Wheat Spikelets Research Articles

An expanded subfamily of G-protein-coupled receptor genes in Fusarium graminearum required for wheat infection.

The cAMP-PKA and MAP kinase pathways are essential for plant infection in the wheat head blight fungus Fusarium graminearum. To identify upstream receptors of these well-conserved signalling pathways, we systematically characterized the 105 G-protein-coupled receptor (GPCR) genes. Although none were required for vegetative growth, five GPCR genes (GIV1-GIV5) significantly upregulated during plant infection were important for virulence. The giv1 mutant was defective in the formation of specialized infection structures known as infection cushions, which was suppressed by application of exogenous cAMP and dominant active FST7 MEK kinase. GIV1 was important for the stimulation of PKA and Gpmk1 MAP kinase by compounds in wheat spikelets. GIV2 and GIV3 were important for infectious growth after penetration. Invasive hyphae of the giv2 mutant were defective in cell-to-cell spreading and mainly grew intercellularly in rachis tissues. Interestingly, the GIV2-GIV5 genes form a phylogenetic cluster with GIV6, which had overlapping functions with GIV5 during pathogenesis. Furthermore, the GIV2-GIV6 cluster is part of a 22-member subfamily of GPCRs, with many of them having in planta-specific upregulation and a common promoter element; however, only three subfamily members are conserved in other fungi. Taken together, F. graminearum has an expanded subfamily of infection-related GPCRs for regulating various infection processes.

Effects of Low Temperature at Booting Stage on Sucrose Metabolism and Endogenous Hormone Contents in Winter Wheat Spikelet.

Low spring temperatures often occur during the winter wheat booting stage, when the young ears are very sensitive to cold. In this study, we used two wheat varieties differing in cold sensitivity (sensitive variety Yangmai 18 and tolerant variety Yannong 19) to examine the effect of low temperature on wheat grain number at booting stage. Low temperature stress was simulated in an artificial climate chamber at 4°C for 60 h in 2016 and at 2, 0, or −2°C for 24 h in morphological assays, showing that the development of wheat spikelets was inhibited and floret growth was delayed following low temperature stress. However, an increase in the sucrose content of young panicles was also observed, and the activity of enzymes involved in sucrose metabolism was dynamically altered. Sucrose phosphate synthase activity was enhanced, and sucrose synthase activity significantly increased after treatment at 4 and 2°C, respectively. However, activities of sucrose synthase and invertase decreased with a reduction in temperature. Gene expression assays further revealed downregulation of TaSuS1 expression and upregulation of TaSuS2, while expression of CWINV was inhibited. Moreover, phytohormone content assays showed an increase in the content of abscisic acid in young wheat ears, but a decrease in the content of auxin and gibberellins. The grain number per spike and 1000-grain weight also showed a downward trend following low temperature stress. Overall, these findings suggest that low temperature at booting induces abscisic acid accumulation in winter wheat, altering the activity of the enzymes involved in sucrose metabolism, which leads to an accumulation of sucrose in the young ears, thereby having a negative effect on wheat production.

Inferring vascular architecture of the wheat spikelet based on resource allocation in the branched headt (bht-A1) near isogenic lines.

Substantial genetic and physiological efforts were made to understand the causal factors of floral abortion and grain filling problem in wheat. However, the vascular architecture during wheat spikelet development is surprisingly under-researched. We used the branched headt near-isogenic lines, FL-bht-A1-NILs, to visualise the dynamics of spikelet fertility and dry matter accumulation in spikelets sharing the same rachis node (henceforth Primary Spikelet, PSt, and Secondary Spikelet, SSt). The experiment was conducted after grouping FL-bht-A1-NILs into two groups, where tillers were consistently removed from one group. Our results show differential spikelet fertility and dry matter accumulation between the PSt and SSt, but also showed a concomitant improvement after de-tillering. This suggests a tight regulation of assimilate supply and dry matter accumulation in wheat spikelets. Since PSt and SSt share the same rachis node, the main vascular bundle in the rachis/rachilla is expected to bifurcate to connect each spikelet/floret to the vascular system. We postulate that the vascular structure in the wheat spikelet might even follow Murray's law, where the wide conduits assigned at the base of the spikelet feed the narrower conduits of the distal florets. We discuss our results based on the two modalities of the vascular network systems in plants.

