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Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B 1 (FB 1 ) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum , and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides , ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB 1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker's yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1 , increased fumonisin resistance. High-level resistance was conferred by FUM18 , but not by FUM17 . Likewise, strong resistance to FB 1 was caused by overexpression of the presumed F. verticillioides "housekeeping" ceramide synthases CER1 , CER2 , and CER3 , located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

Importance: Fumonisins can cause diseases in animals and humans consuming Fusarium -contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food., Competing Interests: There is a conflict of interest due to T. Krska and K. Twaruschek being employed by FFoQSI. The initial stages of this work have also been funded by Biomin Holding GmbH (part of dsm-firmenich, employing Wulf-Dieter Moll) and the final stages by FFoQSI GmbH (Austrian Competence Centre for Feed and Food Quality, Safety & Innovation, project C30-P12-W03: Toxin Inactivation). IP rights have been transferred to FFoQSI, which filed a patent application (PCT/EP2023/062973).

Aegilops tauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusarium graminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomyces cerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae. tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta , so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation ( kmt6 ) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants ( kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15) . Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence.

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum . Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant-pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant-pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, At HSP90.1 and wheat Ta HSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli . In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants., (Copyright © 2019 Torres Acosta, Michlmayr, Shams, Schweiger, Wiesenberger, Mitterbauer, Werner, Merz, Hauser, Hametner, Varga, Krska, Berthiller and Adam.)

Fusarium graminearum is a plant pathogenic fungus which is able to infect wheat and other economically important cereal crop species. The role of ethylene in the interaction with host plants is unclear and controversial. We have analyzed the inventory of genes with a putative function in ethylene production or degradation of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). F. graminearum , in contrast to other species, does not contain a candidate gene encoding ethylene-forming enzyme. Three genes with similarity to ACC synthases exist; heterologous expression of these did not reveal enzymatic activity. The F. graminearum genome contains in addition two ACC deaminase candidate genes. We have expressed both genes in E. coli and characterized the enzymatic properties of the affinity-purified products. One of the proteins had indeed ACC deaminase activity, with kinetic properties similar to ethylene-stress reducing enzymes of plant growth promoting bacteria. The other candidate was inactive with ACC but turned out to be a d-cysteine desulfhydrase. Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, we generated single and double knockout mutants of both genes in the F. graminearum strain PH-1. No statistically significant effect of the gene disruptions on fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H 2 S is dispensable for full virulence.

Deoxynivalenol (DON) is considered to be one of the most important contaminants in cereals and food commodities produced thereof. So far it is not clear i) to which extent DON is degraded during baking and ii) if a degradation results in reduced toxicity. We have elucidated the fate of DON during baking of crackers, biscuits and bread, which were produced from fortified dough and processed under pilot plant conditions. Untargeted stable isotope assisted liquid chromatography (LC) high resolution mass spectrometry was used to determine all extractable degradation products. Targeted LC - tandem mass spectrometry based quantification revealed that DON was partially degraded to isoDON (1.3-3.9%), norDON B (0.2-0.9%) and norDON C (0.3-1.2%). A DON degradation of 6% (crackers), 5% (biscuits) and 2% (bread), respectively, was observed. In vitro translation experiments indicate that isoDON is less toxic than DON., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In filamentous fungi such as Fusarium graminearum , disruption of multiple genes of interest in the same strain (e.g., to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre -loxP system. The fusion genes contain commonly used C-terminal drug resistance markers, either nptII (G418), nat1 (nourseothricin), or hph (hygromycin B). These resistance genes are fused to the sequence encoding Herpes simplex virus thymidine kinase (HSVtk). Despite the presence of the 1 kb HSVtk gene (about ∼30% increase in total marker size), there is only a slight reduction in transformation efficiency on a molar basis. The fusion genes expressed under the Trichoderma pyruvate kinase (PKI) promoter also confer antibiotic resistance in Escherichia coli , allowing straightforward construction of disruption plasmids. For removal of the loxP flanked resistance cassettes, protoplasts of transformants are directly treated with purified Cre recombinase protein. Loss of the HSVtk containing cassette is selected by restoration of resistance to 5-fluoro-2-deoxyuridine (FdU). As a proof of principle, we demonstrated the efficiency of the HSVtk-based marker removal in Fusarium by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the FgPKS12 gene via integration of the loxP -flanked HSVtk -nptII cassette into the promoter or the first intron, thereby generating transformants with a white mycelium phenotype. Using Cre recombinase and FdU, the selection marker was subsequently removed, and the resulting transformants regained red pigmentation despite the remaining loxP site. We also found that it is possible to remove several unselected loxP -flanked cassettes with a single Cre protein treatment, as long as one of them contains a negative selectable HSVtk cassette. The negative selection system can also be used to introduce allele swaps into strains without leaving marker sequences, by first disrupting the gene of interest and then complementing the deletion in situ with genomic DNA containing a different allele.

