Substrate specificities of Fusarium biosynthetic enzymes explain the genetic basis of a mixed chemotype producing both deoxynivalenol and nivalenol-type trichothecenes.

Fusarium species are traditionally grouped into type A and type B trichothecene producers based on structural differences in the mycotoxin they synthesize. The type B trichothecene-producing Fusarium graminearum strains are further divided into 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV) chemotypes. The former two chemotypes, collectively termed a deoxynivalenol (DON) chemotype, evolved from a NIV chemotype by inactivation of FgTri13, which encodes trichothecene C-4 hydroxylase, during the evolutionary process. Despite stable overexpression of FgTri13, however, both 3-acetylnivalenol (3-ANIV) and 3-ADON accumulate equally in shake flask culture of a transgenic 3-ADON chemotype. In this study, we investigated why the "3-ANIV chemotype" could not be obtained using this strategy. When analysis was extended to the transgenic NIV chemotype, in which FgTri7 C-4 acetylase gene was disrupted and FgTri8 deacetylase gene was replaced with the 3-ADON chemotype's orthologue, C-4 unoxygenated 3-ADON, as well as C-4 oxygenated 3-ANIV, accumulated as the end product. A feeding experiment with an ΔFgtri5ΔFgtri3 double gene disruptant, a trichothecene non-producing mutant unable to acetylate C-15 of the trichothecene ring, revealed the importance of the 15-O-acetyl group for efficient C-4 hydroxylation of DON-type trichothecenes. This implies that traditional DON and NIV chemotype diversification is not solely explained by FgTri13, but is also explained by the function of the FgTri8 trichothecene deacetylase gene. None of the crude cell extracts from existing chemotypes showed highly specific C-15 deacetylation activity against 3,15-diacetylnivalenol (3,15-diANIV) without deacetylating C-15 of the C-4 unoxygenated earlier intermediate, 3,15-diacetyldeoxynivalenol. Thus, an unnatural Fusarium trichothecene, 3-ANIV, could only be synthesized as part of a mixture with 3-ADON, unless the esterase encoded by FgTri8 evolves to act on the 15-O-acetyl of 3,15-diANIV with high specificity. We also explain why the transgenic "15-ANIV chemotype", which can be generated through functional inactivation of FgTri7, uses an engineered pathway via 3,15-diANIV, but not 15-ADON, to generate 15-ANIV. Tri genes appear to evolve continuously, and altered functions of trichothecene pathway enzymes result in the generation of new trichothecenes, such as NX-2 and NX-3, which have been recently discovered in field isolates of F. graminearum. As recombination of FgTri8 between existing F. graminearum isolates could give rise to a strain that produces mixtures of DON and NIV-type trichothecenes, it may also be noteworthy to monitor the emergence of a field isolate that invalidates traditional chemotype classification.

Exploring an Artificial Metabolic Route in Fusarium sporotrichioides: Production and Characterization of 7-Hydroxy T-2 Toxin.

An artificial metabolic route to an unnatural trichothecene was designed by taking advantage of the broad substrate specificities of the T-2 toxin biosynthetic enzymes of Fusarium sporotrichioides. By feeding 7-hydroxyisotrichodermin, a shunt pathway metabolite of F. graminearum, to a trichodiene synthase-deficient mutant of F. sporotrichioides, 7-hydroxy T-2 toxin (1) was obtained as the final metabolite. Such an approach may have future applications in the metabolic engineering of a variety of fungal secondary metabolites. The toxicity of 7-hydroxy T-2 toxin was 10 times lower than that of T-2 toxin in HL-60 cells.

Hydroxylations of trichothecene rings in the biosynthesis of Fusarium trichothecenes: evolution of alternative pathways in the nivalenol chemotype.