Self-adversarial Training and Attention for Multi-task Wheat Phenotyping

Abstract. Phenotypic monitoring provides important data support for precision agriculture management. This study proposes a deep learning-based method to gain an accurate count of wheat ears and spikelets. The deep learning networks incorporate self-adversarial training and attention mechanism with stacked hourglass networks. Four stacked hourglass networks follow a holistic attention map to construct a generator of self-adversarial networks. The holistic attention maps enable the networks to focus on the overall consistency of the whole wheat. The discriminator of self-adversarial networks displays the same structure as the generator, which causes adversarial loss to the generator. This process improves the generator’s learning ability and prediction accuracy for occluded wheat ears. This method yields higher wheat ear count in the Annotated Crop Image Database (ACID) data set than the previous state-of-the-art algorithm. Keywords: Attention mechanism, Plant phenotype, Self-adversarial networks, Stacked hourglass.

Occurrence of Fusarium Spp. in Hulls and Grains of Different Wheat Species

The occurrence of selected Fusarium species was studied in wheat sample collections that involved common wheat, einkorn, emmer and spelt genotypes. The study using PCR (Polymerase Chain Reaction) assays aimed to identify the Fusarium species occurring in hulls and grains of evaluated wheat samples separately and to assess the role of hull in Fusarium spp. contamination of the wheat grain. The obtained results suggested that hulls play the role of a protective barrier against Fusarium spp. contamination and reduce the incidence of these fungi in wheat grain. The incidence of Fusarium spp. in grains decreased by 44.54% in F. avenaceum, 45.15% in F. graminearum, 54.83% in F. sporotrichoides, 55.22% in F. culmorum, 57.49% in F. poae and 85.07% in F. equiseti, compared to incidence of Fusarium spp. in hulls of hulled wheat species. In the case of common wheat, the incidence of Fusarium spp. in grains decreased by 21.43% in F. avenaceum, 29.16% in F. graminearum, 32.14% in F. poae, 39.13% in F. sporotrichoides, 40.91% in F. culmorum and 49.99% in F. equiseti. A lower Fusarium spp. occurrence in hulled wheats grain seems to be connected especially with morphology of hulled wheat spikelets with tight and hard hulls, which could be able to reduce spread of fungal hyphae in the spikelet tissue. At the same time, it is possible to suppose that narrow opening of hulled wheats flowers reduces entry of spores to flowers. Genotypes with high resistance to FHB (emmer wheat genotype Rudico, spelt wheat genotypes Rubiota, Alkor, Tauro) were found in hulled wheat species and since their high grain quality parameters and satisfying yields are already known, they could be good alternative to common wheat, mainly in organic cropping systems.

The soft glumes of common wheat are sterile-lemmas as determined by the domestication gene Q

The Q gene in common wheat encodes an APETALA2 (AP2) transcription factor that causes the free threshing attribute. Wheat spikelets bearing several florets are subtended by a pair of soft glumes that allow free liberation of seeds. In wild species, the glumes are tough and rigid, making threshing difficult. However, the nature of these “soft glumes”, caused by the domestication allele Q is not clear. Here, we found that over expression of Q in common wheat leads to homeotic florets at glume positions. We provide phenotypic, microscopy, and marker genes evidence to demonstrate that the soft glumes of common wheat are in fact lemma-like organs, or so-called sterile-lemmas. By comparing the structures subtending spikelets in wheat and other crops such as rice and maize, we found that AP2 genes may play conserved functions in grasses by manipulating vestigial structures, such as floret-derived soft glumes in wheat and empty glumes in rice. Conversion of these seemingly vegetative organs to reproductive organs may be useful in yield improvement of crop species.