The Fusarium metabolite culmorin (1) is receiving increased attention as an "emerging mycotoxin". It co-occurs with trichothecene mycotoxins and potentially influences their toxicity. Its ecological role and fate in plants is unknown. We synthesized sulfated and glucosylated culmorin conjugates as potential metabolites, which are expected to be formed in planta, and used them as reference compounds. An efficient procedure for the synthesis of culmorin sulfates was developed. Diastereo- and regioselective glucosylation of culmorin (1) was achieved by exploiting or preventing unexpected acyl transfer when using different glucosyl donors. The treatment of a wheat suspension culture with culmorin (1) revealed an in planta conversion of culmorin into culmorin-8-glucoside (6) and culmorin acetate, but no sulfates or culmorin-11-glucoside (7) was found. The treatment of wheat cells with the fungal metabolite 11-acetylculmorin (2) revealed its rapid deacetylation, but also showed the formation of 11-acetylculmorin-8-glucoside (8). These results show that plants are capable of extensively metabolizing culmorin.

A new type A trichothecene mycotoxin, NX-2, was previously reported to be produced by North American isolates of the cereal pathogen Fusarium graminearum. Here we describe the isolation and structural characterization of a rearrangement product, called NX2-M1, and related compounds with different acetylation patterns (NX3-M1 and NX4-M1). In the NX-M1 derivatives, the epoxide ring is opened, and a covalent bridge between C-10 and C-12 of the trichothecene backbone is formed. In vitro translation assays showed that NX3-M1 is less toxic for eukaryotic ribosomes than NX-3. NX3-M1 also has a greatly reduced cytotoxic potential on two tested human colon cell lines. Formation of NX3-M1 can therefore be regarded as a detoxification reaction. The related F. graminearum mycotoxin deoxynivalenol (DON), which is frequently occurring worldwide, is very stable during food processing. Testing NX-3 at different pH-values and temperature conditions, as well as under conditions that simulate the storage of infected grains and bread-making process, revealed a strongly reduced stability of NX-3 and concurrent formation of NX3-M1. Although the NX-3 formed in planta is as toxic as DON, the extensive formation of the non-toxic rearrangement product should be taken into account for risk assessment of this emerging food contaminant., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent in Asia but co-exist with deoxynivalenol producers in lower frequency in North America and Europe. Previous studies identified a barley UDP-glucosyltransferase, HvUGT13248, that efficiently detoxifies deoxynivalenol, and when expressed in transgenic wheat results in high levels of type II resistance against deoxynivalenol-producing F. graminearum. Here we show that HvUGT13248 is also capable of converting nivalenol into the non-toxic nivalenol-3-O-β-d-glucoside. We describe the enzymatic preparation of a nivalenol-glucoside standard and its use in development of an analytical method to detect the nivalenol-glucoside conjugate. Recombinant Escherichia coli expressing HvUGT13248 glycosylates nivalenol more efficiently than deoxynivalenol. Overexpression in yeast, Arabidopsis thaliana, and wheat leads to increased nivalenol resistance. Increased ability to convert nivalenol to nivalenol-glucoside was observed in transgenic wheat, which also exhibits type II resistance to a nivalenol-producing F. graminearum strain. Our results demonstrate the HvUGT13248 can act to detoxify deoxynivalenol and nivalenol and provide resistance to deoxynivalenol- and nivalenol-producing Fusarium., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Trichothecenes are a class of structurally diverse mycotoxins with more than 200 naturally occurring compounds. Previously, a new compound, pentahydroxyscirpene (PHS), was reported as a byproduct of a nivalenol producing Fusarium strain, IFA189. PHS contains a hydroxy group at C-8 instead of the keto group of type B trichothecenes. In this work, we demonstrate that IFA189 belongs to the species Fusarium kyushuense using molecular tools. Production of PHS in vitro was also observed for several isolates of other Fusarium species producing nivalenol. Furthermore, we report the formation of 4-acetyl-PHS by F. kyushuense on inoculated rice. Wheat ears of the variety Remus were infected with IFA189 and the in planta production of PHS was confirmed. Natural occurrence of PHS was verified in barley samples from the Czech Republic using a liquid chromatographic-tandem mass spectrometric method validated for this purpose. Toxicity of PHS to wheat ribosomes was evaluated with a coupled in vitro transcription and translation assay, which showed that PHS inhibits protein biosynthesis slightly less than nivalenol and deoxynivalenol., Competing Interests: The authors declare no conflict of interest.

