Design, Synthesis, and Antifungal Activities of Novel Potent Fluoroalkenyl Succinate Dehydrogenase Inhibitors.

The development of new fungicide molecules is a crucial task for agricultural chemists to enhance the effectiveness of fungicides in agricultural production. In this study, a series of novel fluoroalkenyl modified succinate dehydrogenase inhibitors were synthesized and evaluated for their antifungal activities against eight fungi. The results from the in vitro antifungal assay demonstrated that compound 34 exhibited superior activity against Rhizoctonia solani with an EC50 value of 0.04 µM, outperforming commercial fluxapyroxad (EC50 = 0.18 µM) and boscalid (EC50 = 3.07 µM). Furthermore, compound 34 showed similar effects to fluxapyroxad on other pathogenic fungi such as Sclerotinia sclerotiorum (EC50 = 1.13 µM), Monilinia fructicola (EC50 = 1.61 µM), Botrytis cinerea (EC50 = 1.21 µM), and also demonstrated protective and curative efficacies in vivo on rapeseed leaves and tomato fruits. Enzyme activity experiments and protein-ligand interaction analysis by surface plasmon resonance revealed that compound 34 had a stronger inhibitory effect on succinate dehydrogenase compared to fluxapyroxad. Additionally, molecular docking and DFT calculation confirmed that the fluoroalkenyl unit in compound 34 could enhance its binding capacity with the target protein through p-π conjugation and hydrogen bond interactions.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 µg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

The Linker Region Promotes Activity and Binding Efficiency of Modular LPMO towards Polymeric Substrate.

Lytic polysaccharide monooxygenases (LPMOs) mediate oxidative degradation of plant polysaccharides. The genes encoding LPMOs are most commonly arranged with one catalytic domain, while a few are found tethered to additional noncatalytic units, i.e., cellulase linker and carbohydrate-binding module (CBM). The presence of CBM is known to facilitate catalysis by directing the enzymes toward cellulosic polymer, while the role of linkers is poorly understood. Based on limited experimental evidence, linkers are believed to serve merely as flexible spacers between the structured domains. Thus, this study aims to unravel the role of the linker regions present in LPMO sequences. For this, we analyzed the genome of Botrytis cinerea and found 9 genes encoding cellulose lytic monooxygenases (AA9 family), of which BcAA9C was overexpressed in cellulose-inducible conditions. We designed variants of flLPMO (full-length enzyme) with truncation of either linker or CBM to examine the role of linker in activity, binding, and thermal stability of the associated monooxygenase. Biochemical assays predicted that the deletion of linker does not impact the potential of flLPMO for catalyzing the oxidation of Amplex Red, but that it does have a major influence on the capability of flLPMO to degrade recalcitrant polysaccharide substrate. Langmuir isotherm and SEM analysis demonstrated that linker domain aids in polysaccharide binding during flLPMO-mediated deconstruction of plant cell wall. Interestingly, linker domain was also found to contribute toward the thermostability of flLPMO. Overall, our study reveals that linker is not merely a spacer, but plays a key role in LPMO-mediated biomass fibrillation; these findings are broadly applicable to other polysaccharide-degrading enzymes. IMPORTANCE The polysaccharide-disintegrating carbohydrate-active enzymes (CAZymes) are often found with multimodular architecture, where the catalytic domain is connected to an accessory CBM domain with the help of a flexible linker region. So far, the linker has been understood merely as a flexible spacer between the two domains. Therefore, the current study is designed to determine the role of linker in polysaccharide fibrillation. To conceive this study, we have selected LPMO as a model enzyme, as it is not only an industrially relevant enzyme but it also harbors a catalytic domain, linker region, and CBM domain. The present study highlighted the crucial and indispensable role of the linker region in mediating polysaccharide disintegration. Considering its role in binding, thermostability, and activity toward polysaccharide substrate, we propose linker as a potential candidate for future CAZyme engineering.

Unambiguous NMR Structural Determination of (+)-Catechin-Laccase Dimeric Reaction Products as Potential Markers of Grape and Wine Oxidation.

(+)-Catechin-laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.

The Botrytis cinerea Crh1 transglycosylase is a cytoplasmic effector triggering plant cell death and defense response.