Involvement of the two L-lactate dehydrogenase in development and pathogenicity in Fusarium graminearum.

Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD+. In this study, two L-type lactate dehydrogenase genes FgLDHL1 and FgLDHL2 were characterized in an ascomycete fungus Fusarium graminearum, a causal agent of wheat head blight. Both the single-gene deletion mutants of FgLDHL1 or FgLDHL2 exhibited phenotypic defects in vegetative growth, sporulation, spore germination, L-lactate biosynthesis and activity. Additionally, the two L-lactate dehydrogenases were involved in the utilization of carbon sources and maintenance of redox homeostasis during spore germination. Pathogenicity assays showed that ΔFgLDHL1 exhibits reduced virulence on wheat spikelets and on corn stigmas, suggesting that it was indirectly correlated with a reduced level of deoxynivalenol accumulation. These results indicate that FgLDHL1 and FgLDHL2 play multiple roles in the developmental processes and pathogenesis in F. graminearum, and help understand the functional diversity of D-/L-lactate dehydrogenase in phytopathogenic fungi.

A wheat cytochrome P450 enhances both resistance to deoxynivalenol and grain yield.

The mycotoxin deoxynivalenol (DON) serves as a plant disease virulence factor for the fungi Fusarium graminearum and F. culmorum during the development of Fusarium head blight (FHB) disease on wheat. A wheat cytochrome P450 gene from the subfamily CYP72A, TaCYP72A, was cloned from wheat cultivar CM82036. TaCYP72A was located on chromosome 3A with homeologs present on 3B and 3D of the wheat genome. Using gene expression studies, we showed that TaCYP72A variants were activated in wheat spikelets as an early response to F. graminearum, and this activation was in response to the mycotoxic Fusarium virulence factor deoxynivalenol (DON). Virus induced gene silencing (VIGS) studies in wheat heads revealed that this gene family contributes to DON resistance. VIGS resulted in more DON-induced discoloration of spikelets, as compared to mock VIGS treatment. In addition to positively affecting DON resistance, TaCYP72A also had a positive effect on grain number. VIGS of TaCYP72A genes reduced grain number by more than 59%. Thus, we provide evidence that TaCYP72A contributes to host resistance to DON and conclude that this gene family warrants further assessment as positive contributors to both biotic stress resistance and grain development in wheat.

Infection process and wheat response to Fusarium head blight caused by Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most important diseases of wheat in humid and warm areas. It reduces yield and seed quality significantly. Knowledge on fungal infection of wheat spikelets is essential in effective control of FHB disease. Adult plants of the cultivar FALAT and the line ERN92–4 were inoculated at anthesis stage by injection of 10μl spore suspension at concentration of 5 × 104 per ml in medial spikelets using point inoculation method. The number of infected spikelets was counted 7, 14, 21 and 28 days post inoculation (dpi). At 4, 8, 24, 48, 72, 84 and 120 h post inoculation (hpi), parts of infected spikelets were observed with light microscopy and TEM. Disease severity (percentage of infected spikelets per spike) in line ERN92–4 significantly differed from the cultivar FALAT at 14 dpi and 21 dpi. Hyphal colonization and germination of spores in line ERN92–4 spikelets were more than in FALAT spikelets. Light microscopy and TEM observations indicated that mycelium penetrated into the cells through the stoma and spread to other cells via cell wall or plasmodesmata. The fungus exhibited biotrophic and necrotrophic life styles at 24–84 and 120 hpi, respectively. Accumulation of lignin displayed a different pattern during the activation of plant defense responses, and showed brownish sites on the infected florets at 72 and 120 hpi. From 72 hpi, lignin accumulation was observed in both the examined cultivar and line, and elevated lignin accumulation reached by 120 hpi in both treatments. With the exception of the initial stages of infection of florets, defense response of none of the both genotypes was effective in the suppression of disease progress, as, F. graminearum is a necrotrophic fungus; however, lignin and O2− accumulations were increased at 4 to 72 hpi.