Fusarium sporotrichioides genes FsTri11, FsTri13, and FsTri1 encode cytochrome P450 monooxygenases (CYPs) responsible for hydroxylations at C-15, C-4, and C-8 of the trichothecene skeleton, respectively. However, the corresponding genes of nivalenol (NIV)-chemotype Fusarium graminearum remain to be functionally elucidated. In this study, we characterized the roles of these CYPs in NIV biosynthesis. Analyses of the metabolites of the F. graminearum Fgtri11- mutant, a disruptant of FgTri11 encoding isotrichodermin (ITD) C-15 hydroxylase, revealed a small amount of NIV-type trichothecenes suggesting that an alternative C-15 hydroxylase partially complemented FgTRI11p. In contrast, the C-7/C-8 hydroxylations depended solely on FgTRI1p, as suggested by the metabolite profiles of the Fgtri11- Fgtri1- double gene disruptant. Disruption of FgTri1 in both the wild-type and Fgtri13- mutant backgrounds revealed that FgTRI13p exhibits marginal activity toward calonectrin (CAL) and that it was the only C-4 hydroxylase. In addition, feeding experiments demonstrated that the C-4 hydroxylation of a 7-hydroxytrichothecene lacking C-8 ketone was extremely limited. The marginal activity of FgTRI13p toward CAL was advantageous for the C-7/C-8 hydroxylation steps in NIV biosynthesis, as transformation of a C-4 oxygenated trichothecene lacking C-7/C-8 modifications into NIV-type trichothecenes was quite inefficient. The significance of hydroxylation steps in the evolution of Fusarium trichothecenes is discussed.

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay.

BACKGROUND: SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues. RESULTS: To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells. CONCLUSIONS: The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.

4-O-acetylation and 3-O-acetylation of trichothecenes by trichothecene 15-O-acetyltransferase encoded by Fusarium Tri3.

In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50 microM), we observed 3-O-acetylation activity against isotrichodermol that was more than 10(5) times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15-diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.

A screening system for inhibitors of trichothecene biosynthesis: hydroxylation of trichodiene as a target.

Fusarium Tri4 encodes a key cytochrome P450 monooxygenase for hydroxylation of trichodiene early in the biosynthesis of trichothecenes. In this study, we established a system for screening for inhibitors of trichothecene biosynthesis using transgenic Saccharomyces cerevisiae expressing Tri4. For easy evaluation of the TRI4 activity, trichodiene-11-one was used as a substrate and the formation of 2alpha-hydroxytrichodiene-11-one was monitored by HPLC. Using this system, TRI4 proved to be inhibited by various flavones and furanocoumarins. We also found that a catechin-containing commercial beverage product, Catechin Supplement 300 (CS300), inhibited TRI4 activity, at a concentration which did not significantly affect the growth of the transgenic yeast. At an early stage of culture, both flavone and CS300 exhibited a toxin-inhibitory activity against Fusarium graminearum. However, inhibition of trichothecene production was not observed with longer incubation periods at minimum concentrations necessary to inhibit >50% of the TRI4 activity, presumably due to the metabolism by the fungus. The results suggest that this yeast screening system with TRI4 is useful for the rapid identification of lead compounds for the design of trichothecene biosynthesis inhibitors that are resistant to modification by the fungus.

Molecular and genetic studies of fusarium trichothecene biosynthesis: pathways, genes, and evolution.

Trichothecenes are a large family of sesquiterpenoid secondary metabolites of Fusarium species (e.g., F. graminearum) and other molds. They are major mycotoxins that can cause serious problems when consumed via contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene Tri5 encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes (Tri genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the Tri5 gene cluster. At least three pathway genes, Tri101 (separated alone), and Tri1 and Tri16 (located in the Tri1-Tri16 two-gene cluster), were found outside of the Tri5 gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned Tri genes, and the evolution of Tri genes, focusing on Fusarium species.

Involvement of the osmosensor histidine kinase and osmotic stress-activated protein kinases in the regulation of secondary metabolism in Fusarium graminearum.

Fusarium graminearum produces trichothecenes in aerial hyphae, a process which is markedly suppressed by NaCl without a significant effect on fungal growth. Here we report on the involvement of kinases of the two-component osmotic signal transduction pathway in the regulation of secondary metabolism in F. graminearum. While a deletion null mutant of FgOs1 (encoding the osmosensor histidine kinase) (deltaFgOs1) produced a reduced amount of the red pigment aurofusarin and was unaltered in its ability to produce trichothecenes, deletion null mutants of FgOs4 (encoding mitogen-activated protein kinase kinase kinase; MAPKKK), FgOs5 (MAPKK), and FgOs2 (MAPK) showed markedly enhanced pigmentation and failed to produce trichothecenes in aerial hyphae. Also, the transcript levels of PKS12 and GIP2 (aurofusarin biosynthetic pathway and regulatory genes, respectively) were significantly enhanced in the deltaFgOs4, deltaFgOs5, and deltaFgOs2 mutants and were reduced in the deltaFgOs1 mutant. In addition, expression of Tri4 and Tri6 (trichothecene biosynthetic pathway and regulatory genes) and production of trichothecenes in rice medium were markedly reduced in the former three protein kinase mutants. This is the first report demonstrating the involvement of a MAPK in the regulation of secondary metabolism.

