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

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

Abstract 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

Fusarium graminearum hydrophobins: role in fungal growth and plant infection.

Hydrophobins are small fungal-specific proteins which are present at the surface of aerial hyphae and spores. They may play a role 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 by masking the fungal cell wall. The plant pathogen Fusarium graminearum, a necrotrophic fungus which causes Fusarium head blight (FHB) of wheat, barley and other cereal grains, contains five hydrophobin genes. To determine their role in F. graminearum, single and triple mutants of the five genes were produced and characterized. Interestingly, Hyd1 is dramatically upregulated during initial epiphytical growth on wheat paleas and glumes. A reduction in hydrophobicity was observed for the single mutants 06hyd1 and 06hyd3 as well as the triple mutants including 06hyd1 or 06hyd3. Single disruptants 06hyd2, 06hyd4 or 06hyd5 behaved wild type-like. The single mutant 06hydr3 and the triple mutants including 06hydr3 showed a decrease in growth compared to the wild type in a complete or minimal growth medium. Surprisingly, a reduced growth was registered when these mutants were grown under osmotic stress conditions or in the presence of H2O2. In order to verify possible defects in their cell wall, the growth of the mutants is currently under scrutiny in the presence of \u3b2-1,3-glucanase, chitinase and two fungicides. Point inoculation of wheat spikes with all mutants showed no role of the hydrophobins during infection. Preliminary data suggest a reduction in virulence of the Hyd1 mutant after spray inoculation

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