ABSTRACT Tri10, a regulatory gene in trichothecene mycotoxin-producing Fusarium species, is required for trichothecene biosynthesis and the coordinated expression of four trichothecene pathway-specific genes (Tri4, Tri5, Tri6, and Tri101) and the isoprenoid biosynthetic gene for farnesyl pyrophosphate synthetase (FPPS). We showed that six more trichothecene genes (Tri3, Tri7, Tri8, Tri9, Tri11, and Tri12) are regulated by Tri10. We also constructed a cDNA library from a strain of Fusarium sporotrichioides that overexpresses Tri10 (↑Tri10) and used cDNA derived from the ↑Tri10 strain and a non-Tri10-expressing strain (ΔTri10) to differentially screen macroarrays prepared from the cDNA library. This screen identified 15 additional Tri10-regulated transcripts. Four of these transcripts represent Tri1, Tri13, and Tri14 and a gene designated Tri15. Three other sequences are putative orthologs of genes for isoprenoid biosynthesis, the primary metabolic pathway preceding trichothecene biosynthesis. The remaining eight sequences have been designated Ibt (influenced by Tri10) genes. Of the 26 transcripts now known to be positively regulated by Tri10, 22 are positively coregulated by Tri6, a gene that encodes a previously characterized trichothecene pathway-specific transcription factor. These 22 Tri10- and Tri6-coregulated sequences include all of the known Tri genes (except for Tri10), the FPPS gene, and the other three putative isoprenoid biosynthetic genes. Tri6 also regulates a transcript that is not regulated by Tri10. Thus, Tri10 and Tri6 regulate overlapping sets of genes that include a common group of multiple genes for both primary and secondary metabolism.

ABSTRACT 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 .

ABSTRACT 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.

ABSTRACT We report here the characterization of Tri10 , a novel regulatory gene within the trichothecene gene cluster. Comparison of Tri10 genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption of Tri10 in Fusarium sporotrichioides abolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes ( Tri4 , Tri5 , Tri6 , and Tri101 ) and an apparent farnesyl pyrophosphate synthetase ( Fpps ) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes and Fpps . Further analysis revealed that disruption of Tri10 , and to a greater extent the disruption of Tri6, increased sensitivity to T-2 toxin under certain growth conditions. Although Tri10 is conserved in Fusarium graminearum and Fusarium sambucinum and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which Tri10 acts upstream of the cluster-encoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where Tri6 is not required for the transcription of Tri10 but is required to limit the expression of Tri10.

The reactivity of deoxynivalenol (DON) monoclonal antibody 6F5 towards trichothecenes and Fusarium metabolites associated with DON and T-2 toxin biosynthesis was assessed by indirect enzyme-linked immunosorbent assay. Using concentrations required to inhibit antibody binding to a DON hemisuccinate ovalbumin conjugate solid phase by 50% as a basis of comparison, isotrichodermin, 3-acetyl DON, 15-deacetylcalonectrin, and 3,15-dideacetylcalonectrin were 647, 362, 247, and 209% cross-reactive relative to DON, respectively. Isotrichodermol, sambucinol, and 7,8-dihydroxycalonectrin reacted to a lesser extent. The results suggested that the C3, C4, and C15 positions were immunodominant on determining binding to the DON monoclonal antibody. The antibody should be useful in detecting both production of these metabolites in Fusarium cultures as well as their occurrence in foods and feeds.

G. S. Bondy, S. P. McCormick, M. N. Beremand and J. J. Pestka. Murine lymphocyte proliferation impaired by substituted neosolaniols and calonectrins—Fusarium metabolites associated with trichothecene biosynthesis. Toxicon29, 1107–1113, 1991.—The capacity of Fusarium secondary metabolites associated with trichothecene biosynthesis to inhibit murine spleen lymphocyte proliferation was evaluated and compared to that for well known trichothecenes. Activity of these compounds was not specific for B and T lymphocytes since they inhibited [3H]thymidine (TdR) incorporation in unstimulated, Con A- and LPS-stimulated lymphocytes to the same extent. Concentrations of 8-propionyl neosolaniol and 8-butyrylneosolaniol which inhibited [3H]Tdr uptake by 50% (id50s) were 0.95 and 0.34 ng/ml, respectively. The id50 for T-2 toxin was 0.26 ng/ml, indicating that there are minor alterations in 12, 13-epoxytrichothecene toxicity resulting from the replacement of the isovaleryl moiety on C8 of the trichothecene skeleton with other bulky acyl groups. id50 values for 4,15-diacetylnivalenol, fusarenon X, deoxynivalenol and 3-acetyldeoxynivalenol were 25, 38, 120 and 1800 ng/ml, respectively. Comparatively, id50 values for 3,15-dideacetylcalonectrin, 15-deacetylcalonectrin, and 7,8-dihydroxycalonectrin were 390, 2700 and 2400 ng/ml, respectively, indicating that the modified calonectrins had equivalent or less toxicity. Lymphotoxicity of trichothecenes thus decreased upon substitution of acyl groups at the C8 with keto or hydroxy moieties and was also dependent on the nature of substitutions at the C3, C4 and C15 positions. Sambucinol and the trichothecene precursor trichodiene, metabolites which do not contain a 12,13-epoxide, did not inhibit lymphocyte proliferation. The results suggest the need for further assessment of occurrence and in vivo toxicity of Fusarium metabolites, particulary the substituted neosolaniols and calonectrins.

