 8-hydroxycalonectrin, respectively. Transgenic F. verticillioides carrying FgTri1 (FvF8FgTri1) converted isotrichodermin to a mixture of 7-hydroxyisotrichodermin and 8-hydroxyisotrichodermin but converted calonectrin to a mixture of 7-hydroxycalonectrin, 8-hydroxycalonectrin, and 3,15-diacetyldeoxynivalenol. A fourth compound, 7,8-dihydroxycalonectrin, was identified in large-scale F. verticillioides FvF8FgTri1 cultures fed isotrichodermin. Our results indicate that FgTri1 controls both C-7 and C-8 hydroxylation but that FsTri1 controls only C-8 hydroxylation. Our studies also demonstrate that F. verticillioides can metabolize some trichothecenes by adding an acetyl group to C-3 or by removing acetyl groups from C-4 or C-15. In addition, wild-type F. verticillioides can convert 7,8-dihydroxycalonectrin to 3,15-diacetyldeoxynivalenol.     

Trichothecenes accumulated in liquid culture of a mutant of Fusarium sporotrichioides NRRL 3299

A UV-generated mutant of Fusarium sporotrichioides NRRL 3299 was altered in its ability to biosynthesize T-2 toxin, as shown by a rapid screen with monoclonal antibodies to T-2. This stable mutant accumulated two trichothecenes that were not observed in liquid cultures of the parent strain. The two compounds were identified as 3,15-diol 12,13-epoxytrichothec-9-ene and 3,15-diol 12,13-epoxytrichothec-9-ene 3-acetate on the basis of their nuclear magnetic resonance and mass spectra. This is the first report of either of these two compounds as secondary metabolites of F. sporotrichioides and of a trichothecene acetylated at C-3 by this species.     

Trichothecenes accumulated in liquid culture of a mutant of Fusarium sporotrichioides NRRL 3299.

A UV-generated mutant of Fusarium sporotrichioides NRRL 3299 was altered in its ability to biosynthesize T-2 toxin, as shown by a rapid screen with monoclonal antibodies to T-2. This stable mutant accumulated two trichothecenes that were not observed in liquid cultures of the parent strain. The two compounds were identified as 3,15-diol 12,13-epoxytrichothec-9-ene and 3,15-diol 12,13-epoxytrichothec-9-ene 3-acetate on the basis of their nuclear magnetic resonance and mass spectra. This is the first report of either of these two compounds as secondary metabolites of F. sporotrichioides and of a trichothecene acetylated at C-3 by this species.     

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.     

A genetic and biochemical approach to study trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum

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.     

禾谷镰孢菌高产毒菌株的构建*

为研究禾谷镰孢菌Fusarium graminearum Schw.单端孢霉烯族毒素生物合成基因（产毒基因）在寄主体内的表达,作者构建了带报告基因GUS（(-葡糖苷酸酶基因）的质粒pGUSTRI6P5,并通过对野生型菌株的转化获得禾谷镰孢高产毒菌株。该质粒含有由TRI5（禾谷镰孢单端孢霉二烯合酶基因）启动子（TRI5 Prom）驱动的GUS基因编码区、潮霉素B抗性基因和拟枝孢镰孢F. sporotrichioides的产毒调控基因TRI6（FSTRI6）。用pGUSTRI6P5转化野生型菌株GZ3639后,在含潮霉素 B的培养基上选取抗性菌落,单孢分离获单孢菌株（转化子）。在GYEP（葡萄糖-酵母粉-蛋白胨）液体培养基上,转化子B4-1和B16-1的GUS比活力强,15-AcDON（15-乙酰脱氧雪腐镰刀菌烯醇）产量高,且两者呈正相关（相关系数（r）分别为0.9839和0.9523）。B4-1和B16-1两个转化子可作为研究禾谷镰孢与其寄主相互作用的工具菌株。     

Biosynthesis of 3-acetyldeoxynivalenol and sambucinol. Identification of the two oxygenation steps after trichodiene

The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha- and 2beta-) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha- and 2beta-) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha-hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta-hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12, 13-epoxy-9,10-trichoene-2alpha-ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha-hydroxytrichodiene and 12,13-epoxy-9, 10-trichoene-2alpha-ol as natural metabolites in F. culmorum cultures.     

Characterization of Novel Sesquiterpenoid Biosynthesis in Tobacco Expressing a Fungal Sesquiterpene Synthase

The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthase from the fungus Fusarium sporotrichioides, was used to transform tobacco (Nicotiana tabacum). Trichodiene was the sole sesquiterpene synthase product in enzyme reaction mixtures derived from unelicited transformant cell-suspension cultures, and both trichodiene and 5-epi-aristolochene were observed as reaction products following elicitor treatment. Immunoblot analysis of protein extracts revealed the presence of trichodiene synthase only in transformant cell lines producing trichodiene. In vivo labeling with [3H]mevalonate revealed the presence of a novel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated in the transformant cell-suspension cultures. In a trichodiene-producing transformant, the level of 15-hydroxytrichodiene accumulation increased after elicitor treatment. In vivo labeling with [14C]acetate showed that the biosynthetic rate of trichodiene and 15-hydroxytrichodiene also increased after elicitor treatment. Incorporation of radioactivity from [14C]acetate into capsidiol was reduced following elicitor treatment of a trichodiene-producing transformant as compared with wild type. These results demonstrate that sesquiterpenoid accumulation resulting from the constitutive expression of a foreign sesquiterpene synthase is responsive to elicitation and that the farnesyl pyrophosphate present in elicited cells can be utilized by a foreign sesquiterpene synthase to produce high levels of novel sesquiterpenoids.