Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilops tauschii and wheat.

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.

Pyramiding Fusarium head blight resistance QTL from T. aestivum, T. dicoccum and T. dicoccoides in durum wheat.

KEY MESSAGE: FHB resistance of durum wheat was improved by introgression of Fhb1 and resistance genes from emmer wheat and by selection against adverse alleles of elite durum wheat. Durum wheat is particularly susceptible to Fusarium head blight (FHB) and breeding for resistance is impeded by the low genetic variation within the elite gene pool. To extend the genetic basis for FHB resistance in durum wheat, we analyzed 603 durum wheat lines from crosses of elite durum wheat with resistance donors carrying resistance alleles derived from Triticum aestivum, T. dicoccum and T. dicoccoides. The lines were phenotyped for FHB resistance, anthesis date, and plant height in artificially inoculated disease nurseries over 5 years. A broad variation was found for all traits, while anthesis date and plant height strongly influenced FHB severities. To correct for spurious associations, we adjusted FHB scorings for temperature fluctuations during the anthesis period and included plant height as a covariate in the analysis. This resulted in the detection of seven quantitative trait loci (QTL) affecting FHB severities. The hexaploid wheat-derived Fhb1 QTL was most significant on reducing FHB severities, highlighting its successful introgression into several durum wheat backgrounds. For two QTL on chromosomes 1B and 2B, the resistance alleles originated from the T. dicoccum line Td161 and T. dicoccoides accessions Mt. Hermon#22 and Mt. Gerizim#36, respectively. The other four QTL featured unfavorable alleles derived from elite durum wheat that increased FHB severities, with a particularly negative effect on chromosome 6A that simultaneously affected plant height and anthesis date. Therefore, in addition to pyramiding resistance genes, selecting against adverse alleles present in elite durum wheat could be a promising avenue in breeding FHB-resistant durum wheat.

Type II Fusarium head blight susceptibility conferred by a region on wheat chromosome 4D.

Fusarium head blight (FHB) causes significant grain yield and quality reductions in wheat and barley. Most wheat varieties are incapable of preventing FHB spread through the rachis, but disease is typically limited to individually infected spikelets in barley. We point-inoculated wheat lines possessing barley chromosome introgressions to test whether FHB resistance could be observed in a wheat genetic background. The most striking differential was between 4H(4D) substitution and 4H addition lines. The 4H addition line was similarly susceptible to the wheat parent, but the 4H(4D) substitution line was highly resistant, which suggests that there is an FHB susceptibility factor on wheat chromosome 4D. Point inoculation of Chinese Spring 4D ditelosomic lines demonstrated that removing 4DS results in high FHB resistance. We genotyped four Chinese Spring 4DS terminal deletion lines to better characterize the deletions in each line. FHB phenotyping indicated that lines del4DS-2 and del4DS-4, containing smaller deletions, were susceptible and had retained the susceptibility factor. Lines del4DS-3 and del4DS-1 contain larger deletions and were both significantly more resistant, and hence had presumably lost the susceptibility factor. Combining the genotyping and phenotyping results allowed us to refine the susceptibility factor to a 31.7 Mbp interval on 4DS.

QTL mapping and successful introgression of the spring wheat-derived QTL Fhb1 for Fusarium head blight resistance in three European triticale populations.

KEY MESSAGE: The spring wheat-derived QTL Fhb1 was successfully introgressed into triticale and resulted in significantly improved FHB resistance in the three triticale mapping populations. Fusarium head blight (FHB) is a major problem in cereal production particularly because of mycotoxin contaminations. Here we characterized the resistance to FHB in triticale breeding material harboring resistance factors from bread wheat. A highly FHB-resistant experimental line which derives from a triticale × wheat cross was crossed to several modern triticale cultivars. Three populations of recombinant inbred lines were generated and evaluated in field experiments for FHB resistance using spray inoculations during four seasons and were genotyped with genotyping-by-sequencing and SSR markers. FHB severity was assessed in the field by visual scorings and on the harvested grain samples using digital picture analysis for quantifying the whitened kernel surface (WKS). Four QTLs with major effects on FHB resistance were identified, mapping to chromosomes 2B, 3B, 5R, and 7A. Those QTLs were detectable with both Fusarium severity traits. Measuring of WKS allows easy and fast grain symptom quantification and appears as an effective scoring tool for FHB resistance. The QTL on 3B collocated with Fhb1, and the QTL on 5R with the dwarfing gene Ddw1. This is the first report demonstrating the successful introgression of Fhb1 into triticale. It comprises a significant step forward for enhancing FHB resistance in this crop.