Molecular Characterization, Fitness, and Mycotoxin Production of Fusarium asiaticum Strains Resistant to Fludioxonil.

Fludioxonil is used in seedborne disease management of various fungal pathogens, including Fusarium asiaticum, the predominant causal agent of Fusarium head blight in China. In this study, we screened resistant strains from a large number of F. asiaticum strains collected from 2012 to 2016 and found that 4 of 1,000 field strains were highly resistant to fludioxonil. The 50% effective concentration values of the resistant strains and induced mutants ranged from 80 to >400 μg/ml. Compared with field-sensitive strains, all field-collected and laboratory-induced resistant strains exhibited fitness defects in traits including mycelial growth, conidial production, pathogenicity, and sensitivity to osmotic conditions. In the presence of fludioxonil, significantly higher glycerol accumulation was found in sensitive strains but not in resistant individuals. The fludioxonil-resistant strains produced lower amounts of glycerol in liquid culture and lower amounts of trichothecene mycotoxins in rice culture and inoculated wheat spikelets than the fludioxonil-sensitive strains. Sequence analyses of the key genes of the two-component histidine kinase signaling pathway showed various amino acid substitutions in the Os1, Os4, and Os5 genes between field-sensitive and resistant strains or mutants. The results of this study suggest a potential risk of fludioxonil resistance development and a possible influence of resistance mutations on fitness parameters and toxin production in F. asiaticum.

Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms.

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity.

Genes WHEAT FRIZZY PANICLE and SHAM RAMIFICATION 2 independently regulate differentiation of floral meristems in wheat

BackgroundInflorescences of wheat species, spikes, are characteristically unbranched and bear one sessile spikelet at a spike rachis node. Development of supernumerary spikelets (SSs) at rachis nodes or on the extended rachillas is abnormal. Various wheat morphotypes with altered spike morphology, associated with the development of SSs, present an important genetic resource for studies on genetic regulation of wheat inflorescence development.ResultsHere we characterized diploid and tetraploid wheat lines of various non-standard spike morphotypes, which allowed for identification of a new mutant allele of the WHEAT FRIZZY PANICLE (WFZP) gene that determines spike branching in diploid wheat Ttiticum monococcum L. Moreover, we found that the development of SSs and spike branching in wheat T. durum Desf. was a result of a wfzp-A/TtBH-A1 mutation that originated from spontaneous hybridization with T. turgidum convar. сompositum (L.f.) Filat. Detailed characterization of the false-true ramification phenotype controlled by the recessive sham ramification 2 (shr2) gene in tetraploid wheat T. turgidum L. allowed us to suggest putative functions of the SHR2 gene that may be involved in the regulation of spikelet meristem fate and in specification of floret meristems. The results of a gene interaction test suggested that genes WFZP and SHR2 function independently in different processes during spikelet development, whereas another spike ramification gene(s) interact(s) with SHR2 and share(s) common functions.ConclusionsSS mutants represent an important genetic tool for research on the development of the wheat spikelet and for identification of genes that control meristem activities. Further studies on different non-standard SS morphotypes and wheat lines with altered spike morphology will allow researchers to identify new genes that control meristem identity and determinacy, to elucidate the interaction between the genes, and to understand how these genes, acting in concert, regulate the development of the wheat spike.