Crh proteins catalyze crosslinking of chitin and glucan polymers in fungal cell walls. Here, we show that the BcCrh1 protein from the phytopathogenic fungus Botrytis cinerea acts as a cytoplasmic effector and elicitor of plant defense. BcCrh1 is localized in vacuoles and the endoplasmic reticulum during saprophytic growth. However, upon plant infection, the protein accumulates in infection cushions; it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids are sufficient for protein uptake and cell death induction, respectively. BcCrh1 mutant variants that are unable to dimerize lack transglycosylation activity, but are still able to induce plant cell death. Furthermore, Arabidopsis lines expressing the bccrh1 gene exhibit reduced sensitivity to B. cinerea, suggesting a potential use of the BcCrh1 protein in plant immunization against this necrotrophic pathogen.

Compartmentalization of Melanin Biosynthetic Enzymes Contributes to Self-Defense against Intermediate Compound Scytalone in Botrytis cinerea.

In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite.IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.

Design, synthesis and inhibitory activity of novel 2, 3-dihydroquinolin-4(1H)-one derivatives as potential succinate dehydrogenase inhibitors.

Thirty-three new 2, 3-dihydroquinolin-4(1H)-one analogues were designed, synthesized and characterized by IR, 1H NMR, 13C NMR and HRMS. The crystal structures of compounds 2g and 4l were characterized by single crystal X-ray diffraction. Their antifungal activities were determined against five plant pathogenic fungi namely Rhizoctonia solani, Fusarum graminearum, Helminthosporium maydis, Sclerotinia sclerotiorum and Botrytis cinerea. The results indicated that most of them revealed significant antifungal activity at 20 mg/L. Compound 4e showed the strongest antifungal activity against Botrytis cinerea and had better effects than the commercial fungicide fluopyram. Meanwhile, the active compounds were evaluated for their inhibitory activities against succinate dehydrogenase (SDH). The results displayed that they exhibited excellent activity. Compound 4e had better inhibitory activity than fluopyram. The molecular modeling results demonstrated that compound 4e could strongly bind to and interact with the binding sites of SDH. The inhibitory activity of 2, 3-dihydroquinolin-4(1H)-one derivatives against SDH has been reported for the first time.

Botrytis cinerea methyl isocitrate lyase mediates oxidative stress tolerance and programmed cell death by modulating cellular succinate levels.

Fungi lack the entire animal core apoptotic machinery. Nevertheless, regulated cell death with apoptotic markers occurs in multicellular as well as in unicellular fungi and is essential for proper fungal development and stress adaptation. The discrepancy between appearance of an apoptotic-like regulated cell death (RCD) in the absence of core apoptotic machinery is further complicated by the fact that heterologous expression of animal apoptotic genes in fungi affects fungal RCD. Here we describe the role of BcMcl, a methyl isocitrate lyase from the plant pathogenic fungus Botrytis cinerea, in succinate metabolism, and the connection of succinate with stress responses and cell death. Over expression of bcmcl resulted in elevated tolerance to oxidative stress and reduced levels of RCD, which were associated with accumulation of elevated levels of succinate. Deletion of bcmcl had almost no effect on fungal development or stress sensitivity, and succinate levels were unchanged in the deletion strain. Gene expression experiments showed co-regulation of bcmcl and bcicl (isocitrate lyase); expression of the bcicl gene was enhanced in bcmcl deletion and suppressed in bcmcl over expression strains. External addition of succinate reproduced the phenotypes of the bcmcl over expression strains, including developmental defects, reduced virulence, and improved oxidative stress tolerance. Collectively, our results implicate mitochondria metabolic pathways, and in particular succinate metabolism, in regulation of fungal stress tolerance, and highlight the role of this onco-metabolite as potential mediator of fungal RCD.

Expedient Discovery for Novel Antifungal Leads Targeting Succinate Dehydrogenase: Pyrazole-4-formylhydrazide Derivatives Bearing a Diphenyl Ether Fragment.

The pyrazole-4-carboxamide scaffold containing a flexible amide chain has emerged as the molecular skeleton of highly efficient agricultural fungicides targeting succinate dehydrogenase (SDH). Based on the above vital structural features of succinate dehydrogenase inhibitors (SDHI), three types of novel pyrazole-4-formylhydrazine derivatives bearing a diphenyl ether moiety were rationally conceived under the guidance of a virtual docking comparison between bioactive molecules and SDH. Consistent with the virtual verification results of a molecular docking comparison, the in vitro antifungal bioassays indicated that the skeleton structure of title compounds should be optimized as an N'-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide scaffold. Strikingly, N'-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives 11o against Rhizoctonia solani, 11m against Fusarium graminearum, and 11g against Botrytis cinerea exhibited excellent antifungal effects, with corresponding EC50 values of 0.14, 0.27, and 0.52 µg/mL, which were obviously better than carbendazim against R. solani (0.34 µg/mL) and F. graminearum (0.57 µg/mL) as well as penthiopyrad against B. cinerea (0.83 µg/mL). The relative studies on an in vivo bioassay against R. solani, bioactive evaluation against SDH, and molecular docking were further explored to ascertain the practical value of compound 11o as a potential fungicide targeting SDH. The present work provided a non-negligible complement for the structural optimization of antifungal leads targeting SDH.

