Role of Fusarium graminearum cerato-platanin and hydrophobin proteins in fungal growth and plant infection

Cerato-platanins (CPPs) and hydrophobins (HPs) are small secreted non-catalytic cysteine-rich proteins typical of filamentous fungi. CPPs are possibly localized in the fungal cell walls, they are similar to plant expansins having carbohydrate binding/loosening properties with a non-enzymatic mechanism and can have phytotoxic activity. HPs, after being secreted as monomers from the hyphal apexes, cover the fungal surfaces with a hydrophobic layer and may be 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 by masking the fungal cell wall. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease (FHB) of wheat and other cereal grains, contains two genes putatively encoding for proteins with a CP domain, named fgcpp1 and fgcpp2, and five genes encoding for HPs, named FgHyd1-5. In order to verify their contribution during plant infection and fungal growth, single and multiple gene knock-out mutants were produced and characterized. Besides, the two F. graminearum CPPs (FgCPPs) were heterologously expressed to investigate their activity. The FgCPPs seem to be dispensable for fungal virulence but protect fungal cell wall polysaccharides from enzymatic degradation. The FgCPPs show a strong ability to reduce, mainly by a non-enzymatic mechanism, the viscosity of carboxymethyl cellulose (CMC), with higher affinity for substrates with medium/high viscosity and favour fungal cellulase activity. The observation that the double knock-out mutant ∆∆fgcpp1,2 grown on CMC produced more cellulase activity than the wild type suggests that the higher enzymatic activity produced by the mutant could compensate during infection for the absence of the FgCPPs activity on plant cellulose. The F. graminearum Hyd2 and Hyd3 are responsible of the hydrophobicity of aerial hyphae and are involved in adhesion of conidia to the host surface during the early stages of the infection process, as shown by the reduced virulence of the ∆hyd2 and ∆hyd3 on Triticum aestivum observed by spray inoculation. Interestingly, triple ∆hyd1,2,3, ∆hyd2,3,4 and ∆hyd2,3,5 mutants produced a reduced number of mature perithecia and showed defects at the cell wall level, being significantly more inhibited than the wild type by β-1,3-glucanase and more susceptible to tebuconazole, an ergosterol biosynthesis inhibitor fungicide. Based on our results, FgCPPs and FgHyds could be used as targets of new molecules in innovative disease management strategies aimed at increasing fungal susceptibility to plant defense proteins or at reducing fungal ability to adhere to host plant tissues. Furthermore, the ability of FgCPPs to loosen CMC and favor cellulase activity could make these proteins suitable for future potential applications in biofuel production using cellulose rich tissues as substrate

Role of Fusarium graminearum cerato-platanin and hydrophobin proteins in fungal growth and plant infection