Physiological and molecular responses of resistant and susceptible wheat cultivars to Fusarium graminearum mycotoxin extract

Fusarium graminearum, causing Fusarium head blight (FHB), is one of the most important diseases on cereals worldwide leading to a reduction in both grain yield and quality. Currently, there is limited knowledge about the physiological and molecular mechanisms involved in wheat resistance against F. graminearum mycotoxins. Crude culture extract of F. graminearum was used to determine the physiological and molecular responses of wheat cultivars ‘Falat’ and ‘Sumai3‘, susceptible and resistant to FHB, respectively. Point inoculation of crude extract in the wheat spikelets resulted in significant increases in hydrogen peroxide (H2O2) and malondialdehyde (MDA) content in both cultivars, most likely due to oxidative stress. A greater induction level and activity of H2O2, MDA, peroxidase (POX), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) was observed in the resistant versus the susceptible cultivar. In addition, quantitative real-time PCR indicated earlier and greater induction of PAL, pleiotropic drug resistance (PDR) and cytochrome P450 (CYP) gene expression in ‘Sumai3‘ compared with ‘Falat’. Responses associated with the resistant interaction in ‘Sumai3‘ were higher induction of superoxide dismutase (SOD) and POX and decreased activity of catalase, while decreased SOD activity was observed in ‘Falat’. Our data suggest that these responses could explain the higher degree of tolerance of ‘Sumai3‘ against F. graminearum.

Functional regulation of Q by microRNA172 and transcriptional co-repressor TOPLESS in controlling bread wheat spikelet density.

SummaryBread wheat (Triticum aestivum) spike architecture is an important agronomic trait. The Q gene plays a key role in the domestication of bread wheat spike architecture. However, the regulatory mechanisms of Q expression and transcriptional activity remain largely unknown. In this study, we show that overexpression of bread wheat tae‐miR172 caused a speltoid‐like spike phenotype, reminiscent of that in wheat plants with the q gene. The reduction in Q transcript levels in the tae‐miR172 overexpression transgenic bread wheat lines suggests that the Q expression can be suppressed by tae‐miR172 in bread wheat. Indeed, our RACE analyses confirmed that the Q mRNA is targeted by tae‐miR172 for cleavage. According to our analyses, the Q protein is localized in nucleus and confers transcriptional repression activity. Meanwhile, the Q protein could physically interact with the bread wheat transcriptional co‐repressor TOPLESS (TaTPL). Specifically, the N‐terminal ethylene‐responsive element binding factor‐associated amphiphilic repression (EAR) (LDLNVE) motif but not the C‐terminal EAR (LDLDLR) motif of Q protein mediates its interaction with the CTLH motif of TaTPL. Moreover, we show that the N‐terminal EAR motif of Q protein is also essentially required for the transcriptional repression activity of Q protein. Taken together, we reveal the functional regulation of Q protein by tae‐miR172 and transcriptional co‐repressor TaTPL in controlling the bread wheat spike architecture.

Agronomic characterization and genetic analysis of the supernumerary spikelet in tetraploid wheat (Triticum turgidum L.)

The supernumerary spikelets (SS) characters of tetraploid wheat (Triticum turgidum L.) resulting in more spikelets and kernels per spike, thus enhancing sink capacity may contribute to potential wheat yield improvement. In order to investigate the effect of different SS types on agronomic characters and understand the genetic base of SS phenotype in tetraploid wheat, near isogenic lines (NILs), bh-50 with normal spikelets (NS), bh-51 with four-rowed spikelets (FRS), bh-52 with short-ramified spikelets (SRS), and bh-53 with long-ramified spikelets (LRS) in a Triticum durum cv. ZY1286 genetic background were developed by continuous backcrossing. Agronomic characters showed that the SS phenotype lines, bh-51, bh-52 and bh-53 have significant increase in the number of spikelets and grains per spike compared with the NS phenotype line bh-50 (P<0.05), and bh-53 line showed much more increase than those of bh-51 and bh-52. However, bh-53 had the lowest grain weight and the longest spike development stage than those of other spike phenotypes. These results indicated that the different SS types have different effects on the agronomic and spike characters. Genetic analysis through bh-50/bh-51 and bh-51/bh-53 F2 populations showed that a recessive major gene controlled the spike architecture to transform from NS to FRS, and a dominant major gene determined the change of spike phenotype from FRS to RS. DNA sequences of TtBH/WFZP ortholog on chromosome 2AS revealed that a single nucleotide polymorphism (SNP) substitution happened in the open reading frame (ORF) region of all the SS tetraploid wheat accessions, which may lead to the generation of lateral meristems between glume and lemma during the immature spike development. While the fates of the lateral meristems, developing into lateral spikelets or branched spikelets, may be determined by another major gene. Our results presented here may advance our understanding and knowledge of the genes and genetic pathways determining the spike architecture development in wheat.

