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

Espressione eterologa in Pichia pastoris di una xilanasi del fungo patogeno Fusarium graminearum e caratterizzazione preliminare dell'enzima ricombinante.

The fungal pathogen fusarium graminearum is the casual agent of fusarium head blight, a disease affecting some cereal species such as wheat

Cerato platanins (CP) of Fusarium graminearum induce defense responses in plant and are not essential for fungal virulence.

Cerato platanins (CP) belong to a family of small secreted fungal proteins with phytotoxic activity which seem to induce defense responses and necrosis in plants and contribute to Botrytis cinerea and Magnaporthe grisea virulence. In the genome of F. graminearum, a necrotrophic fungal pathogen which causes Fusarium head blight (FHB) disease of wheat, barley and other cereal grains, there are two genes (FGSG_10212 and FGSG_11205) putatively encoding for CP-like proteins that we cloned and heterologously expressed in Pichia pastoris. The recombinant proteins, native and boiled, resulted able to reduce the viscosity of carboxymethyl cellulose, in particular the FgCP11205. Treatments of Arabidopsis thaliana leaves with the two F. graminearum CPs induced necrotic symptoms, accumulation of H2O2 and expression of defense genes, specifically PR1, marker of salicylic acid signaling, and ERF1b, a transcriptional regulator of some ethylene-responsive genes. Being these CPs able to induce defense responses, we tested their effectiveness in increasing the resistance of A. thaliana to the fungal pathogen B. cinerea; treatments with both CPs determined a reduction of lesion size of about 30-40%. The expression of the two cp genes was analyzed by qPCR in the early stages of wheat spike infection and during in vitro growth and only the Fgcp10212 gene resulted strongly transcribed. To verify the contribution of F. graminearum CPs to fungal virulence, single and double gene knock-out mutants were produced and used to infect host plants such as wheat and soybean but their virulence resulted comparable to that of the wild-type strain

Fusarium graminearum SnodProt proteins protect fungal cell wall polysaccharides from enzymatic degradation

The genome of Fusarium graminearum, a necrotrophic fungal pathogen causing Fusarium head blight (FHB) disease of wheat, barley and other cereal grains, contains five genes putatively encoding for proteins similar to cerato platanins, which contribute to the virulence of the fungal pathogens Botrytis cinerea and Magnaporthe grisea. Two of them (FGSG_10212 and FGSG_11205) belong to the class of SnodProt proteins, with reported phytotoxic activity. To verify the contribution of the two F. graminearum SnodProt proteins to the infection process, single and double gene knock-out mutants were produced but no reduction in symptoms severity was observed compared to the wild type strain on both soybean and wheat spikes. Histological analysis performed by fluorescence microscopy on wheat spikelets infected with mutants constitutively expressing the dsRed confirmed that the F. graminearum SnodProt proteins do not contribute to fungal virulence. In particular, the formation of compound appressoria on wheat glumes was unchanged. Looking for other functions of these proteins, the double mutant was characterized by in vitro experiments. Wild type and mutants were similarly inhibited by salt and H2O2 stress. Surprisingly, the mutant grew better than wild type on carboxymethyl cellulose, while no difference was observed on glucose. Furthermore, conidia and mycelium of the mutant were more affected by treatments with chitinase and \u3b2-1,3-glucanase, thus indicating that the F. graminearum SnodProt proteins could protect fungal cell wall polysaccharides from enzymatic degradation

Involvement of the Fusarium graminearum cerato-platanin proteins in fungal growth and plant infection

The genome of Fusarium graminearum, a necrotrophic fungal pathogen causing Fusarium head blight (FHB) disease of wheat, barley and other cereal grains, contains five genes putatively encoding for proteins with a cerato-platanin domain. Cerato-platanins are small secreted cysteine-rich proteins possibly localized in the fungal cell walls and also contributing to the virulence. Two of these F. graminearum proteins (FgCPP1 and FgCPP2) belong to the class of SnodProt proteins which exhibit phytotoxic activity in the fungal pathogens Botrytis cinerea and Magnaporthe grisea. In order to verify their contribution during plant infection and fungal growth, single and double gene knock-out mutants were produced and no reduction in symptoms severity was observed compared to the wild type strain on both soybean and wheat spikes. Histological analysis performed by fluorescence microscopy onwheat spikelets infected with mutants constitutively expressing the dsRed confirmed that FgCPPs do not contribute to fungal virulence. In particular, the formation of compound appressoria on wheat paleas was unchanged. Looking for other functions of these proteins, the double mutant was characterized by in vitro experiments. The mutant was inhibited by salt and H2O2 stress similarly to wild type. Though no growth difference was observed on glucose, the mutant grew better than wild type on carboxymethyl cellulose. Additionally, the mutant's mycelium was more affected by treatments with chitinase and b-1,3- glucanase, thus indicating that FgCPPs could protect fungal cell wall polysaccharides from enzymatic degradation

The activity of the Botrytis cinerea endo-polygalacturonase PG1 is detected in berry skins and is required for full virulence during grape infection.

