Transcriptomic and Ultrastructural Analyses of Pyricularia Oryzae Treated With Fungicidal Peptaibol Analogs of Trichoderma Trichogin.

Eco-friendly analogs of Trichogin GA IV, a short peptaibol produced by Trichoderma longibrachiatum, were assayed against Pyricularia oryzae, the causal agent of rice blast disease. In vitro and in vivo screenings allowed us to identify six peptides able to reduce by about 70% rice blast symptoms. One of the most active peptides was selected for further studies. Microscopy analyses highlighted that the treated fungal spores could not germinate and the fluorescein-labeled peptide localized on the spore cell wall and in the agglutinated cytoplasm. Transcriptomic analysis was carried out on P. oryzae mycelium 3 h after the peptide treatment. We identified 1,410 differentially expressed genes, two-thirds of which upregulated. Among these, we found genes involved in oxidative stress response, detoxification, autophagic cell death, cell wall biogenesis, degradation and remodeling, melanin and fatty acid biosynthesis, and ion efflux transporters. Molecular data suggest that the trichogin analogs cause cell wall and membrane damages and induce autophagic cell death. Ultrastructure observations on treated conidia and hyphae confirmed the molecular data. In conclusion, these selected peptides seem to be promising alternative molecules for developing effective bio-pesticides able to control rice blast disease.

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.

The Fusarium graminearum cerato-platanins loosen cellulose substrates enhancing fungal cellulase activity as expansin-like proteins.

Cerato-platanin proteins (CPPs) are small non-catalytic, cysteine-rich hydrophobic proteins produced by filamentous fungi. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease of wheat and other cereal grains, contains two genes putatively encoding for CPPs. To better characterize their features, the two FgCPPs were heterologously expressed in Pichia pastoris. The recombinant FgCPPs reduced the viscosity of a cellulose soluble derivate (carboxymethyl cellulose, CMC). The same effect was not observed on other polysaccharide substrates such as chitin, 1,3-ß-glucan, xylan and pectin. Indeed, differently from other fungal CPPs and similarly to expansins, FgCPPs are trapped by cellulose and not by chitin, thus suggesting that these proteins interact with cellulose. A double knock-out mutant deleted of both FgCPPs encoding genes produces much more cellulase activity than the corresponding wild type strain when grown on CMC, likely compensating the absence of FgCPPs. This result prompted us to investigate a possible synergistic effect of these proteins with fungal cellulases. The incubation of FgCPPs in the presence of a fungal cellulase (EC 3.2.1.4) determines an increased enzymatic activity on CMC, filter paper and wheat cell walls. The observation that FgCPPs act with a non-hydrolytic mechanism indicates that these proteins favor fungal cellulase activity in an expansin-like manner. Though the disruption of the FgCPP genes had no demonstrable impact on fungal virulence, our experimental data suggest their probable involvement in virulence, thus we refer to them as accessory virulence genes. Our results suggest also that the FgCPPs could be exploited for future biotechnological application in second-generation biofuels production on lignocellulosic biomasses rich in cellulose. Finally, we demonstrate that FgCPPs act as elicitors of defense responses on Arabidopsis leaves, increasing resistance to Botrytis cinerea infections.

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 on wheat 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 ß-1,3-glucanase, thus indicating that FgCPPs could protect fungal cell wall polysaccharides from enzymatic degradation.