Disruption of the GABA shunt affects mitochondrial respiration and virulence in the cereal pathogen Fusarium graminearum.

The cereal pathogen Fusarium graminearum threatens food and feed production worldwide. It reduces the yield and poisons the remaining kernels with mycotoxins, notably deoxynivalenol (DON). We analyzed the importance of gamma-aminobutanoic acid (GABA) metabolism for the life cycle of this fungal pathogen. GABA metabolism in F. graminearum is partially regulated by the global nitrogen regulator AreA. Genetic disruption of the GABA shunt by deletion of two GABA transaminases renders the pathogen unable to utilize the plant stress metabolites GABA and putrescine. The mutants showed increased sensitivity against oxidative stress, GABA accumulation in the mycelium, downregulation of two key enzymes of the TCA cycle, disturbed potential gradient in the mitochondrial membrane and lower mitochondrial oxygen consumption. In contrast, addition of GABA to the wild type resulted in its rapid turnover and increased mitochondrial steady state oxygen consumption. GABA concentrations are highly upregulated in infected wheat tissues. We conclude that GABA is metabolized by the pathogen during infection increasing its energy production, whereas the mutants accumulate GABA intracellularly resulting in decreased energy production. Consequently, the GABA mutants are strongly reduced in virulence but, because of their DON production, are able to cross the rachis node.

A GFP-expressing minigenome of a chrysovirus replicating in fungi.

The Fusarium graminearum virus China 9 (FgV-ch9) is a member of the genus Betachrysovirus in the Chrysoviridae family and causes hypovirulence in its host, Fusarium graminearum, the causal agent of Fusarium head blight. Although insights into viral biology of FgV-ch9 have expanded in recent years, questions regarding the function of virus-encoded proteins, cis-acting elements, and virus transmission are yet to be answered. Therefore, we developed a tool for the establishment of an artificial 6th segment of FgV-ch9, which encodes a GFP gene flanked by the non-translated regions of FgV-ch9 segment 1. Subsequently, we have proved successful encapsidation of this artificial segment into virus particles as well as its horizontal transmission. Expression of GFP was further verified via immunoassay and life cell imaging. Thus far, we were able to establish for the first time a mini-replicon system for segmented dsRNA viruses replicating in fungi.

Fast preparation of high-quality viral dsRNA from fungal tissue by commercial nucleic acid extraction kits.

The genomes of most known mycoviruses consist of double stranded RNA (dsRNA) or single stranded RNA (ssRNA). Therefore, for all aspects of mycovirology, the research is highly dependent on the quality and quantity of RNA either by the extraction of genomic dsRNA or dsRNA as a replicating intermediate. A common procedure to extract dsRNA is its binding on a cellulose matrix after a phenol/chloroform purification step. A commercial kit for dsRNA extraction facilitated the researchers´ daily work, but is not available anymore. To extract nucleic acids in a standardized good quality and quantity from small amounts of starting material, we compared commercial kits for gDNA extraction to the kits for RNA extraction using fungal material with a high and a low virus titer. Here we show that viral dsRNA can be extracted using commercial gDNA kits from fungal tissue with a high and a low virus titer in the same quality and quantity as it was done with the discontinued dsRNA extraction kit.

Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus.

BACKGROUND: Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host-pathogen interaction can be studied in more detail. RESULTS: Here, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus, for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette. CONCLUSIONS: The invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen.

Developing kernel and rachis node induce the trichothecene pathway of Fusarium graminearum during wheat head infection.

The fungal pathogen Fusarium graminearum is the most common agent of Fusarium head blight (FHB) in small grain cereals and cob rot of maize. The threat posed by this fungus is due to a decrease in yield and, additionally, mycotoxin contamination of the harvested cereals. Among the mycotoxins, trichothecenes influence virulence of F. graminearum in a highly complex manner that is strongly host- as well as chemotype-specific. The factors inducing mycotoxin production during plant infection are still unknown. To evaluate the induction of the trichothecene pathway, the green fluorescence protein (GFP) gene was fused to the promoter of the TRI5 gene coding for the trichodiene synthase and integrated into the genome by homologous integration. The resulting mutant contains a fully functional TRI5 gene ensuring virulence on wheat and exhibits GFP driven by the endogenous TRI5 promoter. We are now able to monitor the induction of trichothecenes under real-time conditions. To localize the fungus in the plant tissue, the dsRed gene was integrated under constitutive control of the glycerol-3-phosphate dehydrogenase (gpdA) promoter. We are now able to show that, first, induction of GFP as well as trichothecene production in the reporter strain reflects TRI5 induction and trichothecene production in the wild type; second, expression of TRI5 is inducible during growth in culture; and, third, trichothecene production is not uniformly induced during the onset of infection but is tissue specific during fungal infection of wheat.

Different Hydrophobins of Fusarium graminearum Are Involved in Hyphal Growth, Attachment, Water-Air Interface Penetration and Plant Infection.

Hydrophobins (HPs) are small secreted fungal proteins possibly involved in several processes such as formation of fungal aerial structures, attachment to hydrophobic surfaces, interaction with the environment and protection against the host defense system. The genome of the necrotrophic plant pathogen Fusarium graminearum contains five genes encoding for HPs (FgHyd1-5). Single and triple FgHyd mutants were produced and characterized. A reduced growth was observed when the ΔFghyd2 and the three triple mutants including the deletion of FgHyd2 were grown in complete or minimal medium. Surprisingly, the growth of these mutants was similar to wild-type when grown under ionic, osmotic or oxidative stress conditions. All the mutant strains confirmed the ability to develop conidia and perithecia, suggesting that the FgHyds are not involved in normal development of asexual and sexual structures. A reduction in the ability of hyphae to penetrate through the water-air interface was observed for the single mutants ΔFghyd2 and ΔFghyd3 as well as for the triple mutants including the deletion of FgHyd2 and FgHyd3. Besides, ΔFghyd3 and the triple mutant ΔFghyd234 were also affected in the attachment to hydrophobic surface. Indeed, wheat infection experiments showed a reduction of symptomatic spikelets for ΔFghyd2 and ΔFghyd3 and the triple mutants only when spray inoculation was performed. This result could be ascribed to the affected ability of mutants deleted of FgHyd2 and FgHyd3 to penetrate through the water-air interface and to attach to hydrophobic surfaces such as the spike tissue. This hypothesis is strengthened by a histological analysis, performed by fluorescence microscopy, showing no defects in the morphology of infection structures produced by mutant strains. Interestingly, triple hydrophobin mutants were significantly more inhibited than wild-type by the treatment with a systemic triazole fungicide, while no defects at the cell wall level were observed.

Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence.

Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A.

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.

SUMMARY 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.