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.

Exogenous 1',4'-trans-Diol-ABA Induces Stress Tolerance by Affecting the Level of Gene Expression in Tobacco (Nicotiana tabacum L.).

1',4'-trans-diol-ABA is a key precursor of the biosynthesis of abscisic acid (ABA) biosynthesis in fungi. We successfully obtained the pure compound from a mutant of Botrytis cinerea and explored its function and possible mechanism on plants by spraying 2 mg/L 1',4'-trans-diol-ABA on tobacco leaves. Our results showed that this compound enhanced the drought tolerance of tobacco seedlings. A comparative transcriptome analysis showed that a large number of genes responded to the compound, exhibiting 1523 genes that were differentially expressed at 12 h, which increased to 1993 at 24 h and 3074 at 48 h, respectively. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were primarily enriched in pathways related to hormones and resistance. The DEGs of transcription factors were generally up-regulated and included the bHLH, bZIP, ERF, MYB, NAC, WRKY and HSF families. Moreover, the levels of expression of PYL/PYR, PP2C, SnRK2, and ABF at the ABA signaling pathway responded positively to exogenous 1',4'-trans-diol-ABA. Among them, seven ABF transcripts that were detected were significantly up-regulated. In addition, the genes involved in salicylic acid, ethylene and jasmonic acid pathways, reactive oxygen species scavenging system, and other resistance related genes were primarily induced by 1',4'-trans-diol-ABA. These findings indicated that treatment with 1',4'-trans-diol-ABA could improve tolerance to plant abiotic stress and potential biotic resistance by regulating gene expression, similar to the effects of exogenous ABA.

Antifungal effects of lycorine on Botrytis cinerea and possible mechanisms.

Botrytis cinerea cause postharvest diseases on fruit and lead economic losses. Application of environment-friendly natural compounds is an alternative for synthetic fungicides to control postharvest disease. Lycorine is an indolizidine alkaloid which is widely used for human drug design, however, application of lycorine in controlling postharvest disease and the underlying mechanisms have not been reported. In this study, the effects of lycorine on mycelium growth, spore germination, disease development in apple fruit, cell viability, cell membrane integrity, cell wall deposition, and expression of mitogen-activated protein kinase (MAPK) and GTPase of B. cinerea were investigated. Our results showed that lycorine was effective in controlling postharvest gray mold caused by B. cinerea on apple fruit. In the in vitro tests, lycorine strongly inhibited spore germination and mycelium spreading in culture medium. Investigation via fluorescein diacetate and propidium iodide staining suggested that lycorine could damage the membrane integrity and impair cell viability of B. cinerea. Furthermore, the expression levels of several MAPK and GTPase coding genes were reduced upon the lycorine treatment. Taken together, lycorine is an effective and promising way to control postharvest disease caused by B. cinerea.

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.

Mechanisms of nanotoxicity - biomolecule coronas protect pathological fungi against nanoparticle-based eradication.

Whereas nanotoxicity is intensely studied in mammalian systems, our knowledge of desired or unwanted nano-based effects for microbes is still limited. Fungal infections are global socio-economic health and agricultural problems, and current chemical antifungals may induce adverse side-effects in humans and ecosystems. Thus, nanoparticles are discussed as potential novel and sustainable antifungals via the desired nanotoxicity but often fail in practical applications. In our study, we found that nanoparticles' toxicity strongly depends on their binding to fungal spores, including the clinically relevant pathogen Aspergillus fumigatus as well as common plant pests, such as Botrytis cinerea or Penicillum expansum. Employing a selection of the model and antimicrobial nanoparticles, we found that nanoparticle-spore complex formation is influenced by the NM's physicochemical properties, such as size, identified as a key determinant for our silica model particles. Biomolecule coronas acquired in pathophysiologically and ecologically relevant environments, protected fungi against nanoparticle-induced toxicity as shown by employing antimicrobial ZnO, Ag, or CuO nanoparticles as well as dissolution-resistant quantum dots. Mechanistically, dose-dependent corona-mediated resistance was conferred via reducing the physical adsorption of nanoparticles to fungi. The inhibitory effect of biomolecules on nano-based toxicity of Ag NPs was further verified in vivo, using the invertebrate Galleria mellonella as an alternative non-mammalian infection model. We provide the first evidence that biomolecule coronas are not only relevant in mammalian systems but also for nanomaterial designs as future antifungals for human health, biotechnology, and agriculture.