The mycotoxin deoxynivalenol (DON) is an abundant contaminant of cereal based food and a severe issue for global food safety. We report the discovery of DON-3-sulfate as a novel human metabolite and potential new biomarker of DON exposure. The conjugate was detectable in 70% of urine samples obtained from pregnant women in Croatia. For the measurement of urinary metabolites, a highly sensitive and selective LC-MS/MS method was developed and validated. The method was also used to investigate samples from a duplicate diet survey for studying the toxicokinetics of DON-3-sulfate. To get a preliminary insight into the biological relevance of the newly discovered DON-sulfates, in vitroexperiments were performed. In contrast to DON, sulfate conjugates lacked potency to suppress protein translation. However, surprisingly we found that DON-sulfates enhanced proliferation of human HT-29 colon carcinoma cells, primary human colon epithelial cells (HCEC-1CT) and, to some extent, also T24 bladder cancer cells. A proliferative stimulus, especially in tumorigenic cells raises concern on the potential impact of DON-sulfates on consumer health. Thus, a further characterization of their toxicological relevance should be of high priority.

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease.

Recently, deoxynivalenol-3-sulfate (DON-3-sulfate) was proposed as a major DON metabolite in poultry. In the present work, the first LC-MS/MS based method for determination of DON-3-sulfate, deepoxy-DON-3-sulfate (DOM-3-sulfate), DON, DOM, DON sulfonates 1, 2, 3, and DOM sulfonate 2 in excreta samples of chickens and turkeys was developed and validated. To this end, DOM-3-sulfate was chemically synthesized and characterized by NMR and LC-HR-MS/MS measurements. Application of the method to excreta and chyme samples of four feeding trials with turkeys, chickens, pullets, and roosters confirmed DON-3-sulfate as the major DON metabolite in all poultry species studied. Analogously to DON-3-sulfate, DOM-3-sulfate was formed after oral administration of DOM both in turkeys and in chickens. In addition, pullets and roosters metabolized DON into DOM-3-sulfate. In vitro transcription/translation assays revealed DOM-3-sulfate to be 2000 times less toxic on the ribosome than DON. Biological recoveries of DON and DOM orally administered to broiler chickens, turkeys, and pullets were 74%-106% (chickens), 51%-72% (roosters), and 131%-151% (pullets). In pullets, DON-3-sulfate concentrations increased from jejunum chyme samples to excreta samples by a factor of 60. This result, put into context with earlier studies, indicates fast and efficient absorption of DON between crop and jejunum, conversion to DON-3-sulfate in intestinal mucosa, liver, and possibly kidney, and rapid elimination into excreta via bile and urine.