Chitosan Hydrochloride Decreases Fusarium graminearum Growth and Virulence and Boosts Growth, Development and Systemic Acquired Resistance in Two Durum Wheat Genotypes.

Fusarium head blight (FHB) is a devastating disease for cereals. FHB is managed by fungicides at anthesis, but their efficacy is variable. Conventional fungicides accumulate in the soil and are dangerous for animal and human health. This study assayed the antifungal ability of chitosan hydrochloride against Fusarium graminearum. Chitosan reduced F. graminearum growth and downregulated the transcript of the major genes involved in the cell growth, respiration, virulence, and trichothecenes biosynthesis. Chitosan promoted the germination rate, the root and coleoptile development, and the nitrogen balance index in two durum wheat genotypes, Marco Aurelio (FHB-susceptible) and DBC480 (FHB-resistant). Chitosan reduced FHB severity when applied on spikes or on the flag leaves. FHB severity in DBC480 was of 6% at 21 dpi after chitosan treatments compared to F. graminearum inoculated control (20%). The elicitor-like property of chitosan was confirmed by the up-regulation of TaPAL, TaPR1 and TaPR2 (around 3-fold). Chitosan decreased the fungal spread and mycotoxins accumulation. This study demonstrated that the non-toxic chitosan is a powerful molecule with the potential to replace the conventional fungicides. The combination of a moderately resistant genotype (DBC480) with a sustainable compound (chitosan) will open new frontiers for the reduction of conventional compounds in agriculture.

Exploring and exploiting the genetic variation of Fusarium head blight resistance for genomic-assisted breeding in the elite durum wheat gene pool.

KEY MESSAGE: Genomic selection had a higher selection response for FHB resistance than phenotypic selection, while association mapping identified major QTL on chromosome 3B unaffected by plant height and flowering date. Fusarium head blight (FHB) is one of the most destructive diseases of durum wheat. Hence, minimizing losses in yield, quality and avoiding contamination with mycotoxins are of pivotal importance, as durum wheat is mostly used for human consumption. While growing resistant varieties is the most promising approach for controlling this fungal disease, FHB resistance breeding in durum wheat is hampered by the limited variation in the elite gene pool and difficulties in efficiently combining the numerous small-effect resistance quantitative trait loci (QTL) in the same line. We evaluated an international collection of 228 genotyped durum wheat cultivars for FHB resistance over 3 years to investigate the genetic architecture and potential of genomic-assisted breeding for FHB resistance in durum wheat. Plant height was strongly positively correlated with FHB resistance and led to co-localization of plant height and resistance QTL. Nevertheless, a major QTL on chromosome 3B independent of plant height was identified in the same chromosomal interval as reported for the prominent hexaploid resistance QTL Fhb1, though haplotype analysis highlighted the distinctiveness of both QTL. Comparison between phenotypic and genomic selection for FHB resistance revealed a superior prediction ability of the former. However, simulated selection experiments resulted in higher selection responses when using genomic breeding values for early generation selection. An earlier identification of the most promising lines and crossing parents was feasible with a genomic selection index, which suggested a much faster short-term population improvement than previously possible in durum wheat, complementing long-term strategies with exotic resistance donors.

A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat.

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-ß-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.

Ribosome quality control is a central protection mechanism for yeast exposed to deoxynivalenol and trichothecin.

BACKGROUND: The trichothecene mycotoxins deoxynivalenol (DON) and trichothecin (TTC) are inhibitors of eukaryotic protein synthesis. Their effect on cellular homeostasis is poorly understood. We report a systematic functional investigation of the effect of DON and TTC on the yeast Saccharomyces cerevisiae using genetic array, network and microarray analysis. To focus the genetic analysis on intracellular consequences of toxin action we eliminated the PDR5 gene coding for a potent pleiotropic drug efflux protein potentially confounding results. We therefore used a knockout library with a pdr5Δ strain background. RESULTS: DON or TTC treatment creates a fitness bottleneck connected to ribosome efficiency. Genes isolated by systematic genetic array analysis as contributing to toxin resistance function in ribosome quality control, translation fidelity, and in transcription. Mutants in the E3 ligase Hel2, involved in ribosome quality control, and several members of the Rpd3 histone deacetylase complex were highly sensitive to DON. DON and TTC have similar genetic profiles despite their different toxic potency. Network analysis shows a coherent and tight network of genetic interactions among the DON and TTC resistance conferring gene products. The networks exhibited topological properties commonly associated with efficient processing of information. Many sensitive mutants have a "slow growth" gene expression signature. DON-exposed yeast cells increase transcripts of ribosomal protein and histone genes indicating an internal signal for growth enhancement. CONCLUSIONS: The combination of gene expression profiling and analysis of mutants reveals cellular pathways which become bottlenecks under DON and TTC stress. These are generally directly or indirectly connected to ribosome biosynthesis such as the general secretory pathway, cytoskeleton, cell cycle delay, ribosome synthesis and translation quality control. Gene expression profiling points to an increased demand of ribosomal components and does not reveal activation of stress pathways. Our analysis highlights ribosome quality control and a contribution of a histone deacetylase complex as main sources of resistance against DON and TTC.

