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.     

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.     

Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides

We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.     

Structural and functional characterization of TRI3 trichothecene 15‐O‐acetyltransferase from Fusarium sporotrichioides

Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13‐epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15‐O‐trichothecene acetyltransferase isolated from F. sporotrichioides and the “in vivo” characterization of Δtri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15‐decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15‐decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3‐O‐trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.     

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.     

Structural and functional characterization of the TRI101 trichothecene 3-O-acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum: kinetic insights to combating Fusarium head blight

Fusarium head blight (FHB) is a plant disease with serious economic and health impacts. It is caused by fungal species belonging to the genus Fusarium and the mycotoxins they produce. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by approximately 100-fold but has demonstrated limited resistance to FHB in transgenic cereal. To understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes toward B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three-dimensional structures for the ternary complexes for both of these enzymes with coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein.     

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.     

禾谷镰孢菌高产毒菌株的构建*

为研究禾谷镰孢菌Fusarium graminearum Schw.单端孢霉烯族毒素生物合成基因（产毒基因）在寄主体内的表达,作者构建了带报告基因GUS（(-葡糖苷酸酶基因）的质粒pGUSTRI6P5,并通过对野生型菌株的转化获得禾谷镰孢高产毒菌株。该质粒含有由TRI5（禾谷镰孢单端孢霉二烯合酶基因）启动子（TRI5 Prom）驱动的GUS基因编码区、潮霉素B抗性基因和拟枝孢镰孢F. sporotrichioides的产毒调控基因TRI6（FSTRI6）。用pGUSTRI6P5转化野生型菌株GZ3639后,在含潮霉素 B的培养基上选取抗性菌落,单孢分离获单孢菌株（转化子）。在GYEP（葡萄糖-酵母粉-蛋白胨）液体培养基上,转化子B4-1和B16-1的GUS比活力强,15-AcDON（15-乙酰脱氧雪腐镰刀菌烯醇）产量高,且两者呈正相关（相关系数（r）分别为0.9839和0.9523）。B4-1和B16-1两个转化子可作为研究禾谷镰孢与其寄主相互作用的工具菌株。     

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。     

Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae

Gibberella zeae, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a Tri13 homolog from each chemotype. The Tri13 from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of Fusarium sporotrichioides, whereas the Tri13 from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the Tri13 and Tri7 genes in trichothecene production by G. zeae, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of Tri13 from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1 Tri13 gene alone or in combination with the 88-1 Tri7 gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that Tri13 and Tri7 are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in G. zeae. These functional analyses of the Tri13 and Tri7 genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in G. zeae.     

Use of the volatile trichodiene to reduce Fusarium head blight and trichothecene contamination in wheat

Fusarium graminearum is the primary cause of Fusarium head blight (FHB), one of the most economically important diseases of wheat worldwide. FHB reduces yield and contaminates grain with the trichothecene mycotoxin deoxynivalenol (DON), which poses a risk to plant, human and animal health. The first committed step in trichothecene biosynthesis is formation of trichodiene (TD). The volatile nature of TD suggests that it could be a useful intra or interspecies signalling molecule, but little is known about the potential signalling role of TD during F. graminearum-wheat interactions. Previous work using a transgenic Trichoderma harzianum strain engineered to emit TD (Th + TRI5) indicated that TD can function as a signal that can modulate pathogen virulence and host plant resistance. Herein, we demonstrate that Th + TRI5 has enhanced biocontrol activity against F. graminearum and reduced DON contamination by 66% and 70% in a moderately resistant and a susceptible cultivar, respectively. While Th + TRI5 volatiles significantly influenced the expression of the pathogenesis-related 1 (PR1) gene, the effect was dependent on cultivar. Th + TRI5 volatiles strongly reduced DON production in F. graminearum plate cultures and downregulated the expression of TRI genes. Finally, we confirm that TD fumigation reduced DON accumulation in a detached wheat head assay.     

Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.     

Disruption of TRI101, the Gene Encoding Trichothecene 3-O-Acetyltransferase, from Fusarium sporotrichioides

We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3,15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.     

A fungal symbiont of plant-roots modulates mycotoxin gene expression in the pathogen Fusarium sambucinum

Fusarium trichothecenes are fungal toxins that cause disease on infected plants and, more importantly, health problems for humans and animals that consume infected fruits or vegetables. Unfortunately, there are few methods for controlling mycotoxin production by fungal pathogens. In this study, we isolated and characterized sixteen Fusarium strains from naturally infected potato plants in the field. Pathogenicity tests were carried out in the greenhouse to evaluate the virulence of the strains on potato plants as well as their trichothecene production capacity, and the most aggressive strain was selected for further studies. This strain, identified as F. sambucinum, was used to determine if trichothecene gene expression was affected by the symbiotic Arbuscular mycorrhizal fungus (AMF) Glomus irregulare. AMF form symbioses with plant roots, in particular by improving their mineral nutrient uptake and protecting plants against soil-borne pathogens. We found that that G. irregulare significantly inhibits F. sambucinum growth. We also found, using RT-PCR assays to assess the relative expression of trichothecene genes, that in the presence of the AMF G. irregulare, F. sambucinum genes TRI5 and TRI6 were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We conclude that AMF can modulate mycotoxin gene expression by a plant fungal pathogen. This previously undescribed effect may be an important mechanism for biological control and has fascinating implications for advancing our knowledge of plant-microbe interactions and controlling plant pathogens.     

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.     

Flippases play specific but distinct roles in the development,pathogenicity, and secondary metabolism of Fusarium graminearum

The membrane trafficking system is important for compartmentalization of the biosynthesis pathway and secretion of deoxynivalenol (DON) mycotoxin (a virulence factor) in Fusarium graminearum. Flippases are transmembrane lipid transporters and mediate a number of essential physiological steps of membrane trafficking, including vesicle budding, charging, and protein diffusion within the membrane. However, the roles of flippases in secondary metabolism remain unknown in filamentous fungi. Herein, we identified five flippases (FgDnfA, FgDnfB, FgDnfC1, FgDnfC2, and FgDnfD) in F. graminearum and established their specific and redundant functions in the development and pathogenicity of this phytopathogenic fungus. Our results demonstrate that FgDnfA is critical for normal vegetative growth while the other flippases are dispensable. FgDnfA and FgDnfD were found crucial for the fungal pathogenesis, and a remarkable reduction in DON production was observed in ΔFgDNFA and ΔFgDNFD. Deletion of the FgDNFB gene increased DON production to about 30 times that produced by the wild type. Further analysis showed that FgDnfA and FgDnfD have positive roles in the regulation of trichothecene (TRI) genes (TRI1, TRI4, TRI5, TRI6, TRI12, and TRI101) expression and toxisome reorganization, while FgDnfB acts as a negative regulator of DON synthesis. In addition, FgDnfB and FgDnfD have redundant functions in the regulation of phosphatidylcholine transport, and double deletion of FgDNFB and FgDNFD showed serious defects in fungal development, DON synthesis, and virulence. Collectively, our findings reveal the distinct and specific functions of flippase family members in F. graminearum and principally demonstrate that FgDnfA, FgDnfD, and FgDnfB have specific spatiotemporal roles during toxisome biogenesis.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.