MiR156-Targeted SBP-Box Transcription Factors Interact with DWARF53 to Regulate TEOSINTE BRANCHED1 and BARREN STALK1 Expression in Bread Wheat

Genetic and environmental factors affect bread wheat (Triticum aestivum) plant architecture, which determines grain yield. In this study, we demonstrate that miR156 controls bread wheat plant architecture. We show that overexpression of tae-miR156 in bread wheat cultivar Kenong199 leads to increased tiller number and severe defects in spikelet formation, probably due to the tae-miR156-mediated repression of a group of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Furthermore, we found that the expression of two genes TEOSINTE BRANCHED1 (TaTB1) and BARREN STALK1 (TaBA1), whose orthologous genes in diverse plant species play conserved roles in regulating plant architecture, is markedly reduced in the tae-miR156-OE bread wheat plants. Significantly, we demonstrate that the strigolactone (SL) signaling repressor DWARF53 (TaD53), which physically associates with the transcriptional corepressor TOPLESS, can directly interact with the N-terminal domains of miR156-controlled TaSPL3/17. Most importantly, TaSPL3/17-mediated transcriptional activation of TaBA1 and TaTB1 can be largely repressed by TaD53 in the transient expression system. Our results reveal potential association between miR156-TaSPLs and SL signaling pathways during bread wheat tillering and spikelet development.

Screening of wheat endophytes as biological control agents against Fusarium head blight using two different in vitro tests

In order to find biological control agents (BCAs) for the management of Fusarium head blight (FHB), a major disease on wheat crops worldwide, 86 microorganisms isolated from inner tissues of wheat plants were discriminated for their ability to inhibit the growth of Fusarium graminearum and Fusarium culmorum by in vitro dual culture assays. A group of 22 strains appeared very effective to inhibit F. graminearum (inhibition of 30–51%) and they were also globally effective in controlling F. culmorum (inhibition of 15–53%). Further evaluation of a subselection of strains by screening on detached spikelets in vitro confirmed three species, namely Phoma glomerata, Aureobasidium proteae and Sarocladium kiliense, that have not yet been reported for their efficacy against Fusarium spp., indicating that looking for BCAs toward FHB among wheat endophytes proved to be promising. The efficacy of some strains turned out different between both in vitro screening approaches, raising the importance of finding the most appropriate screening approach for the search of BCAs. This study pointed out the interest of the test on detached wheat spikelets that provided information about a potential pathogenicity, the growth capacity and efficacy of the endophyte strains on the targeted plant, before testing them on whole plants.

Study on early inflorescence development in bread wheat (T. aestivum L.) lines with non-standard SCR-morphotype

Features of wheat (Triticum aestivum L.) inflorescence development define its architecture and have an impact on yield potential. Wheat lines and forms with altered inflorescence morphology are important genetic resources for the study on the genetic mechanisms underlying plant developmental programs and inflorescence architecture; they are also important for practical use to increase productivity. Normally, wheat spikelets are arranged in two parallel rows along the spike axis. The SCR (screwed spike rachis) lines represent a non-standard morphotype, which is characterized by a spiral arrangement of spikelets along the spiked rachis. The study of the early stages of the inflorescence development in SCR-lines using light and scanning electron microscopy revealed that the spiral arrangement of spikelets were not related to changes at the early stage of inflorescence development, and resulted from spiral growth of spike rachis cells at later stages of spike growth. Thus, the spiral arrangement of spikelets in cereal inflorescence may have resulted not only from peculiarities of the mutual arrangement of spikelet meristems (phyllotaxis), but also from cell growth features at later stages of inflorescence growth. It was shown that SCR is inherited as a dominant monogenic trait; its expression can be modified by genotypic background. The SCR-lines characterized using light and scanning electron microscopy represent an important genetic resource for further study of the molecular-genetic mechanisms determining plant architecture. Furthermore, they can be used to develop wheat lines and cultivars with new inflorescence phenotypes.