Stable Isotope-Assisted Evaluation of Different Extraction Solvents for Untargeted Metabolomics of Plants.

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics , which allows for the first time every detected feature to be considered for method evaluation. The efficiency and complementarity of commonly used extraction solvents, namely 1 + 3 (v/v) mixtures of water and selected organic solvents (methanol, acetonitrile or methanol/acetonitrile 1 + 1 (v/v)), with and without the addition of 0.1% (v/v) formic acid were compared. Four different wheat organs were sampled, extracted and analysed by LC-HRMS. Data evaluation was performed with the in-house-developed MetExtract II software and R. With all tested solvents a total of 871 metabolites were extracted in ear, 785 in stem, 733 in leaf and 517 in root samples, respectively. Between 48% (stem) and 57% (ear) of the metabolites detected in a particular organ were found with all extraction mixtures, and 127 of 996 metabolites were consistently shared between all extraction agent/organ combinations. In aqueous methanol, acidification with formic acid led to pronounced pH dependency regarding the precision of metabolite abundance and the number of detectable metabolites, whereas extracts of acetonitrile-containing mixtures were less affected. Moreover, methanol and acetonitrile have been found to be complementary with respect to extraction efficiency. Interestingly, the beneficial properties of both solvents can be combined by the use of a water-methanol-acetonitrile mixture for global metabolite extraction instead of aqueous methanol or aqueous acetonitrile alone.

QCScreen: a software tool for data quality control in LC-HRMS based metabolomics.

BACKGROUND: Metabolomics experiments often comprise large numbers of biological samples resulting in huge amounts of data. This data needs to be inspected for plausibility before data evaluation to detect putative sources of error e.g. retention time or mass accuracy shifts. Especially in liquid chromatography-high resolution mass spectrometry (LC-HRMS) based metabolomics research, proper quality control checks (e.g. for precision, signal drifts or offsets) are crucial prerequisites to achieve reliable and comparable results within and across experimental measurement sequences. Software tools can support this process. RESULTS: The software tool QCScreen was developed to offer a quick and easy data quality check of LC-HRMS derived data. It allows a flexible investigation and comparison of basic quality-related parameters within user-defined target features and the possibility to automatically evaluate multiple sample types within or across different measurement sequences in a short time. It offers a user-friendly interface that allows an easy selection of processing steps and parameter settings. The generated results include a coloured overview plot of data quality across all analysed samples and targets and, in addition, detailed illustrations of the stability and precision of the chromatographic separation, the mass accuracy and the detector sensitivity. The use of QCScreen is demonstrated with experimental data from metabolomics experiments using selected standard compounds in pure solvent. The application of the software identified problematic features, samples and analytical parameters and suggested which data files or compounds required closer manual inspection. CONCLUSIONS: QCScreen is an open source software tool which provides a useful basis for assessing the suitability of LC-HRMS data prior to time consuming, detailed data processing and subsequent statistical analysis. It accepts the generic mzXML format and thus can be used with many different LC-HRMS platforms to process both multiple quality control sample types as well as experimental samples in one or more measurement sequences.

Deoxynivalenol-sulfates: identification and quantification of novel conjugated (masked) mycotoxins in wheat.

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.

Tracing the metabolism of HT-2 toxin and T-2 toxin in barley by isotope-assisted untargeted screening and quantitative LC-HRMS analysis.

An extensive study of the metabolism of the type A trichothecene mycotoxins HT-2 toxin and T-2 toxin in barley using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is reported. A recently developed untargeted approach based on stable isotopic labelling, LC-Orbitrap-MS analysis with fast polarity switching and data processing by MetExtract software was combined with targeted LC-Q-TOF-MS(/MS) analysis for metabolite structure elucidation and quantification. In total, 9 HT-2 toxin and 13 T-2 toxin metabolites plus tentative isomers were detected, which were successfully annotated by calculation of elemental formulas and further LC-HRMS/MS measurements as well as partly identified with authentic standards. As a result, glucosylated forms of the toxins, malonylglucosides, and acetyl and feruloyl conjugates were elucidated. Additionally, time courses of metabolite formation and mass balances were established. For absolute quantification of those compounds for which standards were available, the method was validated by determining apparent recovery, signal suppression, or enhancement and extraction recovery. Most importantly, T-2 toxin was rapidly metabolised to HT-2 toxin and for both parent toxins HT-2 toxin-3-O-ß-glucoside was identified (confirmed by authentic standard) as the main metabolite, which reached its maximum already 1 day after toxin treatment. Graphical Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

Automated LC-HRMS(/MS) approach for the annotation of fragment ions derived from stable isotope labeling-assisted untargeted metabolomics.