Cerato-platanins (CPPs) and hydrophobins (HPs) are small secreted non-catalytic cysteine-rich proteins typical of filamentous fungi. CPPs are possibly localized in the fungal cell walls, they are similar to plant expansins having carbohydrate binding/loosening properties with a non-enzymatic mechanism and can have phytotoxic activity. HPs, after being secreted as monomers from the hyphal apexes, cover the fungal surfaces with a hydrophobic layer and may be 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 by masking the fungal cell wall. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease (FHB) of wheat and other cereal grains, contains two genes putatively encoding for proteins with a CP domain, named fgcpp1 and fgcpp2, and five genes encoding for HPs, named FgHyd1-5. In order to verify their contribution during plant infection and fungal growth, single and multiple gene knock-out mutants were produced and characterized. Besides, the two F. graminearum CPPs (FgCPPs) were heterologously expressed to investigate their activity. The FgCPPs seem to be dispensable for fungal virulence but protect fungal cell wall polysaccharides from enzymatic degradation. The FgCPPs show a strong ability to reduce, mainly by a non-enzymatic mechanism, the viscosity of carboxymethyl cellulose (CMC), with higher affinity for substrates with medium/high viscosity and favour fungal cellulase activity. The observation that the double knock-out mutant ∆∆fgcpp1,2 grown on CMC produced more cellulase activity than the wild type suggests that the higher enzymatic activity produced by the mutant could compensate during infection for the absence of the FgCPPs activity on plant cellulose. The F. graminearum Hyd2 and Hyd3 are responsible of the hydrophobicity of aerial hyphae and are involved in adhesion of conidia to the host surface during the early stages of the infection process, as shown by the reduced virulence of the ∆hyd2 and ∆hyd3 on Triticum aestivum observed by spray inoculation. Interestingly, triple ∆hyd1,2,3, ∆hyd2,3,4 and ∆hyd2,3,5 mutants produced a reduced number of mature perithecia and showed defects at the cell wall level, being significantly more inhibited than the wild type by β-1,3-glucanase and more susceptible to tebuconazole, an ergosterol biosynthesis inhibitor fungicide. Based on our results, FgCPPs and FgHyds could be used as targets of new molecules in innovative disease management strategies aimed at increasing fungal susceptibility to plant defense proteins or at reducing fungal ability to adhere to host plant tissues. Furthermore, the ability of FgCPPs to loosen CMC and favor cellulase activity could make these proteins suitable for future potential applications in biofuel production using cellulose rich tissues as substrate.Le cerato-platanine (CPP) e le idrofobine (HP) sono piccole proteine tipiche dei funghi filamentosi non catalitiche, secrete e ricche di cisteina. Le CPP sono probabilmente localizzate nelle pareti delle cellule fungine, sono simili alle espansine delle piante che hanno proprietà di allentamento dei carboidrati con meccanismo non enzimatico e possono avere attività fitotossica. Le HP, dopo essere state secrete come monomeri dagli apici delle ife fungine, coprono le superfici fungine con uno strato idrofobo e possono essere coinvolte in diversi processi come la formazione di strutture aeree fungine, l'attaccamento a superfici idrofobiche, l'interazione con l'ambiente e la protezione contro il sistema di difesa dell’ospite mascherando la parete cellulare dei funghi. Il genoma di Fusarium graminearum, l'agente causale della fusariosi della spiga (Fusarium head blight, FHB) di frumento e altri cereali, contiene due geni codificanti per proteine con un dominio CP, denominati fgcpp1 e fgcpp2, e cinque geni codificanti per HP, denominati FgHyd1 -5. Al fine di verificare il loro contributo durante l'infezione in pianta e la crescita fungina, sono stati prodotti e caratterizzati mutanti knock-out singoli e multipli dei geni in analisi. Inoltre, le due CPP di F. graminearum (FgCPP) sono stati espressi eterologamente per investigare la loro attività. Gli FgCPP sembrano essere superflui per la virulenza del fungo ma proteggono i polisaccaridi delle pareti cellulari fungine dalla degradazione enzimatica. Le FgCPP mostrano una forte capacità di ridurre, principalmente mediante un meccanismo non enzimatico, la viscosità della carbossimetilcellulosa (CMC), con maggiore affinità per substrati con medio/alta viscosità e favorire l'attività della cellulasi fungina. L'osservazione che il doppio mutante knock-out ΔΔfgcpp1,2 cresciuto su CMC ha prodotto più attività cellulasica rispetto al fungo wild type suggerisce che l'attività enzimatica più elevata prodotta dal mutante potrebbe compensare durante l'infezione l'assenza dell'attività delle FgCPP sulla cellulosa vegetale. L’Hyd2 e Hyd3 di F. graminearum sono responsabili dell'idrofobicità delle ife aeree e sono coinvolti nell'adesione dei conidi alla superficie ospite durante le prime fasi del processo di infezione, come dimostrato dalla ridotta virulenza di Δhyd2 e Δhyd3 su Triticum aestivum osservato mediante inoculazione a spruzzo delle spighe. È interessante notare che i tripli mutanti Δhyd1,2,3, Δhyd2,3,4 e Δhyd2,3,5 hanno prodotto un numero ridotto di periteci maturi e hanno mostrato difetti a livello della parete cellulare, essendo significativamente più inibito dalla β-1,3-glucanasi rispetto al fungo wild type e più suscettibili al tebuconazolo, un fungicida inibitore della biosintesi dell'ergosterolo. Sulla base dei nostri risultati, FgCPP e FgHyd potrebbero essere utilizzati come bersagli di nuove molecole in strategie innovative di gestione della malattia volte ad aumentare la suscettibilità fungina alle proteine di difesa delle piante o a ridurre la capacità fungina di aderire ai tessuti delle piante ospiti. Inoltre, la capacità delle FgCPP di allentare la CMC e di favorire l'attività delle cellulasi potrebbe rendere queste proteine idonee per potenziali applicazioni future coinvolte nella produzione di biocarburanti utilizzando come substrato tessuti ricchi in cellulosa

Fusarium graminearum cerato-platanin proteins weaken cellulosic materials and enhance cellulase activity in an expansin-like manner