The ubiquitous filamentous fungus Fusarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F. graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX-2) was characterized by liquid chromatography-tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX-2 as 3α-acetoxy-7α,15-dihydroxy-12,13-epoxytrichothec-9-ene. Compared with the well-known 3-acetyl-deoxynivalenol (3-ADON), it lacks the keto group at C-8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10-fold higher contamination with its deacetylated form, named NX-3, (up to 540 mg kg(-1) ) compared with NX-2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga Chlamydomonas reinhardtii. NX-3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX-2 is far less toxic, similar to 3-ADON. Genetic analysis revealed a different TRI1 allele in the N-isolates, which was verified to be responsible for the difference in hydroxylation at C-8., (© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

We report the identification of deoxynivalenol-3-sulfate and deoxynivalenol-15-sulfate as two novel metabolites of the trichothecene mycotoxin deoxynivalenol in wheat. Wheat ears which were either artificially infected with Fusarium graminearum or directly treated with the major Fusarium toxin deoxynivalenol (DON) were sampled 96 h after treatment. Reference standards, which have been chemically synthesized and confirmed by NMR, were used to establish a liquid chromatography-electrospray ionization (LC-ESI)-MS/MS-based "dilute and shoot" method for the detection, unambiguous identification, and quantification of both sulfate conjugates in wheat extracts. Using this approach, detection limits of 0.003 mg/kg for deoxynivalenol-3-sulfate and 0.002 mg/kg for deoxynivalenol-15-sulfate were achieved. Matrix-matched calibration was used for the quantification of DON-sulfates in the investigated samples. In DON-treated samples, DON-3-sulfate was detected in the range of 0.29-1.4 mg/kg fresh weight while DON-15-sulfate concentrations were significantly lower (range 0.015-0.061 mg/kg fresh weight). In Fusarium-infected wheat samples, DON-3-sulfate was the only detected sulfate conjugate (range 0.022-0.059 mg/kg fresh weight). These results clearly demonstrate the potential of wheat to form sulfate conjugates of DON. In order to test whether sulfation is a detoxification reaction in planta, we determined the ability of the sulfated DON derivatives to inhibit in vitro protein synthesis of wheat ribosomes. The results demonstrate that both DON-sulfates can be regarded as detoxification products. DON-15-sulfate was about 44× less inhibitory than the native toxin, and no toxicity was observed for DON-3-sulfate in the tested range.

Intra- and extracellular iron-chelating siderophores produced by fungal non-ribosomal peptide synthetases have been shown to be involved in reproductive and pathogenic developmental processes and in iron and oxidative stress management. Here we report individual and combined contributions of three of these metabolites to developmental success of the destructive cereal pathogen Fusarium graminearum. In previous work, we determined that deletion of the NPS2 gene, responsible for intracellular siderophore biosynthesis, results in inability to produce sexual spores when mutants of this homothallic ascomycete are selfed. Deletion of the NPS6 gene, required for extracellular siderophore biosynthesis, does not affect sexual reproduction but results in sensitivity to iron starvation and oxidative stress and leads to reduced virulence to the host. Building on this, we report that double mutants lacking both NPS2 and NPS6 are augmented in all collective phenotypes of single deletion strains (i.e., abnormal sexual and pathogenic development, hypersensitivity to oxidative and iron-depletion stress), which suggests overlap of function. Using comparative biochemical analysis of wild-type and mutant strains, we show that NPS1, a third gene associated with siderophore biosynthesis, is responsible for biosynthesis of a second extracellular siderophore, malonichrome. nps1 mutants fail to produce this metabolite. Phenotypic characterization reveals that, although single nps1 mutants are like wild-type with respect to sexual development, hypersensitivity to ROS and iron-depletion stress, and virulence to the host, triple nps1nps2nps6 deletion strains, lacking all three siderophores, are even more impaired in these attributes than double nps2nps6 strains. Thus, combinatorial mutants lacking key iron-associated genes uncovered malonichrome function. The intimate connection between presence/absence of siderophores and resistance/sensitivity to ROS is central to sexual and pathogenic development.

Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism.

