Biomedical subjects

Frances Trail

Publications and source records attributed to Frances Trail.

11 recordsLinked to original sources

PubMed2006

Microarray analysis of transcript accumulation during perithecium development in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).

Gibberella zeae (anamorph Fusarium graminearum) is the causal agent of Fusarium head blight (FHB) of wheat and barley in the United States. Ascospores forcibly discharged from mature fruiting bodies, the perithecia, serve as the primary inoculum for FHB epidemics. To identify genes important for perithecium development and function, a cDNA microarray that covered 11% of the G. zeae genome was constructed. The microarray was used to measure changes in transcription levels of genes expressed during three successive stages of perithecium development. When compared with vegetative mycelia, 651 (31%) cDNA clones showed changes in transcript levels in at least one of the three developmental stages. During perithecium development, 263 (13%) cDNA clones showed temporal changes in transcript profiles. Transcripts that showed the greatest changes in levels in maturing perithecia belonged to genes in the FunCat main functional categories of cell rescue, metabolism, cell type differentiation, energy, and cellular transport. For genes related to metabolism and cell type differentiation, transcripts showed the highest levels in immature perithecia, whereas for cellular transport-related genes, transcripts showed the highest levels in mature perithecia. This study represents the first large-scale investigation of both spatial and temporal changes in transcript levels during perithecium development. It provides clear evidence that the sexual development in fungi is a complex, multigenic process and identifies genes involved in sexual development of this agriculturally important fungus.

Expressed Sequence Tags↗

PubMed2006

Development of a Fusarium graminearum Affymetrix GeneChip for profiling fungal gene expression in vitro and in planta.

Recently the genome sequences of several filamentous fungi have become available, providing the opportunity for large-scale functional analysis including genome-wide expression analysis. We report the design and validation of the first Affymetrix GeneChip microarray based on the entire genome of a filamentous fungus, the ascomycetous plant pathogen Fusarium graminearum. To maximize the likelihood of representing all putative genes (approximately 14,000) on the array, two distinct sets of automatically predicted gene calls were used and integrated into the online F. graminearum Genome DataBase. From these gene sets, a subset of calls was manually annotated and a non-redundant extract of all calls together with additional EST sequences and controls were submitted for GeneChip design. Experiments were conducted to test the performance of the F. graminearum GeneChip. Hybridization experiments using genomic DNA demonstrated the usefulness of the array for experimentation with F. graminearum and at least four additional pathogenic Fusarium species. Differential transcript accumulation was detected using the F. graminearum GeneChip with treatments derived from the fungus grown in culture under three nutritional regimes and in comparison with fungal growth in infected barley. The ability to detect fungal genes in planta is surprisingly sensitive even without efforts to enrich for fungal transcripts. The Plant Expression Database (PLEXdb, http://www.plexdb.org) will be used as a public repository for raw and normalized expression data from the F. graminearum GeneChip. The F. graminearum GeneChip will help to accelerate exploration of the pathogen-host pathways that may involve interactions between pathogenicity genes in the fungus and disease response in the plant.

Computational Biology↗

PubMed2006

Genomic analysis of host-pathogen interaction between Fusarium graminearum and wheat during early stages of disease development.

Fusarium graminearum strains responsible for causing the plant disease Fusarium head blight vary greatly in their ability to cause disease and produce mycotoxins on wheat. With the goal of understanding fungal gene expression related to pathogenicity, three cDNA libraries were created by suppression subtractive hybridization using wheat heads inoculated with a highly aggressive strain and either water or a less aggressive strain of this pathogen. Eighty-four fungal genes expressed during initial disease development were identified. The probable functions of 49 of these genes could be inferred by bioinformatic analysis. Thirty-five ESTs had no known homologues in current databases and were not identified by ab initio gene prediction methods. These ESTs from infected wheat heads probably represent F. graminearum genes that previously were not annotated. Four genes represented in one of these libraries were selected for targeted gene replacement, leading to the characterization of a two-component response regulator homologue involved in pathogenicity of the fungus. The mutants for this gene showed reduced sporulation and delayed spread of Fusarium head blight on wheat.

Computational Biology↗

PubMed2006

Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae.

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.

Gene Deletion↗

PubMed2005

Ejection mechanics and trajectory of the ascospores of Gibberella zeae (anamorph Fuarium graminearum).