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.

Mutant strains of Fusarium sporotrichioides NRRL 3299 deficient in the ability to synthesize T-2 toxin were examined on solid rice medium. Five novel alicyclic trichothecenes were isolated: 11 alpha-hydroxytrichodiene; tricho-9-ene-2 alpha,3 alpha,11 alpha-triol; tricho-9-ene-2 alpha,3 alpha,8 alpha,11 alpha-tetraol; tricho-9-ene-2 alpha,3 alpha,8 beta,11 alpha-tetraol; and tricho-9-ene-2 alpha,3 alpha,11 alpha,16-tetraol.

Many plant pathogenic fusaria produce trichothecenes, which are potent phytotoxins and inhibitors of protein synthesis in eukaryotes. In this study we show that a wild-type, trichothecene-producing strain of F. sporotrichioides, NRRL 3299, is pathogenic on Pastinaca sativa (parsnip) roots and produces T-2 toxin in planta. Parsnip roots infected with this strain also accumulate high levels of fungitoxic furanocoumarins, mainly xanthotoxin and angelicin. Analysis of serial sections of infected roots showed that furanocoumarin concentrations decease sharply at the infection boundary to levels apparently insufficient to completely block fungal growth or trichothecene production. The involvement of trichothecenes in pathogenesis was investigated by using three presiously isolated, complementary mutants of NRRL 3299, blocked at different steps in T-2 toxin biosynthesis. T-2 toxin production is restored when these mutants are grown together pairwise and each mutant accumulates different end products. In this study the ability of these mutants to infect parsnips correlates with their ability to produce certain trichothecenes. Thus, the mutant that accumulates 4,15-diacetoxyscirpenol, a trichothecene nearly as toxic as T-2 toxin, is as pathogenic as the wild-type parent. In contrast, neither the mutant that accumulates the less toxic calonectrin analogues nor the mutant that accumulates the nontoxic trichothecene precursor, trichodiene, is pathogenic. Furthermore, coinocul ation of these latter two mutants on parsnip roots results in infection.

The regulation of trichodiene synthase (TS) and its relationship to trichothecene biosynthesis was investigated in Fusarium sporotrichioides NRRL 3299 and Gibberella pulicaris R-6380. Cultures were analyzed for the presence of TS activity, trichothecenes, and immunodetectable TS polypeptide over a time period of 144 h. Enzyme activity increased from barely detectable to maximum levels over a period of 3 h for F. sporotrichioides , while in G. pulicaris , a steady increase was observed over 144 h. Increases in TS activity of 50-fold for F. sporotrichioides and 10-fold for G. pulicaris R-6380 preceded by several hours the detection of trichothecenes. Immunoblot analysis employing polyclonal serum specific for the enzyme from F. sporotrichioides showed that increases in the levels of TS polypeptide corresponded to the observed changes in enzyme activity for both organisms. These data indicate that the regulation of TS activity is accomplished through increases in its cellular concentration and that TS may serve as a useful indicator of trichothecene biosynthetic activity.

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.

The biosynthetic pathway for trichothecenes in the filamentous fungus Fusarium sporotrichioides NRRL 3299 has been further characterized. Experiments using the techniques of mutational analysis and the incorporation of radiolabeled precursors indicated that leucine is a direct precursor to the isovalerate moiety present in the trichothecene, T-2 toxin. Analysis of trichothecene production in a UV-induced leucine auxotroph also revealed the existence of a branched biosynthetic pathway which results in the coproduction of T-2 toxin and the T-2 toxin analogs neosolaniol, 8-isobutyryl-neosolaniol, and 8-propionyl-neosolaniol. Leucine limitation imposed by the leucine auxotroph simultaneously led to underproduction of T-2 toxin and overproduction of these T-2 toxin analogs, which are produced in small amounts by the wild-type parent. Furthermore, it was shown that the ratio of T-2 toxin to T-2 toxin analogs produced by the leucine auxotroph can be modulated by the concentration of leucine in the medium. These results suggest that the four trichothecenes mentioned above are derived from a common intermediate and that there is competition for this intermediate among the branched pathways leading to these four cometabolites.

Trichothecene-toxin-producing isolates of Gibberella pulicaris (anamorph: Fusarium sambucinum) were mated on water agar containing steam-sterilized mulberry (Morus alba) twigs. Mature ascospores developed at 15 C following several weeks of incubation under an alternating 12 hr/25 C light and 12 hr/20 C dark schedule. Segregation patterns among random ascospore progeny from parents differing in diacetoxyscirpenol production and other traits were examined. Protoperithecium formation and red pigmentation were monogenically controlled. Preliminary analyses of progeny from crosses among toxin-producing and nonproducing isolates indicated that ability to produce toxin is controlled by genes at several loci. We conclude that trichothecene toxin biosynthesis in G. pulicaris is amenable to classical genetic analysis. This is the first documented genetic system in a trichothecene-producing fungus.

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.

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.