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

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

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.     

Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum

Based on the intergenic sequences of Tri5-Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.     

A novel gene cluster in Fusarium graminearum contains a gene that contributes to butenolide synthesis

The development of expressed sequence tag (EST) databases, directed transformation and a sequenced genome has facilitated the functional analysis of Fusarium graminearum genes. Extensive analysis of 10,397 ESTs, derived from thirteen cDNA libraries of F. graminearum grown under diverse conditions, identified a novel cluster of eight genes (gene loci fg08077-fg08084) located within a 17kb region of genomic sequence contig 1.324. The expression of these genes is concomitantly up-regulated under growth conditions that promote mycotoxin production. Gene disruption and add-back experiments followed by metabolite analysis of the transformants indicated that one of the genes, fg08079, is involved in butenolide synthesis. The mycotoxin butenolide is produced by several Fusarium species and has been suggested, but not proven, to be associated with tall fescue toxicoses in grazing cattle. This is the first report of the identification of a gene involved in the biosynthetic pathway of butenolide.     

Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum

High-throughput MS/MS was used to identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads. In vitro secreted proteins were collected from the culture filtrates, and in planta proteins were collected by vacuum infiltration. A total of 289 proteins (229 in vitro and 120 in planta) were identified with high statistical confidence. Forty-nine of the in planta proteins were not found in any of the in vitro conditions. The majority (91-100%) of the in vitro proteins had predicted signal peptides, but only 56% of the in planta proteins. At least 13 of the nonsecreted proteins found only in planta were single-copy housekeeping enzymes, including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta but not in vitro secretome might indicate that significant fungal lysis occurs during pathogenesis. On the other hand, several of the proteins lacking signal peptides that were found in planta have been reported to be potent immunogens secreted by animal pathogenic fungi, and therefore could be important in the interaction between F. graminearum and its host plants.     

Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.     

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F.?poae isolates. A total of 125 F.?poae, four F.?cerealis, two F.?culmorum, one F.?langsethiae, one F.?sporotrichioides and seven F.?graminearum, plus F.?austroamericanum, F.?meridionale, F.?graminearum sensu stricto and F.?cortaderiae from the NRRL collection were analysed, and only F.?poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F.?poae and other Fusarium species with a negative result for the specific reaction ( F.?graminearum, F.?oxysporum, F.?chlamydosporum, F.?sporotrichioides, F.?equiseti and F.?acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F.?poae isolates.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

中国小麦赤霉病菌优势种——禾谷镰刀菌产毒素能力的研究

试验测定了分离自中国小麦赤霉病常发生地区病麦穗上的47个禾谷镰刀菌(Fusariumgraminearum)菌株的产毒紊能力。结果表明,它们可以产生25种包括单端孢霉烯族化合物(Trichothecenes)、倍半萜类化合物(sesquiterpenes)、赤霉烯酮(Zearalenone)和丁烯羟酸内酯(Butenolide)等类的已知次生代谢物。这些菌株属于化学型 I,其中,来自我国温暖麦区的菌株都属化学型IA (deoxynivalenol,3-acetyl),并在气候冷凉地区发现化学型IB(de-oxynivalenol,15-acetyl)菌株。     

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.     

Comparative Proteomic Profiling of Methicillin‐Susceptible and Resistant Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin‐susceptible and methicillin‐resistant S. aureus (MSSA; MRSA) recovered from non‐healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty‐two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.