Identification of the Sesquiterpene Cyclase Involved in the Biosynthesis of (+)-4-Epi-eremophil-9-en-11-ol Derivatives Isolated from Botrytis cinerea.

Cultivation of the phytopathogenic fungus Botrytis cinerea using sublethal amounts of copper sulfate yielded a cryptic sesquiterpenoids family, which displayed the basic chemical structure of (+)-4-epi-eremophil-9-ene. The biosynthesis pathway was established, and the route involved the likely transformation of the diphosphate of farnesyl (FDP), to give a cis-fused eudesmane cation, through (S)-hedycaryol, finally yielding the (+)-4-epi-eremophil-9-enol derivatives. An expression study of genes that code for the sesquiterpene cyclases (STC), including the recently reported gene Bcstc7 present in the B. cinerea genome, was performed in order to establish the STC involved in this biosynthesis. The results showed a higher expression level for the Bcstc7 gene with respect to the other stc1-5 genes in both wild-type strains, B05.10 and Botrytis cinerea UCA992. Deletion of the Bcstc7 gene eliminated (+)-4-epi-eremophilenol biosynthesis, which could be re-established by complementing the null mutant with the Bcstc7 gene. Chemical analysis suggested that STC7 is the principal enzyme responsible for the key step of cyclization of FDP to eremophil-9-en-11-ols. Furthermore, a thorough study of the two wild-types and the complemented mutant revealed four new eremophilenol derivatives whose structures are reported here.

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Generation of Stilbene Antimicrobials against Multiresistant Strains of Staphylococcus aureus through Biotransformation by the Enzymatic Secretome of Botrytis cinerea.

The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Botrytis cinerea. Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of S. aureus with moderate human cell line cytotoxicity.

Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici.

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species.

Laccases 2 & 3 as biomarkers of Botrytis cinerea infection in sweet white wines.

Botrytized sweet wines are made with berries infected by the fungus Botrytis cinerea. The aim of this study was to identify biomarkers of B. cinerea infection in sweet wines with a focus on laccases which are exocellular oxidase enzymes produced by this fungus during fruit contamination. Total proteins from six commercial sweet wines, including three naturally botrytized wines and three non-botrytized wines were analysed by LC-QTOF-MS. Five laccases, namely laccase-1-BcLCC1, laccase-2-BcLCC2, laccase-3-BcLCC7, laccase-8-BcLCC8 and laccase-12-BcLCC12, were identified in both types of wine. Then, a targeted proteomic approach by LC-MRM was used to semi-quantify laccase-2-BcLCC2 and laccase-3-BcLCC7, in the six samples. LC-MRM targeted analysis of the two enzymes allowed the discrimination of botrytized versus non-botrytized sweet white wines.

Amino Acid Polymorphism in Succinate Dehydrogenase Subunit C Involved in Biological Fitness of Botrytis cinerea.

Succinate dehydrogenase (SDH) is an important respiratory enzyme which participates in the tricarboxylic acid cycle and oxidative phosphorylation. A previous study of the baseline sensitivity of Botrytis cinerea against SDH inhibitors (SDHIs) showed that intrinsic sensitivity of the small population against the SDHIs exhibited significant differences. In the sequencing assay, we found five kinds of amino acid polymorphism in SDH subunit C (SdhC) of B. cinerea isolates which were never exposed to the SDHIs. To validate that amino acid polymorphism in the SdhC of B. cinerea confers intrinsic sensitivity against the SDHIs, the replacement mutants containing each kind of amino acid polymorphism of SdhC exhibited phenotype differences in intrinsic sensitivity to SDHIs, mycelial growth, sporulation, virulence, oxidative stress response, and carbon source utilization. These results indicated that SdhC of B. cinerea experienced positive selection during evolution and resulted in amino acid polymorphism which is involved in intrinsic sensitivity to SDHIs and biological fitness.