The septins FaCdc3 and FaCdc12 are required for cytokinesis and affect asexual and sexual development, lipid metabolism and virulence in Fusarium asiaticum.

Septins are a highly conserved family of GTP-binding proteins that contribute to many cellular and metabolic functions, including cell polarity, cytokinesis, cell morphogenesis and pathogenesis. In this study, we characterized the septins FaCdc3 and FaCdc12 in the filamentous fungus Fusarium asiaticum. The functions of FaCdc3 and FaCdc12 were evaluated by constructing deletion mutants of FaCdc3 and FaCdc12, designated ΔFaCdc3-5 and ΔFaCdc12-71, respectively. The deletion mutants exhibited a reduced rate of mycelial growth, increased aerial hyphae formation, irregularly shaped hyphae, reduced conidiation and a lack of sexual reproduction in wheat kernels. Histochemical analysis revealed that the conidia and hyphae of ΔFaCdc3-5 and ΔFaCdc12-71 formed large lipid droplets (LDs). ΔFaCdc3-5 and ΔFaCdc12-71 also exhibited increased resistance to agents that induce osmotic stress and damage the cell membrane and cell wall. In addition, the hyphae and conidia of the two mutants formed fewer septa than those of the wild-type and exhibited aberrant nuclear distribution. Pathogenicity assays showed that ΔFaCdc3-5 and ΔFaCdc12-71 exhibited reduced virulence on wheat spikelets, which was indirectly correlated with a reduced level of deoxynivalenol accumulation. All of these defects were restored by genetic complementation of the two mutants with the parental FaCdc3 and FaCdc12. These results indicate that FaCdc3 and FaCdc12 play a critical role in various cellular processes in F. asiaticum.

Glutathione-Conjugates of Deoxynivalenol in Naturally Contaminated Grain Are Primarily Linked via the Epoxide Group.

A glutathione (GSH) adduct of the mycotoxin 4-deoxynivalenol (DON), together with a range of related conjugates, has recently been tentatively identified by LC-MS of DON-treated wheat spikelets. In this study, we prepared samples of DON conjugated at the 10- and 13-positions with GSH, Cys, CysGly, γ-GluCys and N-acetylcysteine (NAC). The mixtures of conjugates were used as standards for LC-HRMS analysis of one of the DON-treated wheat spikelet samples, as well as 19 Norwegian grain samples of spring wheat and 16 grain samples of oats that were naturally-contaminated with DON at concentrations higher than 1 mg/kg. The artificially-contaminated wheat spikelets contained conjugates of GSH, CysGly and Cys coupled at the olefinic 10-position of DON, whereas the naturally-contaminated harvest-ripe grain samples contained GSH, CysGly, Cys, and NAC coupled mainly at the 13-position on the epoxy group. The identities of the conjugates were confirmed by LC-HRMS comparison with authentic standards, oxidation to the sulfoxides with hydrogen peroxide, and examination of product-ion spectra from LC-HRMS/MS analysis. No γ-GluCys adducts of DON were detected in any of the samples. The presence of 15-O-acetyl-DON was demonstrated for the first time in Norwegian grain. The results indicate that a small but significant proportion of DON is metabolized via the GSH-conjugation pathway in plants. To our knowledge, this is the first report of in vivo conjugation of trichothecenes via their epoxy group, which has generally been viewed as unreactive. Because conjugation at the 13-position of DON and other trichothecenes has been shown to be irreversible, this type of conjugate may prove useful as a biomarker of exposure to DON and other 12,13-epoxytrichothecenes.