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.     

Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture

Fusarium head blight caused by Fusarium graminearum is a disease of cereal crops that not only reduces crop yield and quality but also results in contamination with trichothecenes such as nivalenol and deoxynivalenol (DON). To analyze the trichothecene induction mechanism, effects of 12 carbon sources on the production of DON and 3-acetyldexynivalenol (3ADON) were examined in liquid cultures incubated with nine strains of 3ADON-producing F. graminearum. Significantly high levels of trichothecene (DON and 3ADON) production by sucrose, 1-kestose and nystose were commonly observed among all of the strains tested. On the other hand, the levels of trichothecene biosynthesis induced by the other carbon sources were strain-specific. Tri4 and Tri5 expressions were up-regulated in the sucrose-containing medium but not in glucose. Trichothecene accumulation in the sucrose-containing medium was not repressed by the addition of glucose, indicating that trichothecene production was not regulated by carbon catabolite repression. These findings suggest that F. graminearum recognizes sucrose molecules, activates Tri gene expression and induces trichothecene biosynthesis.     

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.     

The velvet gene, FgVe1, affects fungal development and positively regulates trichothecene biosynthesis and pathogenicity in Fusarium graminearum

Trichothecenes are a group of toxic secondary metabolites produced mainly by Fusarium graminearum (teleomorph: Gibberella zeae) during the infection of crop plants, including wheat, maize, barley, oats, rye and rice. Some fungal genes involved in trichothecene biosynthesis have been shown to encode regulatory proteins. However, the global regulation of toxin biosynthesis is still enigmatic. In addition to the production of secondary metabolites belonging to the trichothecene family, F. graminearum produces the red pigment aurofusarin. The gene regulation underlying the production of aurofusarin is not well understood. The velvet gene (veA) is conserved in various genera of filamentous fungi. Recently, the veA gene from Aspergillus nidulans has been shown to be the key component of the velvet complex regulating development and secondary metabolism. Using blast analyses, we identified the velvet gene from F. graminearum, FgVe1. Disruption of FgVe1 causes several phenotypic effects. However, the complementation of this mutant with the FgVe1 gene restores the wild-type phenotypes. The in vitro phenotypes include hyperbranching of the mycelium, suppression of aerial hyphae formation, reduced hydrophobicity of the mycelium and highly reduced sporulation. Our data also show that FgVe1 modulates the production of the aurofusarin pigment and is essential for the expression of Tri genes and the production of trichothecenes. Pathogenicity studies performed on flowering wheat plants indicate that FgVe1 is a positive regulator of virulence in F. graminearum.     

PCR Analysis of the Tri13 gene to Determine the Genetic Potential of Fusarium graminearum Isolates from Iran to Produce Nivalenol and Deoxynivalenol

Fusarium graminearum trichothecene producing isolates can be broadly divided into two chemotypes based on the production of the 8- ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). Functional Tri13 gene required for the production of NIV and 4- acetyl NIV, whereas in the isolates producing DON and its acetylated derivates, this gene is nonfunctional. In this study, a total of 57 isolates from different fields of Mazandaran province, Iran were identified as F. graminearum using classical methods and species specific primers. In order to assess the potential of isolates to produce NIV or DON, we used PCR to determine whether isolates carried a functional or nonfunctional Tri13 gene. Out of the 57 tested F. graminearum isolates with Tri13 PCR assays, 46 yielded an amplicon similar to the size predicted for nivalenol production, while 11 yielded an amplicon similar to the size predicted for deoxynivalenol production. From regions where more than one F. graminearum isolate was obtained, isolates were not exclusively of a single chemotype. It seems that genetic diversity among the isolates has relation with geographical region and wheat cultivar. The assay can provide information about the distribution of Tri13 haplotype that can be used in tracing of trichothecene contaminated samples.     

Trichothecene nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes

The trichothecene 3-O-acetyltransferase gene (FgTri101) required for trichothecene production by Fusarium graminearum is located between the phosphate permease gene (pho5) and the UTP-ammonia ligase gene (ura7). We have cloned and sequenced the pho5-to-ura7 regions from three trichothecene nonproducing Fusarium (i.e., F. oxysporum, F. moniliforme, and Fusarium species IFO 7772) that belong to the teleomorph genus Gibberella. BLASTX analysis of these sequences revealed portions of predicted polypeptides with high similarities to the TRI101 polypeptide. While FspTri101 (Fusarium species Tri101) coded for a functional 3-O-acetyltransferase, FoTri101 (F. oxysporum Tri101) and FmTri101 (F. moniliforme Tri101) were pseudogenes. Nevertheless, F. oxysporum and F. moniliforme were able to acetylate C-3 of trichothecenes, indicating that these nonproducers possess another as yet unidentified 3-O-acetyltransferase gene. By means of cDNA expression cloning using fission yeast, we isolated the responsible FoTri201 gene from F. oxysporum; on the basis of this sequence, FmTri201 has been cloned from F. moniliforme by PCR techniques. Both Tri201 showed only a limited level of nucleotide sequence similarity to FgTri101 and FspTri101. The existence of Tri101 in a trichothecene nonproducer suggests that this gene existed in the fungal genome before the divergence of producers from nonproducers in the evolution of Fusarium species.     

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.     

Exogenous H(2)O(2) and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum

Effect of exogenous H(2)O(2) and catalase was tested in liquid cultures of the deoxynivalenol and 15-acetyldeoxynivalenol-producing fungus Fusarium graminearum. Accordingly to previous results, H(2)O(2) supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15-acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase-treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.     

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.     

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.     

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.     

禾谷镰刀菌降解毒素基因Tri101的克隆和序列分析

禾谷镰刀菌Tri101基因编码的单端孢酶烯3-O-乙酰转移酶可通过加乙酰基的形式使禾谷镰刀菌产生的单族毒素(如DON)转变为较低的毒性。本研究利用RT-PCR技术从禾谷镰刀菌0623中扩增并克隆了Tri101基因的cDNA片段,测序结果表明,Tri101基因核苷酸序列阅读框架全长1356bp(GenBank序列号:GQ907236),编码451个氨基酸的多肽,推测分子量为49.45kD,等电点为5.14。氨基酸序列同源性比对结果表明,它与Kimura报道的禾谷镰刀菌Tri101氨基酸序列同源性最高,为99.56%,与其它13种镰刀菌的Tri101氨基酸序列的同源性分别为97.91%-75.68%。系统进化树分析结果表明,Fusarium graminearium0623与Fusarium sporotrichioides属于同一进化枝且与Fusarium asiaticum有较近的亲缘关系,而与F.oxysporum、F.moniliforme、F.nygamai、F.nisikadoi和F.decemcellulare的亲缘关系较远。     

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.