Late-Stage C-H Functionalization of Nicotinamides for the Expedient Discovery of Novel Antifungal Leads.

Encouraged by the successful flexible modifications of the succinate dehydrogenase inhibitors, antifungal activity guided by the divergent synthesis of nicotinamides of the prevalidated pharmacophore 2-(2-oxazolinyl)aniline was conducted. The work highlighted the first utilization of the late-stage C-H functionalization assisted by the innate pharmacophore for the discovery of promising agrochemicals. New synthetic methodology and antifungal exploration of alkoxylated nicotinamides were accomplished. Fifty-five functionalized nicotinamides of 7 types were rationally designed and efficiently prepared through C-H functionalization, which facilitated the acquirement of four N-para aryloxylated nicotinamides (E3, E13, E19, and E22) as potential antifungal candidates against Botrytis cinerea, with the EC50 values lower than 5 mg/L. In vivo/vitro biotest, molecular docking, and structural analysis reconfirmed the novelty and practical potential of the antifungal candidates E3 and E19. This operationally simple platform will provide various "polar parts" and offer intriguing opportunities for the optimization of the carboxamide fungicides and structure-related pharmaceuticals.

Alternative Oxidase Is Involved in the Pathogenicity, Development, and Oxygen Stress Response of Botrytis cinerea.

Alternative oxidase (AOX) is a ubiquinol terminal oxidase that is involved in fungal mitochondrial oxidative phosphorylation. In this study, we analyzed the roles of AOX in Botrytis cinerea by generating BcAOX deletion mutants. The mutants exhibited defects in mycelial growth, sporulation, spore germination, and virulence. Furthermore, the sensitivity of the mutants to quinone outside inhibitor fungicides and oxidative stress were increased. All phenotypic variations could be restored in the complemented strain. In summary, these results showed that BcAOX is involved in the regulation for vegetative development, adaptation to environmental stress, and virulence of B. cinerea.

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea.

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

The study of intracellular and secreted high-molecular-mass protease(s) of Trichoderma spp., and their responses to conidiation stimuli.

We continued our study of high-molecular-mass proteases (HMMPs) using several strains of the genus Trichoderma, and other filamentous fungi (Botrytis cinerea, Aspergillus niger, Fusarium culmorum, and Penicillium purpurogenum). We found that five Trichoderma strains secreted HMMPs into the media after induction with bovine serum albumin. Botrytis cinerea and F. culmorum secreted proteases in the absence of inducer, while A. niger or P. purpurogenum did not secrete proteolytic activity (PA). The activity of HMMPs secreted by or intracellularly located in Trichoderma spp. represents the predominant part of cellular PA, according to zymogram patterns. This observation allowed the study of HMMPs' physiological role(s) independent from the secretion. In studying conidiation, we found that illumination significantly stimulated PA in Trichoderma strains. In the T. atroviride IMI 206040 strain, we demonstrated that this stimulation is dependent on the BLR1 and BLR2 receptors. No stimulation of PA was observed when mechanical injury was used as an elicitor of conidiation. Compounds used as inhibitors or activators of conidiation exerted no congruent effects on both PA and conidiation. These results do not favour a direct role of HMMPs in conidiation. Probably, HMMP activity may be involved in the process of the activation of metabolism during vegetative growth, differentiation, and aging-related processes.

Depression of Fungal Polygalacturonase Activity in Solanum lycopersicum Contributes to Antagonistic Yeast-Mediated Fruit Immunity to Botrytis.

The acquisition of susceptibility to necrotrophy over the course of ripening is one of the critical factors limiting shelf life. In this study, phytopathology and molecular biology were employed to explore the roles of pectinase in fruit susceptibility and ripening. Solanum lycopersicum fruit softened dramatically from entirely green to 50% red, which was accompanied by a continuously high expressed SlPG2 gene. The necrotrophic fungus Botrytis cinerea further activated the expression of SlPGs and SlPMEs to accelerate cell wall disassembly, while most of the polygalacturonase inhibitor proteins encoding genes expression were postponed in ripe fruit following the pathogen attack. Pectin induced the antagonistic yeast to secrete pectinolytic enzymes to increase fruit resistance against gray mold. The activities of pathogenic pectinase of B. cinerea were correspondingly depressed in the pectin-inducible yeast enzyme elicited ripe fruit. These data suggest that pectinase is a molecular target for regulation of disease resistance during fruit ripening.