Structure elucidation of biological compounds is still a major bottleneck of untargeted LC-HRMS approaches in metabolomics research. The aim of the present study was to combine stable isotope labeling and tandem mass spectrometry for the automated interpretation of the elemental composition of fragment ions and thereby facilitate the structural characterization of metabolites. The software tool FragExtract was developed and evaluated with LC-HRMS/MS spectra of both native (12)C- and uniformly (13)C (U-(13)C)-labeled analytical standards of 10 fungal substances in pure solvent and spiked into fungal culture filtrate of Fusarium graminearum respectively. Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment. The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-(13)C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion. Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies. Moreover, as demonstrated for a fungal culture sample, FragExtract also assists the characterization of unknown metabolites, which are not contained in databases, and thus exhibits a significant contribution to untargeted metabolomics research.

Isolation and structure elucidation of pentahydroxyscirpene, a trichothecene Fusarium mycotoxin.

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.

Functional characterization of two clusters of Brachypodium distachyon UDP-glycosyltransferases encoding putative deoxynivalenol detoxification genes.

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.

Quantitative trait loci-dependent analysis of a gene co-expression network associated with Fusarium head blight resistance in bread wheat (Triticum aestivum L.).

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe is one of the most prevalent diseases of wheat (Triticum aestivum L.) and other small grain cereals. Resistance against the fungus is quantitative and more than 100 quantitative trait loci (QTL) have been described. Two well-validated and highly reproducible QTL, Fhb1 and Qfhs.ifa-5A have been widely investigated, but to date the underlying genes have not been identified. RESULTS: We have investigated a gene co-expression network activated in response to F. graminearum using RNA-seq data from near-isogenic lines, harboring either the resistant or the susceptible allele for Fhb1 and Qfhs.ifa-5A. The network identified pathogen-responsive modules, which were enriched for differentially expressed genes between genotypes or different time points after inoculation with the pathogen. Central gene analysis identified transcripts associated with either QTL within the network. Moreover, we present a detailed gene expression analysis of four gene families (glucanases, NBS-LRR, WRKY transcription factors and UDP-glycosyltransferases), which take prominent roles in the pathogen response. CONCLUSIONS: A combination of a network-driven approach and differential gene expression analysis identified genes and pathways associated with Fhb1 and Qfhs.ifa-5A. We find G-protein coupled receptor kinases and biosynthesis genes for jasmonate and ethylene earlier induced for Fhb1. Similarly, we find genes involved in the biosynthesis and metabolism of riboflavin more abundant for Qfhs.ifa-5A.

Isotope-assisted screening for iron-containing metabolites reveals a high degree of diversity among known and unknown siderophores produced by Trichoderma spp.

Due to low iron availability under environmental conditions, many microorganisms excrete iron-chelating agents (siderophores) to cover their iron demands. A novel screening approach for the detection of siderophores using liquid chromatography coupled to high-resolution tandem mass spectrometry was developed to study the production of extracellular siderophores of 10 wild-type Trichoderma strains. For annotation of siderophores, an in-house library comprising 422 known microbial siderophores was established. After 96 h of cultivation, 18 different iron chelators were detected. Four of those (dimerum acid, fusigen, coprogen, and ferricrocin) were identified by measuring authentic standards. cis-Fusarinine, fusarinine A and B, and des-diserylglycylferrirhodin were annotated based on high-accuracy mass spectral analysis. In total, at least 10 novel iron-containing metabolites of the hydroxamate type were found. On average Trichoderma spp. produced 12 to 14 siderophores, with 6 common to all species tested. The highest number (15) of siderophores was detected for the most common environmental opportunistic and strongly fungicidic species, Trichoderma harzianum, which, however, did not have any unique compounds. The tropical species T. reesei had the most distinctive pattern, producing one unique siderophore (cis-fusarinine) and three others that were present only in T. harzianum and not in other species. The diversity of siderophores did not directly correlate with the antifungal potential of the species tested. Our data suggest that the high diversity of siderophores produced by Trichoderma spp. might be the result of further modifications of the nonribosomal peptide synthetase (NRPS) products and not due to diverse NRPS-encoding genes.

Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat.

An untargeted screening strategy for the detection of biotransformation products of xenobiotics using stable isotopic labelling (SIL) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) is reported. The organism of interest is treated with a mixture of labelled and non-labelled precursor and samples are analysed by LC-HRMS. Raw data are processed with the recently developed MetExtract software for the automated extraction of corresponding peak pairs. The SIL-assisted approach is exemplified by the metabolisation of the Fusarium mycotoxin deoxynivalenol (DON) in planta. Flowering ears were inoculated with 100 µg of a 1 + 1 (v/v) mixture of non-labelled and fully labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides the known DON and DON-3-glucoside, which were confirmed by measurement of authentic standards, eight further DON-biotransformation products were found by the untargeted screening approach. Based on a mass deviation of less than ±5 ppm and MS/MS measurements, one of these products was annotated as DON-glutathione (GSH) conjugate, which is described here for the first time for wheat. Our data further suggest that two DON-GSH-related metabolites, the processing products DON-S-cysteine and DON-S-cysteinyl-glycine and five unknown DON conjugates were formed in planta. Future MS/MS measurements shall reveal the molecular structures of the detected conjugates in more detail.

Transgenic Arabidopsis thaliana expressing a barley UDP-glucosyltransferase exhibit resistance to the mycotoxin deoxynivalenol.

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of small grain cereal crops. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON). DON inhibits protein synthesis in eukaryotic cells and acts as a virulence factor during fungal pathogenesis, therefore resistance to DON is considered an important component of resistance against FHB. One mechanism of resistance to DON is conversion of DON to DON-3-O-glucoside (D3G). Previous studies showed that expression of the UDP-glucosyltransferase genes HvUGT13248 from barley and AtUGt73C5 (DOGT1) from Arabidopsis thaliana conferred DON resistance to yeast. Over-expression of AtUGt73C5 in Arabidopsis led to increased DON resistance of seedlings but also to dwarfing of transgenic plants due to the formation of brassinosteroid-glucosides. The objectives of this study were to develop transgenic Arabidopsis expressing HvUGT13248, to test for phenotypic changes in growth habit, and the response to DON. Transgenic lines that constitutively expressed the epitope-tagged HvUGT13248 protein exhibited increased resistance to DON in a seed germination assay and converted DON to D3G to a higher extent than the untransformed wild-type. By contrast to the over-expression of DOGT1 in Arabidopsis, which conjugated the brassinosteriod castasterone with a glucoside group resulting in a dwarf phenotype, expression of the barley HvUGT13248 gene did not lead to drastic morphological changes. Consistent with this observation, no castasterone-glucoside formation was detectable in yeast expressing the barley HvUGT13248 gene. This barley UGT is therefore a promising candidate for transgenic approaches aiming to increase DON and Fusarium resistance of crop plants without undesired collateral effects.

Advanced backcross QTL mapping of resistance to Fusarium head blight and plant morphological traits in a Triticum macha × T. aestivum population.

While many reports on genetic analysis of Fusarium head blight (FHB) resistance in bread wheat have been published during the past decade, only limited information is available on FHB resistance derived from wheat relatives. In this contribution, we report on the genetic analysis of FHB resistance derived from Triticum macha (Georgian spelt wheat). As the origin of T. macha is in the Caucasian region, it is supposed that its FHB resistance differs from other well-investigated resistance sources. To introduce valuable alleles from the landrace T. macha into a modern genetic background, we adopted an advanced backcross QTL mapping scheme. A backcross-derived recombinant-inbred line population of 321 BC(2)F(3) lines was developed from a cross of T. macha with the Austrian winter wheat cultivar Furore. The population was evaluated for Fusarium resistance in seven field experiments during four seasons using artificial inoculations. A total of 300 lines of the population were genetically fingerprinted using SSR and AFLP markers. The resulting linkage map covered 33 linkage groups with 560 markers. Five novel FHB-resistance QTL, all descending from T. macha, were found on four chromosomes (2A, 2B, 5A, 5B). Several QTL for morphological and developmental traits were mapped in the same population, which partly overlapped with FHB-resistance QTL. Only the 2BL FHB-resistance QTL co-located with a plant height QTL. The largest-effect FHB-resistance QTL in this population mapped at the spelt-type locus on chromosome 5A and was associated with the wild-type allele q, but it is unclear whether q has a pleiotropic effect on FHB resistance or is closely linked to a nearby resistance QTL.