Genetically engineered Fusarium as a tool to evaluate the effects of environmental factors on initiation of trichothecene biosynthesis.

Fusarium graminearum was engineered for expression of enhanced green fluorescent protein gene (egfp) as a reporter regulated in a manner similar to Tri5, a key pathway gene in trichothecene biosynthesis. Using the transgenic fungus, it was found that the reporter gene was induced to express in aerial hyphae developed on trichothecene noninducing medium YG solidified by agar. Unexpectedly, the transcriptional activation of egfp was markedly suppressed by adding NaCl that does not significantly affect fungal growth. As suggested by these findings, wild-type F. graminearum that formed aerial hyphae on YG agar plates produced trichothecenes and the production was effectively suppressed by adding 1% NaCl to the agar. To evaluate the effects of abiotic stress on the expression of trichothecene biosynthesis (Tri) genes, a sensitive plate assay was established using GYEP medium (which very weakly induces trichothecene production) solidified with gellan gum. Using this assay, triazole fungicides were shown to cause transcriptional activation of egfp at sublethal concentrations. Indeed, trichothecene production significantly increased when F. graminearum was grown in rice medium (which moderately induces trichothecene) amended with low doses of tebuconazole. The real-time monitoring system described here may help predict the risks of trichothecene contamination by the fungus under various environmental conditions.

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.

Screening of putative oxygenase genes in the Fusarium graminearum genome sequence database for their role in trichothecene biosynthesis.

In the biosynthesis of type B trichothecenes, four oxygenation steps remain to have genes functionally assigned to them. On the basis of the complete genome sequence of Fusarium graminearum, expression patterns of all oxygenase genes were investigated in Fusarium asiaticum (F. graminearum lineage 6). As a result, we identified five cytochrome P450 monooxygenase (CYP) genes that are specifically expressed under trichothecene-producing conditions and are unique to the toxin-producing strains. The entire coding regions of four of these genes were identified in F. asiaticum. When expressed in Saccharomyces cerevisiae, none of the oxygenases were able to transform trichodiene-11-one to expected products. However, one of the oxygenases catalyzed the 2beta-hydroxylation rather than the expected 2alpha-hydroxylation. Targeted disruption of the five CYP genes did not alter the trichothecene profiles of F. asiaticum. The results are discussed in relation to the presence of as-yet-unidentified oxygenation genes that are necessary for the biosynthesis of trichothecenes.

A wheat xylanase inhibitor gene, Xip-I, but not Taxi-I, is significantly induced by biotic and abiotic signals that trigger plant defense.

XIP-I and TAXI-I are wheat (Triticum aestivum L) grain proteins that inhibit microbial xylanases used in food processing. Although their biochemical properties and structural features were established recently, very little is known about their expression and their family members in wheat plants. To clarify the role of these xylanase inhibitor proteins in plant defense, we examined the expression of the XIP-type genes in response to a variety of biotic and abiotic signals. Although Xip-I was not expressed in flowering spikelets inoculated with Fusarium graminearum, transcription of Xip-I was greatly enhanced in Erysiphe graminis-infected leaves. Thus, unlike Taxi-I, Xip-I is pathogen-inducible, and unlike Taxi-III and Taxi-IV, its expression depends on the type of the pathogen and/or infected tissue. Xip-I was expressed when the leaves were wounded, and its expression was significantly elevated by treatment with methyl jasmonate (MeJA). The different expression profiles of XIP- and TAXI-type genes suggest distinct roles in plant defense.

Concordant evolution of trichothecene 3-O-acetyltransferase and an rDNA species phylogeny of trichothecene-producing and non-producing fusaria and other ascomycetous fungi.