Identification of Brassicadiene, a Diterpene Hydrocarbon Attractive to the Invasive Stink Bug Bagrada hilaris, from Volatiles of Cauliflower Seedlings, Brassica oleracea var. botrytis.

Brassicadiene, a novel tricyclic diterpene hydrocarbon, was identified by a combination of mass spectrometry, microchemical tests, and analysis of NMR spectra. The compound constitutes >90% of the volatile organic compounds produced by cauliflower seedlings, Brassica oleracea var. botrytis. The invasive stink bug Bagrada hilaris is strongly attracted to brassicadiene, providing a mechanism for this herbivore, which specializes on cruciferous plants, to locate its hosts in a nutrient-rich and vulnerable stage.

Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation.

We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment. Opto-Rac1 is a fusion of wildtype human Rac1 small GTPase to the C-terminal region of BcLOV4, a LOV (light-oxygen-voltage) photoreceptor that rapidly binds the plasma membrane upon blue-light activation via a direct electrostatic interaction with anionic membrane phospholipids. Translocation of the fused wildtype Rac1 effector permits its activation by GEFs (guanine nucleotide exchange factors) and consequent actin polymerization and lamellipodia formation, unlike in existing single-chain systems that operate by allosteric photo-switching of constitutively active Rac1 or the heterodimerization-based (i.e. two-component) membrane recruitment of a Rac1-activating GEF. Opto-Rac1 induction of lamellipodia formation was spatially restricted to the patterned illumination field and was efficient, requiring sparse stimulation duty ratios of ∼1-2% (at the sensitivity threshold for flavin photocycling) to cause significant changes in cell morphology. This work exemplifies how the discovery of LOV proteins of distinct signal transmission modes can beget new classes of optogenetic tools for controlling cellular function.

Botryotins A-H, Tetracyclic Diterpenoids Representing Three Carbon Skeletons from a Deep-Sea-Derived Botryotinia fuckeliana.

Eight unprecedent diterpenoids, botryotins A-H (1-8), were obtained from Botryotinia fuckeliana. They represent three novel carbon skeletons with 6/6/5/5 (1), 6/6/5/6 (2-6), and 6/6/6/5 (7 and 8) tetracyclic scaffolds. Their structures were determined by detailed spectroscopic analysis and chemical derivatization as well as quantum chemical calculation of the ECD and OR data. Botryotin A (1) exhibited a moderate antiallergic effect (IC50 = 0.2 mM). A plausible biosynthetic pathway for 1-8 was proposed.

Behavioral evidence for contextual olfactory-mediated avoidance of the ubiquitous phytopathogen Botrytis cinerea by Drosophila suzukii.

Herbivorous insects may benefit from avoiding the smell produced by phytopathogens infecting plant host tissue if the infected tissue reduces insect fitness. However, in many cases the same species of phytopathogen can also infect host plant tissues that do not directly affect herbivore fitness. Thus, insects may benefit from differentiating between pathogen odors emanating from food and nonfood tissues. This is based on the hypothesis that unnecessarily staying attentive to pathogen odor from nonfood tissue may incur opportunity costs associated with not responding to other important survival functions. In this study adults of Drosophila suzukii Matsumura, an invasive larval frugivore, showed reduced attraction to the odor of raspberry fruit, a food tissue, when infected with Botrytis cinerea Pers., a ubiquitous phytopathogen, in favor of odors of uninfected raspberry fruit. Moreover, D. suzukii oviposited fewer eggs on infected raspberry fruit relative to uninfected raspberry fruit. Larval survival and adult size after eclosion were significantly reduced when reared on B. cinerea-infected raspberry relative to uninfected fruit. Interestingly, when the behavioral choice experiment was repeated using Botrytis-infected vs. -uninfected strawberry leaves, a nonfood tissue, in combination with fresh raspberry fruit, odor from B. cinerea-infected leaves did not reduce D. suzukii attraction to raspberries relative to raspberries with uninfected leaves. These behavioral results illustrate the important role context can play in odor-mediated interactions between insects, plants and microbes. We discuss implications of our findings for developing a repellent that can be useful for the management of D. suzukii.

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.

Aphidicolin Chemistry of the Deep-Sea-Derived Fungus Botryotinia fuckeliana MCCC 3A00494.

Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been discovered. However, none have shown a stronger effect. Herein, we report 71 new (aphidicolins A1-A71, 1-71) and eight known (72-79) aphidicolin congeners from Botryotinia fuckeliana MCCC 3A00494, a fungus isolated from the western Pacific Ocean (-5572 m). The structures of 1-71 were determined through extensive spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher's method, and the ECD exciton chirality method. Compounds 54-57 and 58-64 are novel 6/6/5/6/5 pentacyclic aphidicolins featuring tetrahydrofuran and dihydrofuran rings, respectively, while compounds 65-71 are rare noraphidicolins. Aphidicolin A8 (8) significantly induced apoptosis in T24 (IC50 = 2.5 µM) and HL-60 (IC50 = 6.1 µM) cancer cells by causing DNA damage. By docking its structure to the human DNA polymerase α binding pocket, 8 was found to form tight intermolecular contacts, elaborating aphidicolin A8 as a potently cytotoxic lead compound.

Detoxification Mechanism of 8,8-Dimethyl-3-[(R-phenyl)amino]-1,4,5(8H)-naphthalentrione Derivatives by Botrytis cinerea.

The effect of 8,8-dimethyl-3-[(R-phenyl)amino]-1,4,5(8H)-naphthalentrione derivatives (compounds 1⁻13) on the mycelial growth of Botrytis cinerea was evaluated. The fungitoxic effect depended on the substituent and its position in the aromatic ring. Compounds substituted with halogens in meta and/or para positions (compounds 3, 4, 5 and 7), methyl (compounds 8 and 9), methoxyl (compounds 10 and 11), or ethoxy-carbonyl groups (compound 12) presented higher antifungal activity than compound 1, which had an unsubstituted aromatic ring. In addition, compounds with halogens in the ortho position, such as compounds 2 and 6, and a substitution with an acetyl group in the para position (compound 13) were less active. The role of the ABC efflux pump Bctr B-type as a defense mechanism of B. cinerea against these naphthalentrione derivatives was analyzed. This pump could be involved in the detoxification of compounds 2, 6, and 13. On the contrary, this mechanism would not participate in the detoxification of compounds 1, 7, 9 and 12. Finally, the biotransformation of compound 7 by B. cinerea was studied. A mixture of two biotransformed products was obtained. One of them was compound 7A, which is reduced at C1 and C4, compared to compound 7. The other product of biotransformation, 7B, is oxidized at C7.

In-process quality control of wine by planar chromatography versus micro planar chromatography.

High-performance thin-layer chromatography (HPTLC) methods were developed to determine glycerol, gluconic acid, amino acids and sugars for in-process quality control of wine. Twenty wine samples (Pfalz region, Germany) were diluted with methanol and used for quantitative analysis without any further sample preparation. The developed amino acid method provided quantitative and characteristic fingerprints of wine varieties. The amino acid assignments were verified by HPTLC-MS. The developed gluconic acid method was primarily used to control the threshold, indicating a Botrytis cinerea infection of grapes. However, this method also enabled the detection of further organic acids like malic, tartaric and citric acids. A glycerol method was developed for control of the grape must fermentation (spontaneous/regular) and for fraud detection (glycerol adulteration). The HPTLC results of the sugar contents in the wine samples were similar to those of the well-known Luff-Schoorl method. The combined use of these developed HPTLC methods allowed the fermentation control (e.g., alcoholic and malolactic fermentation) and the monitoring of the grapes' overall health status. Without modification, the HPTLC methods for sugar and amino acid analysis could be transferred to circular micro planar chromatography (µ-PLC), showing its potential and benefits in terms of an inexpensive alternative for wineries and distributors.

Proteomic analysis of the inhibitory effect of oligochitosan on the fungal pathogen, Botrytis cinerea.

BACKGROUND: The fungal pathogen Botrytis cinerea infects a broad range of horticultural plants worldwide, resulting in significant economic losses. A derivative of chitosan, oligochitosan, has been reported to be an eco-friendly alternative to synthetic fungicides. RESULTS: Oligochitosan can greatly inhibit B. cinerea spore germination and induce protein carbonylation. To further investigate the molecular mechanism underlying the inhibitory effect, a comparative proteome analysis was conducted of oligochitosan-treated versus non-treated B. cinerea spores. The cellular proteins were obtained from B. cinerea spore samples and subjected to two-dimensional gel electrophoresis. In total, 21 differentially expressed proteins (DEPs) were identified. Three DEPs were up-regulated in the oligochitosan-treated versus the untreated spores, including scytalone dehydratase and a serine carboxypeptidase III precursor. By contrast, seven DEPs, including Hsp 88 and cell division cycle protein 48, were down-regulated by oligochitosan treatment. Notably, 10 DEPs, including phosphatidylserine decarboxylase proenzyme and ATP-dependent molecular chaperone HSC82, were only detected in the control spores, whereas one DEP, a non-annotated predicted protein, was only detected in the oligochitosan-treated spores. CONCLUSION: Oligochitosan may affect the spore germination of B. cinerea by impairing protein function. These findings have practical implications with respect to the use of oligochitosan for controlling fungal pathogens. © 2018 Society of Chemical Industry.