Since wind speed drops to zero at a surface, forced ejection should facilitate spore dispersal. But for tiny spores, with low mass relative to surface area, high ejection speed yields only a short range trajectory, so pernicious is their drag. Thus, achieving high speeds requires prodigious accelerations. In the ascomycete Gibberella zeae, we determined the launch speed and kinetic energy of ascospores shot from perithecia, and the source and magnitude of the pressure driving the launch. We asked whether the pressure inside the ascus suffices to account for launch speed and energy. Launch speed was 34.5 ms-1, requiring a pressure of 1.54 MPa and an acceleration of 870,000 g--the highest acceleration reported in a biological system. This analysis allows us to discount the major sugar component of the epiplasmic fluid, mannitol, as having a key role in driving discharge, and supports the role of potassium ion flux in the mechanism.

Gibberella↗

PubMed2005

Role of cis-acting sites NorL, a TATA box, and AflR1 in nor-1 transcriptional activation in Aspergillus parasiticus.

The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus Aspergillus. nor-1 encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (AflR1). Proteins in Aspergillus parasiticus cell extracts and AflR expressed in Escherichia coli do not bind to A. parasiticus AflR1 in vitro, so it was not clear if this site was required for nor-1 expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in nor-1 expression in A. parasiticus and identified additional cis-acting sites required for maximum nor-1 transcriptional activation. Deletion and substitution of AflR1 in the nor-1 promoter in A. parasiticus nor-1::GUS reporter strains showed that this site is required for nor-1 transcriptional activation in vivo. Substitution of a putative TATA box in the nor-1 promoter resulted in nondetectable beta-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel cis-acting site, designated NorL, between residues -210 and -238 that was required for maximum nor-1 transcriptional activation in A. parasiticus grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific NorL-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the NorL site appears only once in the aflatoxin gene cluster, its association with the nor-1 promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.

Aflatoxins↗

PubMed2005

Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.

Amino Acid Sequence↗

PubMed2003

Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum).

Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world. To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX. Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression. Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the "unclassified" category. Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries. Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N. crassa.

Expressed Sequence Tags↗

PubMed2002

Purification and characterization of mannitol dehydrogenase and identification of the corresponding cDNA from the head blight fungus, Gibberella zeae (Fusarium graminearum).

The mannitol-2-dehydrogenase (MtDH) from Gibberella zeae was purified and the corresponding cDNA identified. Purification of MtDH was accomplished using a combination of ammonium sulfate fractionation, anion exchange and dye-ligand chromatography. Final purification was achieved following electroelution from a native gel. Molecular mass determination based on SDS-PAGE indicated that the denatured protein was 29 kDa. Native protein mass was determined to be 110 kDa using gel permeation chromatography, indicating a tetrameric form. The pH optima for mannitol oxidation and fructose reductase activities were 9.0, and 7.0, respectively. Activity with sorbitol as the substrate was 21% of activity with mannitol. Kinetic parameters were determined by direct-linear plots of enzyme activity vs. substrate concentrations. Fructose concentrations above 600 mM and NADPH concentrations above 0.3 mM caused substrate inhibition. Comparisons of predicted amino acid sequences of several fungal MtDHs indicated high conservation within the phyla. A possible role for MtDH in generation of turgor pressure for forcible ascospore discharge is discussed.

Amino Acid Sequence↗

PubMed2002

Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.

Aflatoxins↗

PubMedDate not supplied

The development and differentiation of Gibberella zeae (anamorph: Fusarium graminearum) during colonization of wheat.

Worldwide, one of the most devastating pathogens of small grains is the head blight fungus, Gibberella zeae. Ascospore-laden perithecia of this fungus develop on mature cereal crops and crop debris and provide the primary inoculum of the disease. We characterize the process of colonization of wheat tissue that leads to perithecium production. Stems were colonized systemically and extensively following inoculation of the wheat head. Haploid mycelia moved down the vascular system and pith and then colonized the stem tissue radially. Dikaryotic hyphae developed at two distinct stages: in the xylem, in support of radial hyphal growth and in the chloremchyma, in support of perithecium development. Perithecium formation was initiated in association with stomatesand silica cells. Vascular occlusions prevented mycelia from colonizing the stem in 25% of inoculated plants. Implications of these findings are discussed for developing resistant cultivars and improving chemical control of the disease.

Fusarium↗