The cereal pathogen Fusarium graminearum species complex (e.g. Fusarium asiaticum, previously referred to as F. graminearum lineage 6) produces the mycotoxin trichothecene in infected grains. The fungus has a gene for self-defence, Tri101, which is responsible for 3-O-acetylation of the trichothecene skeleton in the biosynthetic pathway. Recently, trichothecene non-producers Fusarium oxysporum and Fusarium fujikuroi (teleomorph Gibberella fujikuroi) were shown to have both functional (Tri201) and non-functional (pseudo-Tri101) trichothecene 3-O-acetyltransferase genes in their genome. To gain insight into the evolution of the trichothecene genes in Gibberella species, the authors examined whether or not other (pseudo-)biosynthesis-related genes are found near Tri201. However, sequence analysis of a 12 kb region containing Tri201 did not result in identification of additional trichothecene (pseudo-)genes in F. oxysporum. In a further attempt to find other trichothecene (pseudo-)genes from the non-producer, the authors examined whether or not the non-trichothecene genes flanking the ends of the core trichothecene gene cluster (i.e. the Tri5 cluster) comprise a region of synteny in Gibberella species. However, it was not possible to isolate trichothecene (pseudo-)genes from F. oxysporum (in addition to the previously identified pseudo-Tri101), because synteny was not observed for this region in F. asiaticum and F. oxysporum. In contrast to this unsuccessful identification of additional trichothecene (pseudo-)genes in the non-producer, a functional trichothecene 3-O-acetyltransferase gene could be identified in fusaria other than Gibberella: Fusarium decemcellulare and Fusarium solani; and in an ascomycete from a different fungal genus, Magnaporthe grisea. Together with the recent functional identification of Saccharomyces cerevisiae ScAYT1, these results are suggestive of a different evolutionary origin for the trichothecene 3-O-acetyltransferase gene from other biosynthesis pathway genes. The phylogeny of the 3-O-acetyltransferase was mostly concordant with the rDNA species phylogeny of these ascomycetous fungi.

Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene.

Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, is converted to a non-estrogenic product by a detoxifying enzyme of Clonostachys rosea. Previously, we investigated whether recombinant Saccharomyces cerevisiae carrying this detoxification gene, zhd101, can remove 2 microg ml(-1) of ZEN in a liquid culture. Although the transgenic yeasts eliminated most of the ZEN, they also converted a significant amount to a poor substrate, beta-zearalenol, which remained in the medium. In this study, we synthesized a codon-optimized zhd101 gene and investigated whether the transgenic yeast strain can overcome the problem of insufficient detoxification of ZEN. Importantly, within 48 h of incubation at 28 degrees C or 8 h of incubation at 37 degrees C, the transgenic yeasts completely eliminated 2 microg ml(-1) of ZEN in the medium without accumulating even a trace amount of beta-zearalenol. The result suggests that incomplete ZEN detoxification attributed to the action of an endogenous yeast beta-reductase can be overcome by simply increasing the expression of the detoxifying gene.

Expression in cereal plants of genes that inactivate Fusarium mycotoxins.

Trichothecene 3-O-acetyltransferase (encoded by Tri101) inactivates the virulence factor of the cereal pathogen Fusarium graminearum. Zearalenone hydrolase (encoded by zhd101) detoxifies the oestrogenic mycotoxin produced by the same pathogen. These genes were introduced into a model monocotyledon rice plant to evaluate their usefulness for decontamination of mycotoxins. The strong and constitutive rice Act1 promoter did not cause accumulation of TRI101 protein in transgenic rice plants. In contrast, the same promoter was suitable for transgenic production of ZHD101 protein; so far, five promising T0 plants have been generated. Low transgenic expression of Tri101 was suggested to be increased by addition of an omega enhancer sequence upstream of the start codon.

Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus.

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz. a promoter of adenylate cyclase interacting protein 1-like gene expressed in conidia, germ tubes, and appressoria, and a promoter of putative membrane-associated or secreted protein gene specifically expressed in appressoria. Although targeted knockout mutants of either gene failed to show detectable phenotypic alterations under laboratory conditions, these ESTs should be useful for identification of genes expressed during infection stages.

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.

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.