Marian N. Beremand

Identification of New Genes Positively Regulated by Tri10 and a Regulatory Network for Trichothecene Mycotoxin Production

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.

Murine lymphocyte proliferation impaired by substituted neosolaniols and calonectrins--Fusarium metabolites associated with trichothecene biosynthesis

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, particularly the substituted neosolaniols and calonectrins.

Trichothecene biosynthesis inGibberella pulicaris: Inheritance of C−8 hydroxylation

SummaryNaturally occurring strains ofGibberella pulicaris (Fusarium sambucinum) produce different kinds and levels of trichothecene toxins. Progeny from crosses between strains which produce trichothecenes with an oxygen-containing group at C−8 (C8+) and those that do not (C8−) can segregate in a 1∶1 ratio for this trait. These results define a genetic locus, which we have designatedTox1. The segregation patterns observed for progeny obtained from crosses between high-toxin producers and low-toxin producers indicate that the level of toxin production is determined by several loci. One gene which controls quantitative aspects of toxin production segregates independently of both theTox1 locus and another locus which controls toxin levels. These results suggest that multiple, unlinked nuclear loci are involved in the control of trichothecene biosynthesis.

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production inGibberella pulicaris (Fusarium sambucinum)

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production inG. pulicaris are described in the present study. A highly fertile, isogenic line was constructed forG. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.

Reactivity of deoxynivalenol (Vomitoxin) monoclonal antibody towards putative trichothecene precursors and shunt metabolites

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.