Pentahydroxyscirpene, a novel trichothecene-type compound, was isolated from Fusarium-inoculated rice. The structure of pentahydroxyscirpene was elucidated by 1D and 2D NMR spectroscopy and X-ray single-crystal diffraction. The conformation in solution was determined by NOESY experiments supported by quantum chemical calculations. In vitro toxicity tests showed that pentahydroxyscirpene inhibits protein synthesis as do other trichothecenes.

Many untargeted LC-ESI-HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on non-labelled and highly 13 C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U- 13 C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC-HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87-135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U- 13 C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples.

Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe-S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe-S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.

Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248., (2013 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY JOHN WILEY & SONS LTD AND BSPP.)

Plant small-molecule UDP-glycosyltransferases (UGT) glycosylate a vast number of endogenous substances but also act in detoxification of metabolites produced by plant-pathogenic microorganisms. The ability to inactivate the Fusarium graminearum mycotoxin deoxynivalenol (DON) into DON-3-O-glucoside is crucial for resistance of cereals. We analyzed the UGT gene family of the monocot model species Brachypodium distachyon and functionally characterized two gene clusters containing putative orthologs of previously identified DON-detoxification genes from Arabidopsis thaliana and barley. Analysis of transcription showed that UGT encoded in both clusters are highly inducible by DON and expressed at much higher levels upon infection with a wild-type DON-producing F. graminearum strain compared with infection with a mutant deficient in DON production. Expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae revealed that only two B. distachyon UGT encoded by members of a cluster of six genes homologous to the DON-inactivating barley HvUGT13248 were able to convert DON into DON-3-O-glucoside. Also, a single copy gene from Sorghum bicolor orthologous to this cluster and one of three putative orthologs of rice exhibit this ability. Seemingly, the UGT genes undergo rapid evolution and changes in copy number, making it difficult to identify orthologs with conserved substrate specificity.

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Trichothecenes are an important class of mycotoxins that act as potent protein synthesis inhibitors in eukaryotic organisms. The compound 4,15-diacetoxyscirpenol is highly toxic for plants and animals. Potatoes are especially prone to be contaminated with 4,15-diacetoxyscirpenol after infection with Fusarium sambucinum. In the current study, the reduction of 4,15-diacetoxyscirpenol during thermal treatment in aqueous solution was monitored. A new derivative was detected and named DAS-M1. After isolation, DAS-M1 was characterized with LC-HR-MS and LC-MS/MS and structurally elucidated with (1)H, (13)C, and 2D NMR. Potatoes were inoculated with F. sambucinum, and the infected potatoes were cooked at 100 or 121 °C, respectively. A reduction of 4,15-diacetoxyscirpenol from about 26% (1 h at 100 °C) to 100% (4 h at 121 °C) was detected by means of LC-MS/MS analysis. The effects of different pH values on the reduction of 4,15-diacetoxyscirpenol were investigated, showing higher conversion rates at more acidic pH values. In addition, the toxicity of 4,15-diacetoxyscirpenol and DAS-M1 was compared in vitro using a wheat germ transcription/translation assay and in vivo on Saccharomyces cerevisiae. The results show that the inhibitory effect of DAS-M1 on yeast growth is about 50 times lower and inhibition of protein synthesis is about 100 times lower than that of 4,15-diacetoxyscirpenol.

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe(2+) homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe(2+) utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe(2+) which was driven by the external Fe(2+) concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Delta failed to show this rapid Fe(2+) uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe(2+) uptake rates. Cu(2+) was transported at similar rates as Fe(2+), while other divalent cations, such as Zn(2+) and Cd(2+) apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe(2+) across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.

Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.

To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress.

