Inactivation of a cytochrome P-450 is a determinant of trichothecene diversity in Fusarium species

Species of the genus Fusarium produce a great diversity of agriculturally important trichothecene toxins that differ from each other in their pattern of oxygenation and esterification. T-2 toxin, produced by Fusarium sporotrichioides, and nivalenol (NIV), produced by some strains of F. graminearum, contain an oxygen at the C-4 position. Deoxynivalenol (DON), produced by other strains of F. graminearum, lacks a C-4 oxygen. NIV and DON are identical except for this difference, whereas T-2 differs from these trichothecenes at three other carbon positions. Sequence and Northern analyses of the F. sporotrichioides genomic region upstream of the previously described core trichothecene gene cluster have extended the cluster by two genes: TRI13 and TRI14. TRI13 shares significant similarity with the cytochrome P-450 class of enzymes, but TRI14 does not share similarity with any previously characterized proteins. Gene disruption and fermentation studies in F. sporotrichioides indicate that TRI13 is required for the addition of the C-4 oxygen of T-2 toxin, but that TRI14 is not required for trichothecene biosynthesis. PCR and sequence analyses indicate that the TRI13 homolog is functional in NIV-producing strains of F. graminearum but nonfunctional in DON-producing strains of the fungus. These genetic observations are consistent with chemical observations that biosynthesis of T-2 toxin and NIV requires a C-4 hydroxylase while biosynthesis of DON does not.     

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.     

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.     

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 genetic basis for 3-ADON and 15-ADON trichothecene chemotypes in Fusarium

Certain Fusarium species cause head blight of wheat and other small grains worldwide and produce trichothecene mycotoxins. These mycotoxins can induce toxicoses in animals and humans and can contribute to the ability of some fusaria to cause plant disease. Production of the trichothecene 3-acetyldeoxynivalenol (3-ADON) versus 15-acetyldeoxynivalenol (15-ADON) is an important phenotypic difference within and among some Fusarium species. However, until now, the genetic basis for this difference in chemotype has not been identified. Here, we identified consistent DNA sequence differences in the coding region of the trichothecene biosynthetic gene TRI8 in 3-ADON and 15-ADON strains. Functional analyses of the TRI8 enzyme (Tri8) in F. graminearum, the predominant cause of wheat head blight in North America and Europe, revealed that Tri8 from 3-ADON strains catalyzes deacetylation of the trichothecene biosynthetic intermediate 3,15-diacetyldeoxynivalenol at carbon 15 to yield 3-ADON, whereas Tri8 from 15-ADON strains catalyzes deacetylation of 3,15-diacetyldeoxynivalenol at carbon 3 to yield 15-ADON. Fusarium strains that produce the trichothecene nivalenol have a Tri8 that functions like that in 15-ADON strains. TRI3, which encodes a trichothecene carbon 15 acetyltransferase, was found to be functional in all three chemotypes. Together, our data indicate that differential activity of Tri8 determines the 3-ADON and 15-ADON chemotypes in Fusarium.     

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。     

The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes

We report for the first time the complete structure and sequence of the trichothecene biosynthesis gene cluster (i.e. Tri5-cluster) from Fusarium graminearum F15, a strain that produces 3-acetyldeoxynivalenol (3-ADON). A putative tyrosinase and polysaccharide deacetylase gene flank the Tri5-cluster: the number of pathway genes between them is less than half the total number of steps necessary for 3-ADON biosynthesis. In comparison with partial Tri5-cluster sequences of strains with 15-acetyldeoxynivalenol and 4-acetylnivalenol chemotypes, the Tri5-cluster from strain F15 contains three genes that are apparently unnecessary for the biosynthesis of 3-ADON (i.e. Tri8 and Tri3, which are expressed, and pseudo-Tri13, which is not expressed). In addition, the Tri7 gene was missing from the cluster. Recombinant TRI3 protein showed limited trichothecene C-15 acetylase activity. In contrast, recombinant TRI8 protein displayed no C-3 deacetylase activity, suggesting that the loss or alteration of function contribute directly to the chemotype difference.     

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.     

A simple method with liquid chromatography/tandem mass spectrometry for the determination of the six trichothecene mycotoxins in rice medium

A selective and speedy LC-MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC-MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76-106% with a spiked level at 1-6 microg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.     

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.     

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.     

Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis

Fusarium Tri4 encodes a cytochrome P450 monooxygenase (CYP) for hydroxylation at C-2 of the first committed intermediate trichodiene (TDN) in the biosynthesis of trichothecenes. To examine whether this CYP further participates in subsequent oxygenation steps leading to isotrichotriol (4), we engineered Saccharomyces cerevisiae for de novo production of the early intermediates by introducing cDNAs of Fusarium graminearum Tri5 (FgTri5 encoding TDN synthase) and Tri4 (FgTri4). From a culture of the engineered yeast grown on induction medium (final pH 2.7), we identified two intermediates, 2alpha-hydroxytrichodiene (1) and 12,13-epoxy-9,10-trichoene-2alpha-ol (2), and a small amount of non-Fusarium trichothecene 12,13-epoxytrichothec-9-ene (EPT). Other intermediates isotrichodiol (3) and 4 were identified in the transgenic yeasts grown on phosphate-buffered induction medium (final pH 5.5-6.0). When Trichothecium roseum Tri4 (TrTri4) was used in place of FgTri4, 4 was not detected in the culture. The three intermediates, 1, 2, and 3, were converted to 4,15-diacetylnivalenol (4,15-diANIV) when fed to a toxin-deficient mutant of F. graminearum with the FgTri4+ genetic background (viz., by introducing a FgTri5- mutation), but were not metabolized by an FgTri4- mutant. These results provide unambiguous evidence that FgTri4 encodes a multifunctional CYP for epoxidation at C-12,13, hydroxylation at C-11, and hydroxylation at C-3 in addition to hydroxylation at C-2.     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

长江流域禾谷镰孢菌群部分菌株系统发育学、产毒素化学型及致病力研究

从采集自长江流域引起小麦赤霉病的禾谷镰孢菌群(Fusarium graminearum clade)菌株中选取了31株,扩增并测定了这些菌株的EF-1α(translation elongation factor)、PHO(phosphate permease)基因序列,利用相关软件进行了系统发育分析。对这些菌株的产毒素化学型进行了分子检测。同时,用两个小麦品种(扬麦158和安农8455)测定了菌株的致病力。系统发育分析表明绝大多数菌株与F.asiaticum聚为一枝,只有一个菌株11027与F.graminearum聚类。30株F.asiaticum中有24株产脱氧雪腐镰孢菌烯醇(deoxynivalenol,DON)和3-乙酰脱氧雪腐镰孢菌烯醇(3-acetyldeoxynivalenol,3-AcDON),另外6株产雪腐镰孢菌烯醇(Nivalenol,NIV)。一株F.graminearum菌株11027产脱氧雪腐镰孢菌烯醇(DON)和15-乙酰脱氧雪腐镰孢菌烯醇(15-acetyldeoxynivalenol,15-AcDON)。在扬麦158上,菌株间的致病力分化较为明显,产NIV毒素的菌株致病力普遍较弱,强致病力的菌株都产3-AcDON毒素。结果表明在我国长江流域,产3-AcDON毒素的F.asiaticum是引起小麦赤霉病的优势种群,中抗赤霉病的小麦品种扬麦158可以有效评价菌株的致病力强弱。     

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.     

Sphaerodes mycoparasitica biotrophic mycoparasite of 3-acetyldeoxynivalenol- and 15-acetyldeoxynivalenol-producing toxigenic Fusarium graminearum chemotypes

Fusarium spp. are economically important crop pathogens and causal agents of Fusarium head blight (FHB) of cereals worldwide. Of the FHB pathogens, Fusarium graminearum 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) are the most aggressive mycotoxigenic chemotypes, threatening food and feed quality as well as animal and human health. The objective of the study was to evaluate host specificity and fungal-fungal interactions of Sphaerodes mycoparasitica- a recently described mycoparasite - with F. graminearum 3- and 15-ADON strains by employing in vitro, microscopic and PCR techniques. Results obtained in this study show that the germination of mycoparasite ascospore in the presence of F. graminearum 3- and 15-ADON filtrates was greatly improved compared with Fusarium proliferatum and Fusarium sporotrichioides filtrates, suggesting a compatible interaction. Using quantitative real-time PCR with Fusarium-specific (Fg16N) and trichothecene Tri5 (Tox5-1/2)-specific primer sets, S. mycoparasitica was found to reduce the amount of F. graminearum 3-ADON and 15-ADON DNAs under separate coinoculation assays. Sphaerodes mycoparasitica was not only able to germinate in the presence of F. graminearum filtrates, but also established biotrophic mycoparasitic relations with two F. graminearum chemotypes and suppressed Fusarium growth.