The transcriptome of Fusarium graminearum during the infection of wheat.

Fusarium graminearum causes head blight disease in wheat and barley. To help understand the infection process on wheat, we studied global gene expression of F. graminearum in a time series from 24 to 196 h after inoculation, compared with a noninoculated control. The infection was rapid and, after 48 h, over 4,000 fungal genes were expressed. The number of genes expressed increased over time up to 96 h (>8,000 genes), and then declined at the 144- and 192-h post-inoculation time points. After subtraction of genes found expressed on complete medium, during carbon or nitrogen starvation, and on barley, only 355 were found exclusively expressed in wheat, mostly genes with unknown function (72.6%). These genes were mainly found in single-nucleotide polymorphism-enriched islands on the chromosomes, suggesting a higher evolutionary selection pressure. The annotated genes were enriched in functional groups predicted to be involved in allantoin and allantoate transport, detoxification, nitrogen, sulfur and selenium metabolism, secondary metabolism, carbohydrate metabolism, and degradation of polysaccharides and ester compounds. Several putative secreted virulence factors were also found expressed in wheat.

Global gene regulation by Fusarium transcription factors Tri6 and Tri10 reveals adaptations for toxin biosynthesis.

Trichothecenes are isoprenoid mycotoxins produced in wheat infected with the filamentous fungus Fusarium graminearum. Some fungal genes for trichothecene biosynthesis (Tri genes) are known to be under control of transcription factors encoded by Tri6 and Tri10. Tri6 and Tri10 deletion mutants were constructed in order to discover additional genes regulated by these factors in planta. Both mutants were greatly reduced in pathogenicity and toxin production and these phenotypes were largely restored by genetic complementation with the wild-type gene. Transcript levels for over 200 genes were altered > or = twofold for Deltatri6 or Deltatri10 mutants including nearly all known Tri genes. Also reduced were transcript levels for enzymes in the isoprenoid biosynthetic pathway leading to farnesyl pyrophosphate, the immediate molecular precursor of trichothecenes. DNA sequences 5' to isoprenoid biosynthetic genes were enriched for the Tri6p DNA binding motif, YNAGGCC, in F. graminearum but not in related species that do not produce trichothecenes. To determine the effect of trichothecene metabolites on gene expression, cultures were treated with trichodiene, the first metabolic intermediate specific to the trichothecene biosynthetic pathway. A total of 153 genes were upregulated by added trichodiene and were significantly enriched for genes likely involved in cellular transport. Differentially regulated genes will be targeted for functional analysis to discover additional factors involved in toxin biosynthesis, toxin resistance and pathogenesis.

Conidial germination in the filamentous fungus Fusarium graminearum.

The ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight disease of wheat and barley. To understand early developmental stages of this organism, we followed the germination of macroconidia microscopically to understand the timing of key events. These events, recorded after suspension of spores in liquid germination medium, included spore swelling at 2h, germination tube emergence and elongation from conidia at 8h and hyphal branching at 24h. To understand changes in gene expression during these developmental changes, RNA was isolated from spores and used to interrogate the F. graminearum Affymetrix GeneChip. RNAs corresponding to 5813 genes were detected in fresh spores and 5146, 5249 and 5993, respectively, in spores incubated in germination medium after 2, 8 or 24h (P<0.001). Gene expression data were used to predict the cellular and physiological state of each developmental stage for known processes. Predictions were confirmed microscopically for several previously unreported developmental events such as manifestation of peroxisomes in fresh spores and nuclear division resulting in binuclear cells within macroconidia prior to spore germination. Knowledge of stage-specific gene expression and changes in gene expression levels between developmental stages are an important first step to understanding the molecular mechanisms responsible for spore germination and development.

The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

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.

Properties of unpaired DNA required for efficient silencing in Neurospora crassa.

The presence of unpaired copies of a gene during meiosis triggers silencing of all copies of the gene in the diploid ascus cell of Neurospora. This phenomenon is called meiotic silencing and on the basis of genetic studies appears to be a post-transcriptional gene silencing (PTGS) mechanism. Previously, meiotic silencing was defined to be induced by the presence of a DNA region lacking an identical segment in the homologous chromosome. However, the determinants of unpaired DNA remained a mystery. Using the Ascospore maturation-1 (Asm-1) gene, we defined what needs to be "unpaired" to silence a gene. For efficient silencing, an unpaired region of DNA needs to be of a sufficient size and contain homology to the reporter transcript. The greater the size of the loop and the larger the homology to the reporter transcript, the better the resulting meiotic silencing is. Conversely, regions not containing homology to the transcript, e.g., intergenic regions, did not silence the reporter. Surprisingly, unpaired fragments lacking a canonical promoter silenced the reporter. Additionally, we detected the unpairing-dependent loss of a transcript during meiotic silencing. Our observations further support a PTGS mechanism for meiotic silencing and offer insight into the evolutionary consequences resulting from this novel meiotic checkpoint.

Unpaired genes do not silence their paired neighbors.

During meiotic chromosome pairing, a loop of unpaired DNA induces the silencing of all paired and unpaired homologous DNA via meiotic silencing, an RNA-mediated post-transcriptional gene-silencing mechanism. To test the effect of unpaired DNA on adjacent genes, we constructed strains containing the DNA of a transformation marker integrated immediately downstream of the Ascospore maturation-1 ( Asm-1) gene and tested whether this unpaired DNA silences asm-1(+). We conclude that unpaired downstream DNA has no effect on Asm-1 expression during meiosis or ascospore development, which suggests that the silencing signal produced by unpaired DNA does not propagate onto adjacent paired regions.