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Title: HETEROLOGOUS EXPRESSION OF TWO TRICHOTHECENE P450 GENES IN FUSARIUM VERTICILLIOIDES

Author
item McCormick, Susan
item Alexander, Nancy
item Proctor, Robert

Submitted to: Canadian Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/11/2005
Publication Date: 2/25/2006
Citation: McCormick, S.P., Alexander, N.J., Proctor, R. H. 2006. Heterologous expression of two trichothecene P450 genes in Fusarium verticillioides. Canadian Journal of Microbiology. 52:220-226.

Interpretive Summary: Trichothecenes are toxic substances produced by the wheat disease fungus Gibberella zeae. We are interested in reducing or eliminating these toxins from wheat. In an effort to accomplish that goal, we are interested in understanding the biology of the fungus with regard to trichothecene production. We know that a group of about 15 genes are involved in the production of the trichothecene vomitoxin and in this report we concentrate on the function of one of those genes and its role in vomitoxin production. One way we study the functions of these genes is to isolate a single gene and introduce it into another fungus that does not make trichothecene toxins. We transferred two forms of the Tri1 gene from two related toxin-producing species into Fusarium verticillioides in order to find out which steps each controlled. Our studies showed that G. zeae has a unique form of this gene that adds of two oxygens and that both oxygens are required for vomitoxin production. A better understanding of this vomitoxin gene may lead to novel strategies for reducing or eliminating these toxins from wheat.

Technical Abstract: Fusarium graminearum and F. sporotrichioides produce the trichothecene mycotoxins 15-acetyldeoxynivalenol (15-ADON) 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. In order to determine the function of an apparent Tri1 homolog from F. graminearum (FgTri1), both FsTri1 and FgTri1 genes were heterologously expressed in the trichothecene-nonproducing species F. verticillioides with the promoter from a fumonisin biosynthetic gene. Transgenic F. verticillioides carrying the FsTri1 converted exogenous isotrichodermin to 8-hydroxyisotrichodermin and calonectrin to 8-hydroxycalonectrin. Transgenic F. verticillioides carrying FgTri1 converted isotrichodermin into a mixture of 7-hydroxyisotrichodermin and 8-hydroxyisotrichodermin while calonectrin was converted into a mixture of 7-hydroxycalonectrin, 8-hydroxycalonectrin, and 3,15-diADON. A fourth compound, 7,8-dihydroxycalonectrin was identified in large scale cultures. Our results indicate that FgTri1 controls both C-7 and C-8 hydroxylation but that FsTri1 controls only C-8 hydroxylation. Our studies also demonstrated that F. verticillioides can metabolize some trichothecenes by adding an acetyl group to C-3 or removing acetyl groups from C-4 or C-15. In addition, wildtype F. verticillioides can convert 7,8-dihydroxycalonectrin to 3,15-diacetyldeoxynivalenol (3,15-diADON).