Cerato-platanin proteins (CPPs) belong to a family of small secreted non-catalytic fungal proteins with phytotoxic activity. CPPs have been recently classified as expansin-like proteins because of structural and functional features related to plant expansins, small secreted proteins able to loosen and disrupt the non-covalent bonding networks of plant cell wall polysaccharides without enzymatic activity. 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 (FgCPPs). To characterize their role, the two proteins have been heterologously expressed in yeast. Enzymatic assays have shown the ability of the recombinant FgCPPs to reduce the viscosity of a cellulose soluble derivate (carboxymethyl cellulose, CMC) mainly with a non-enzymatic activity. Indeed, differently from other fungal CPPs and similarly to expansins, FgCPPs seem trapped by cellulose and not by chitin, thus suggesting that they could interact with cellulose. The incubation of CMC with a cellulase in presence or absence of the two recombinant proteins has shown that the FgCPPs enhance cellulase activity. A double knock-out mutant deleted of both FgCPPs encoding genes produces higher cellulase activity when grown on CMC, thus suggesting that the absence of FgCPPs forces the fungus to produce more cellulase activity to compensate for the lack of expansin-like activity. Finally, the preliminary demonstration that the FgCPPs act also loosening filter paper, a natural insoluble cellulose, could suggest a possible future biotechnological application in second-generation biofuels production from agricultural lignocellulosic biomasses rich in cellulose

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-\u3b2-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 produce smuch 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-likemanner. 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

Twenty years of research on cerato-platanin family proteins: clues, conclusions, and unsolved issues

Twenty years of research on cerato-platanin family proteins (CPPs) have led to some clear conclusions: CPPs are exclusively present in the fungal kingdom and possess an outstanding capacity to stimulate the immune system of plants. Recent discoveries have highlighted remarkable structural and functional similarities between CPPs and expansins, a class of non-enzymatic proteins found in both plants and microbes possessing loosening ability on the cell wall structure. Nevertheless, the determination of a biological role for CPPs in fungi is becoming a complicated puzzle to solve, since experimental data are often divergent and point to functional diversification. A general consensus appears however possible: CPPs from pathogenic and beneficial fungi may be considered as microbe-associated molecular patterns (MAMPs) and likely play a dual role, exerting functions in the fungal cell wall and/or in plant colonization. In this review, which celebrates 20 y of research on CPPs, we trace the history of these proteins and highlight experimental evidence and still unsolved issues

Characterization of some industrially relevant polysaccharide monooxygenases and related enzymes