Modelling the growth of pear postharvest fungal isolates at different temperatures.

The effect of temperature on the mycelium growth kinetics of four postharvest fungal isolates (i.e., Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer) was assessed. A cardinal model with inflection (CMI) was used to describe the effect of the temperature on the growth rate (µ) and the lag time (λ) of each isolate. Cardinal temperature values such as Tmin, Tmax and Topt were estimated and isolates were sorted according to their growth rate and lag time duration. Additionally, model validation was performed on a medium prepared from mashed pear pulp and on artificially wound-inoculated pear fruits. P. expansum was shown to be the most psychotrophic fungus with the lowest estimated Tmin = -8.78. Model validation on pear pulp agar showed growth rate over-prediction in the case of R. stolonifer and B. cinerea but a good correlation in the case of P. expansum and A. alternata. In vivo experiments on pear fruits showed discrepancies from the synthetic and the simulated counterparts for all the fungi with the only exception of P. expansum.

Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids.

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Structural and biosynthetic studies on eremophilenols related to the phytoalexin capsidiol, produced by Botrytis cinerea.

A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopathogenic fungi. The biosynthesis of these compounds has been studied using 2H and 13C labelled acetate.

Discrimination and growth tracking of fungi contamination in peaches using electronic nose.

A non-destructive method for detection of fungal contamination in peaches using an electronic nose (E-nose) is presented. Peaches were inoculated with three common spoilage fungi, Botrytis cinerea, Monilinia fructicola and Rhizopus stolonifer and then stored for various periods. E-nose was then used to analyze volatile compounds generated in the fungi-inoculated peaches, which was then compared with the growth data (colony counts) of the fungi. The results showed that changes in volatile compounds in fungi-inoculated peaches were correlated with total amounts and species of fungi. Terpenes and aromatic compounds were the main contributors to E-nose responses. While principle component analysis (PC1) scores were highly correlated with fungal colony counts, Partial Least Squares Regression (PLSR) could effectively be used to predict fungal colony counts in peach samples. The results also showed that the E-nose had high discrimination accuracy, demonstrating the potential use of E-nose to discriminate among fungal contamination in peaches.

Antioxidant capacity of phenolic compounds on human cell lines as affected by grape-tyrosinase and Botrytis-laccase oxidation.

Phenolic components (PCs) are well-known for their positive impact on human health. In addition to their action as radical scavengers, they act as activators for the intrinsic cellular antioxidant system. Polyphenol oxidases (PPOs) such as tyrosinase and laccase catalyze the enzymatic oxidation of PCs and thus, can alter their scavenging and antioxidative capacity. In this study, oxidation by tryosinase was shown to increase the antioxidant capacity of many PCs, especially those that lack adjacent aromatic hydroxyl groups. In contrast, oxidation by laccase tended to decrease the antioxidant capacity of red wine and distinct PCs. This was clearly demonstrated for p-coumaric acid and resveratrol, which is associated with many health benefits. While oxidation by tyrosinase increased their antioxidant activity laccase treatment resulted in a decreased activity and also of that for red wines.

Generation of Antifungal Stilbenes Using the Enzymatic Secretome of Botrytis cinerea.

The protein secretome of Botrytis cinerea was used to perform the biotransformation of resveratrol, pterostilbene, and a mixture of both. Metabolite profiling by UHPLC-HRMS revealed the presence of compounds with unusual molecular formula, suggesting the existence of new products. To isolate these products, the reactions were scaled-up, and 21 analogues were isolated and fully characterized by NMR and HRESIMS analyses. The reaction with pterostilbene afforded five new compounds, while the reaction with a mixture of pterostilbene and resveratrol afforded seven unusual stilbene dimers. The antifungal properties of these compounds were evaluated using in vitro bioassays against Plasmopara viticola. The cytological effects of the isolated antifungal compounds on the ultrastructure of P. viticola were also evaluated.