We report here on the role of open reading frame (ORF) SPCC1183.04c of Schizosaccharomyces pombe in mitochondrial RNA metabolism. A mutant deleted for this ORF on chromosome III accumulates mitochondrial transcripts with the exception of the cob mRNA. A detailed Northern blot analysis showed that the effect results from a decrease in RNA degradation but not from RNA processing deficiencies. Overexpression of the SPCC1183.04c gene in a S. pombe wild-type strain is characterized by slow growth at 37 degrees C on non-fermentable carbon sources and a significant reduction of steady-state levels of mitochondrial transcripts. A NCBI BLASTP search with the amino acid sequence deduced from the S. pombe gene identified significant similarity to a number of proteins in fungi (e.g. Ascomycota, Basidiomycota) and in some non-fungal eukaryotes (e.g. ciliate, slime mold, red algae). By heterologous expression of SPCC1183.04c in a Saccharomyces cerevisiae pet127Delta strain, we demonstrate that the fission yeast protein and Pet127p from S. cerevisiae function similarly: The fission yeast gene complemented the respiratory defect associated with the pet127Delta allele and partially restored the RNA processing phenotype. Although it lacks any recognizable targeting signal, the S. pombe protein is imported into S. cerevisiae mitochondria in vivo. We conclude from our results that the fission yeast SPCC1183.04c gene is a member of a new protein family that functions to stimulate mitochondrial RNA degradation, a function that is conserved within the mitochondria of lower eukaryotes but seems to have been replaced by alternative pathways in metazoans and higher plants.

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Delta mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Delta mitochondria have increased cation contents, particularly K+ and low-membrane-potential Deltapsi. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K+/H+ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K+ homeostasis and organelle volume control.

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.

Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders. The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation. In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C. Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression. Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes. These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria.

We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA.

Disruption of the nuclear MRS2 gene (mrs2-1 mutation) causes a strong pet- phenotype in strains with mitochondrial group II introns, and a leaky pet- phenotype in strains without group II introns. MRS3 and MRS4, the genes for two mitochondrial-solute carrier proteins, can suppress both phenotypes when present in high-copy-number plasmids. In order to search for further multicopy suppressors of the mrs2-1 mutant phenotype, an yeast genomic DNA library, MW90, was constructed in YEp351 from a strain deleted for the MRS2, MRS3 and MRS4 genes. Ten different Sau3A DNA fragments that act as multicopy suppressors of the mrs2-1 respiratory-deficient phenotype were isolated from this library. Some of the newly isolated genes suppress the pet- phenotypes of mrs2-1 cells in strains with and without mitochondrial group II introns. Other genes, however, are suppressors only for the mitochondrial intron-less strains. This supports the notion that the MRS2 gene product is bifunctional i.e., it is essential for the splicing of group II introns and is also involved in processes of mitochondrial biogenesis other than RNA splicing.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.

When present in high copy number plasmids, the nuclear genes MRS3 and MRS4 from Saccharomyces cerevisiae can suppress the mitochondrial RNA splicing defects of several mit- intron mutations. Both genes code for closely related proteins of about Mr 32,000; they are 73% identical. Sequence comparisons indicate that MRS3 and MRS4 may be related to the family of mitochondrial carrier proteins. Support for this notion comes from a structural analysis of these proteins. Like the ADP/ATP carrier protein (AAC), the mitochondrial phosphate carrier protein (PiC) and the uncoupling protein (UCP), the two MRS proteins have a tripartite structure; each of the three repeats consists of two hydrophobic domains that are flanked by specific amino acid residues. The spacing of these specific residues is identical in all domains of all proteins of the family, whereas spacing between the hydrophobic domains is variable. Like the AAC protein, the MRS3 and MRS4 proteins are imported into mitochondria in vitro and without proteolytic cleavage of a presequence and they are located in the inner mitochondrial membrane. In vivo studies support this mitochondrial localization of the MRS proteins. Overexpression of the MRS3 and MRS4 proteins causes a temperature-dependent petite phenotype; this is consistent with a mitochondrial function of these proteins. Disruption of these genes affected neither mitochondrial functions nor cellular viability. Their products thus have no essential function for mitochondrial biogenesis or for whole yeast cells that could not be taken over by other gene products. The findings are discussed in relation to possible functions of the MRS proteins in mitochondrial solute translocation and RNA splicing.

A gene bank of a yeast wild type DNA in the high copy number vector YEp13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a -1 bp deletion in the first intron of the mitochondrial COB gene (bI1). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bI1, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.