INTRODUCTION AND AIMS By 2030, global rice production is expected to increase to meet the demand of a growing world population. However, rice is severely affected by blast disease, caused by the fungus Magnaporthe oryzae, which can reduce total annual rice production by 10-30% and, if not controlled, can cause complete loss of production. In the early stages of the infection process, M. oryzae forms an infection structure called appressorium to break the plant cuticle and it 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 is to identify M. oryzae PMOs and related enzymes active on polysaccharides and lignin essential for pathogenesis on rice. A screening of natural molecules or inhibitor proteins will then be performed to identify those effective in inhibiting the essential fungal enzymes, thus reducing growth and infection rate of M. oryzae. The final aim of this objective is to develop new methods to control rice blast disease in order to increase food production. Rice straw is the main by-product of rice-producing areas and is a potential resource for biofuel production. Worldwide, about 400 million tons of rice straw are annually produced, with about 30-40 million tons/year in Vietnam alone, but more than 95% is burned, resulting in hazardous airborne emissions. Several polysaccharide and lignin-degrading enzymes like PMOs have been applied in the biofuel industry. When mixed to the typical cellulose-hydrolyzing enzymes, PMOs could dramatically change the enzyme technology involved in biomass degradation, reducing biofuel production costs. However, the activity of PMOs on rice straw needs to be further characterized. The second objective of the project is to heterologously express the M. oryzae PMOs and related enzymes to characterize their enzymatic activities on polysaccharides and lignin, with the aim of developing new enzyme mixtures for rice straw degradation to be used in biofuel production. Aim 1: Characterization of the pathogenetic mechanism of Magnaporthe oryzae to develop new rice blast disease control measures Candidate M. oryzae genes encoding PMOs and related polysaccharides and lignin hydrolyzing enzymes have been identified by an in silico analysis of the fungal genome, completely sequenced. Their expression during the infection process, and particularly during appressorium formation, have been characterized by transcriptomic analysis. Reverse genetics approach: the knock-outs of the most expressed genes (two PMOs, two beta-1,3-glucanases, one ligninase, one chitin synthase and one chitinase) are in progress by targeted homologous recombination. Screening of mutants: infection of rice seedlings and plants will be performed to determine the contribution of the fungal enzymes of interest to fungal virulence. The ability of mutants to form appressorium will also be observed by light microscopy. Natural bioactive compounds and protein inhibitors potentially inhibiting the M. oryzae enzymes shown to play important roles for fungal virulence will be identified by in silico simulation of protein structures. A screening by in vitro bioassays for their ability to inhibit or reduce M. oryzae growth and its virulence on rice seedlings and plants will then be performed. Bioassays will also be performed to evaluate the inhibition potential of bioactive compounds and protein inhibitors on appressorium formation. Effective inhibitors of the target enzymes will then be tested on rice field trials. Rice transgenic plants expressing proteins able to inhibit some target enzymes of M. oryzae will be also created. Aim 2: Application of Magnaporthe oryzae PMOs and related enzymes to rice straw degradation and biofuel production Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species. They have recently been found to degrade recalcitrant polysaccharides, including chitin, cellulose and starch with an unprecedented oxidative mechanism. Polysaccharides are known to be degraded by hydrolytic enzymes, termed collectively as glycoside hydrolases (GHs). The available structures of PMOs reveal a conserved fold and a highly conserved monocopper active site on a flat protein surface. PMOs likely act directly on the substrate surface, bypassing the energy-intensive step of removing polysaccharide chains from the insoluble substrates required in GHs. The new ends created by PMOs can be subsequently processed by various GHs, including exo- and endo-glucanases, resulting in enhanced overall polysaccharide degradation. Genes encoding for M. oryzae PMOs and related polysaccharides and lignin degrading enzymes, previously identified by transcriptomic analysis, will be obtained from M. oryzae RNA from infected rice tissues and the corresponding enzymes of interest will be heterologously expressed using the Pichia pastoris yeast system. The recombinant PMOs will then be purified and characterized at their mononuclear copper active site by UV/Vis, rRaman, Electron Paramagnetic Resonance (EPR) and X-Ray Absorption (XAS). The recombinant enzymes will also be purified at a larger scale (pilot scale) and tested for their enzymatic activity by bioassays performed on different substrates. Since PMOs, like other glycoside hydrolases, have different activities on different types of materials, the purified recombinant enzymes will be tested alone or in mixture in order to optimize the digestion of rice straw. In addition, optimization of ratios of M. oryzae PMOs and commercialized polysaccharide hydrolysis enzymes for degradation of rice straw will be performed. The final aim is the development of a novel rice straw degrading enzyme technology. PERSPECTIVES The expected results, that is the characterization of the role and enzymatic activities of M. oryzae PMOs and related enzymes in order to identify new methods to control rice blast disease and to develop new enzyme mixtures for rice straw degradation and biofuel production, will be exploited for production of scientific publications, dissemination purposes and transfer of new agricultural and industrial technologies. In particular, the PMOs or polysaccharide hydrolase enzymes identified in the project will be tested at the trial scale at bioethanol production factories; the bioactive compounds identified in the project and able to inhibit or control rice blast disease will be applied in rice field trials. The expected impact and benefits of these activities are enhancing the use of rice straw for bioethanol production, with consequent reduction of dependence on fossil fuels and rice straw burning, and the effective inhibition or control of the rice blast disease, with consequent increase of rice productivity and improvement of rice farmers’ economic conditions. Another expected result of the proposed research is training several students: in particular, visits at the Vietnam and Italian laboratories will enable the training of young researchers and the future development of new research objectives aimed at improving the agricultural and industrial systems of both countries

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

Transcriptomic and ultrastructural analyses of Pyricularia oryzae treated with fungicidal peptaibols analogs of Trichoderma trichogin

none10siEco-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 furtherstudies.Microscopyanalyseshighlightedthatthetreatedfungalsporescouldnot 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, twothirds 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 induceautophagiccelldeath.Ultrastructureobservationsontreatedconidiaandhyphae 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.openSella L., Govind R., Caracciolo R., Quarantin A., Vu V.V., Tundo S., Nguyen H.M., Favaron F., Musetti R., De Zotti M.Sella, L.; Govind, R.; Caracciolo, R.; Quarantin, A.; Vu, V. V.; Tundo, S.; Nguyen, H. M.; Favaron, F.; Musetti, R.; De Zotti, M

Novel peptide-based 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. New antimicrobial peptides, analogs of the natural peptaibol trichogin GA IV, have been synthesized and tested in vitro against several P. oryzae strains from different geographic origin. Trichogin GA IV is the main component of the mixture of peptide congeners produced by Trichoderma longibrachiatum as part of its defence mechanism against other microorganisms. Its primary sequence is as follows: Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol (where Aib is alpha-aminoisobutyric acid, Oct is the n-octanoyl group and Lol is the 1,2-aminoalcohol leucinol). All analogs were synthesized by SPPS using a cost-effective strategy. In this presentation, the synthesis and conformational analysis of the peptides are presented, together with the results obtained with our in vitro screening, that has allowed the identification of several peptide sequences very effective in inhibiting spore germination and fungal growth. Those sequences are currently being tested in vivo to confirm their efficacy in protecting rice from the blast disease. This work 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

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