The necrotrophic fungal pathogen Botrytis cinerea is the causal agent of grey mould or Botrytis bunch rot in grapes. During the infection process this fungus secretes several cell-wall degrading enzymes, in particular endo-polygalacturonases (PGs) which are involved in the depolymerization of pectin, the main constituent of primary cell wall and middle lamella. The genome of B. cinerea contains six endo-PG encoding genes. Aim of the present work was to characterize the role of these enzymes during infection of grape berries. First, we studied the expression of the corresponding genes on berries of cv. Pinot blanc and Pinot noir. Among the genes analyzed by qPCR, only Bcpg2 was not expressed, while Bcpg1, encoding a basic isoform, showed the highest transcript levels at all time points analyzed and in both cvs. We also analyzed the PG activity produced by B. cinerea during grape berries infection by loading on a gel activity assay grinded berry skins: the BcPG1 isoform was detected but only as a weak band compared to other PGs produced by the fungus. However, since the BcPG1 has been previously demonstrated to be an important virulence factor in several host tissues although its role has never been investigated on grape berries, we performed infection experiments with a \u394BcPG1 knock-out mutant. A 20% reduction of symptoms caused by the \u394BcPG1 mutant was observed on both Pinot cv. and also on cv. Italia table grape, thus indicating that BcPG1 is required for full virulence on grape berries

Characterization of the role played by Magnaporthe oryzae polysaccharide monooxygenases and related enzymes during infection of rice.

In 2030 the global rice production is expected to increase to meet the demand of the growing world population. However, rice is severely affected by the blast disease caused by the fungus Magnaporthe oryzae, which can reduce by 10-30% the total annual rice production. In the early stages of the infection process, M. oryzae forms an infection structure called appressorium to break the plant cuticle and expresses many polysaccharide and lignin degrading enzymes; among them, polysaccharide monooxygenases (PMOs) degrade polysaccharides by an oxidative mechanism and could be important virulence factors for the fungus. The first objective of the project, which is part of the Scientific and Technological Cooperation Agreement between the Italian Ministry of Foreign Affairs and International Cooperation and the Department of International Cooperation of the Ministry of Science and Technology of Vietnam, is to identify the role played by M. oryzae PMOs and related enzymes during pathogenesis, with the final aim to develop new methods to control rice blast disease. In particular, candidate M. oryzae genes encoding PMOs and related enzymes have been identified by an in silico analysis of the fungal genome and their expression during the infection process, and particularly during appressorium formation, have been characterized by transcriptomic analysis. Knock-out mutants of the most expressed genes will be generated and their virulence will be evaluated on rice plants. Besides, the role played by the target enzymes on appressorium formation will be also evaluated

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

Looking for novel control measures against the rice fungal pathogen Pyricularia oryzae.

The filamentous fungus Pyricularia oryzae is the main causal agent of the rice blast disease, which accounts for 10-30% yield losses per year globally. The objective of this project, which is part of the Scientific and Technological Cooperation Agreement between the Italian Ministry of Foreign Affairs and International Cooperation and the Department of International Cooperation of the Ministry of Science and Technology of Vietnam, is to identify new pathogen targets and new molecules to control rice blast disease. P. oryzae uses a large number of degrading enzymes active on cell wall polysaccharides and lignin to penetrate and invade the rice plant tissues. Since these enzymes could be potential targets for plant inhibitors, candidate P. oryzae genes encoding enzymes particularly expressed during the infection process have been deleted from the fungal genome and the characterization of the obtained mutants is in progress to identify enzymes essential for fungal virulence on rice. In addition, new ecofriendly antimicrobial peptides, analogs of the natural Trichoderma longibrachiatum peptaibol, have been synthesized and tested in vitro against several P. oryzae strains from different geographic origin. The screening has allowed to identify some peptides very effective in inhibiting spore germination and fungal growth that could be used in vivo to confirm their efficacy in protecting rice from the blast disease