Accumulation of 4-Deoxy-7-hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-Type FgTri1 Gene.

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain's genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Impact of nitrogen metabolism-associated culture pH changes on regulation of Fusarium trichothecene biosynthesis: revision of roles of polyamine agmatine and transcription factor AreA.

Fusarium graminearum produces trichothecene mycotoxins in infected grains and axenic liquid culture. A proposed regulatory model of trichothecene biosynthesis was examined in relation to nitrogen utilization. First, we showed that an important factor for the stimulation of trichothecene biosynthesis was not the occurrence of agmatine as a specific inducer molecule, but rather continuous acidification of the liquid culture medium arising from agmatine catabolism. When the pH of the L-Gln synthetic medium was frequently adjusted to the pH of the agmatine culture, trichothecene productivity of the L-Gln culture was equal to that of the agmatine culture. For efficient trichothecene biosynthesis, the culture pH should be lowered at an appropriate time point during the early growth stage. Second, we re-evaluated the role of the nitrogen regulatory GATA transcription factor AreA in trichothecene biosynthesis. Since Tri6 encodes a transcription factor indispensable for trichothecene biosynthesis, all fifteen AreA-binding consensus sequences in the Tri6 promoter were mutated. The mutant could catabolize L-Phe as the sole nitrogen source; furthermore, the pH profile of the synthetic L-Phe medium (initial pH 4.2) was the same as that of the wild-type (WT) strain. Under such conditions, the promoter mutant exhibited approximately 72% of the trichothecene productivity compared to the WT strain. Thus, F. graminearum AreA (FgAreAp) is dispensable for the functioning of the Tri6 promoter, but it contributes to the increased production of mycotoxin under mildly acidic conditions to some extent. Further investigations on the culture pH revealed that extremely low pH bypasses the function of FgAreAp.

Identification of amino acids negatively affecting Fusarium trichothecene biosynthesis.

Nitrogen sources in media have a significant impact on the onset of secondary metabolism in fungi. For transcriptional activation of many nitrogen catabolic genes, an AreA transcription factor is indispensable. This also holds true for Fusarium graminearum that produces trichothecenes, an important group of mycotoxin, in axenic culture. Despite the presence of numerous consensus AreA-binding sites in the promoters of Tri genes in the trichothecene cluster core region, the effect of medium amino acids on trichothecene biosynthesis is poorly understood. In this study, we examined the effect of certain amino acids, which were predicted to activate AreA function and increase Tri gene transcription, on trichothecene production in liquid culture. By frequent monitoring and adjustments in the pH of the culture medium, including replacement of the spent medium with fresh medium, we demonstrate the suppressive effects of the amino acids, used as the sole nitrogen source, on trichothecene biosynthesis. When the medium pH was maintained at 4.0, Gly, L-Ser, and L-Thr suppressed trichothecene production by F. graminearum. Enhanced trichothecene-inducing effects were observed when the medium pH was 3.5, with only L-Thr suppressing trichothecene synthesis.

Identification of a trichothecene production inhibitor by chemical array and library screening using trichodiene synthase as a target protein.

Trichothecene mycotoxins often accumulate in apparently normal grains of cereal crops. In an effort to develop an agricultural chemical to reduce trichothecene contamination, we screened trichothecene production inhibitors from the compounds on the chemical arrays. By using the trichodiene (TDN) synthase tagged with hexahistidine (rTRI5) as a target protein, 32 hit compounds were obtained from chemical library of the RIKEN Natural Product Depository (NPDepo) by chemical array screening. At 10µgmL-1, none of the 32 chemicals inhibited trichothecene production by Fusarium graminearum in liquid culture. Against the purified rTRI5 enzyme, however, NPD10133 [progesterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine] showed weak inhibitory activity at 10µgmL-1 (18.7µM). For the screening of chemicals inhibiting trichothecene accumulation in liquid culture, 20 analogs of NPD10133 selected from the NPDepo chemical library were assayed. At 10µM, only NPD352 [testosterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine methyl ester] inhibited rTRI5 activity and trichothecene production. Kinetic analysis suggested that the enzyme inhibition was of a mixed-type. The identification of NPD352 as a TDN synthase inhibitor lays the foundation for the development of a more potent inhibitor via systematic introduction of wide structural diversity on the gonane skeleton and amino acid residues.

Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi.

Tailor-made TALEN system for highly efficient targeted gene replacement in the rice blast fungus.

Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. Recently, engineered nucleases that can generate DNA double-strand breaks (DSBs) at a specific site in the desired locus within genome are utilized in a rapidly developing genome editing technology via DSBs repair. However, the use of engineered nucleases in filamentous fungi has not been validated. In this study, we demonstrated that tailor-made transcriptional activator-like effector nucleases (TALENs) system, Platinum-Fungal TALENs (PtFg TALENs), could improve the efficiency of homologous recombination-mediated targeted gene replacement by up to 100% in the rice blast fungus Pyricularia oryzae. This high-efficiency PtFg TALEN has great potential for basic and applied biological applications in filamentous fungi.

Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage.

Transposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungus Pyricularia oryzae. Genetic and physical interaction studies revealed that RecA domain-containing proteins, including P. oryzae homologs of Rad51, Rad55, and Rad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly, P. oryzae mutants of specific RNA silencing components (MoDCL1 and MoAGO2) were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

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.

Inhibition of Fusarium trichothecene biosynthesis by yeast extract components extractable with ethyl acetate.

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tri6, the presence of the ethyl acetate extractable-substances suppressed 15-ADON production. These results suggested that both Tri6p-independent initial activation of Tri6 expression and subsequent Tri6p-dependent activation of Tri expression were affected by the hydrophobic substances in the yeast extract products.

Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100-1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.

Identification and Characterization of Small Molecule Compounds That Modulate Trichothecene Production by Fusarium graminearum.

From the RIKEN Natural Products Depository (NPDepo) chemical library, we identified small molecules that alter trichothecene 15-acetyldeoxynivalenol (15-ADON) production by Fusarium graminearum. Among trichothecene production activators, a furanocoumarin NPD12671 showed the strongest stimulatory activity on 15-ADON production by the fungus cultured in a 24-well plate. NPD12671 significantly increased the transcription of Tri6, a transcription factor gene necessary for trichothecene biosynthesis, in both trichothecene-inducing and noninducing culture conditions. Dihydroartemisinin (DHA) was identified as the most effective inhibitor of trichothecene production in 24-well plate culture; DHA inhibited trichothecene production (>50% inhibition at 1 µM) without affecting fungal mass by suppressing Tri6 expression. To determine the effect of DHA on trichothecene pathway Tri gene expression, we generated a constitutively Tri6-overexpressing strain that produced 15-ADON in YG_60 medium in Erlenmeyer flasks, conditions under which no trichothecenes are produced by the wild-type. While 5 µM DHA failed to inhibit trichothecene biosynthesis by the overexpressor in trichothecene-inducing YS_60 culture, trichothecene production was suppressed in the YG_60 culture. Regardless of a high Tri6 transcript level in the constitutive overexpressor, the YG_60 culture showed reduced accumulation of Tri5 and Tri4 mRNA upon treatment with 5 µM DHA. Deletion mutants of FgOs2 were also generated and examined; both NPD12671 and DHA modulated trichothecene production as they did in the wild-type strain. These results are discussed in light of the mode of actions of these chemicals on trichothecene biosynthesis.

Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD.

A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants.

Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.

Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae.

To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.

Construction and characterization of a copy number-inducible fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018.

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was >34kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.

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.

Reduced contamination by the Fusarium mycotoxin zearalenone in maize kernels through genetic modification with a detoxification gene.

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6 degrees C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28 degrees C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (aw) and temperature; e.g., 16.9 microg of ZEN was removed per gram of seed within 48 h at an aw of 0.90 at 20 degrees C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.

Identification of multiple highly similar XIP-type xylanase inhibitor genes in hexaploid wheat.

In hexaploid wheat, Xip-I is the only XIP-type xylanase inhibitor gene whose expression and function have been characterized in detail. Here we demonstrate the existence of new XIP-type genes with the identification of Xip-R1 and Xip-R2 in the root cDNAs. Southern blot analysis with the Xip-R1 probe revealed that XIP-type genes comprised a significantly greater gene family than previously speculated on in studies with the Xip-I probe. The transcript level of Xip-R genes was increased upon an inoculation with Erysiphe graminis in the leaves, but not with Fusarium graminearum in the spikelets. RT-PCR with the RNA samples followed by extensive sequencing of the cloned amplified products revealed the presence of 12 highly similar Xip-R genes. Among these genes, Xip-R1 was the only predominant Xip-R family member induced to express in response to E. graminis. XIP-R1 was located in the apoplastic space and inhibited family 11 xylanases, but the protein did not show chitinolytic activity. These results suggest that hexaploid wheat has a large family of XIPs in its genome, but that only some of them are expressed for plant defense in limited tissues.

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.

Transgenic rice plants expressing trichothecene 3-O-acetyltransferase show resistance to the Fusarium 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.