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.

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.

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.

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.

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.

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.

Translocation from nuclei to cytoplasm is necessary for anti A-PCD activity and turnover of the Type II IAP BcBir1.

Type II inhibitors of apoptosis (IAPs) belong to a subgroup of IAP-related proteins. While IAPs are restricted to animals, Type II IAPs are found in other phyla, including fungi. BcBir1, a Type II IAP from Botrytis cinerea has anti apoptotic-like programmed cell death (A-PCD) activity, which is important for pathogenicity of this fungus. Here we report on the role of sub-cellular localization of BcBir1 in protein turnover and anti A-PCD activity. Expression of BcBir1 in Saccharomyces cerevisiae had no effect on sensitivity of the yeast cells to A-PCD-inducing conditions, whereas expression of a truncated N' part reduced sensitivity of the cells to these conditions. The full-length BcBir1 protein was detected only in the yeast nucleus, whereas the N' part was observed both in the nucleus and cytoplasm. In B. cinerea, BcBir1 was mainly nuclear under optimal conditions, whereas under A-PCD-inducing conditions it shuttled to the cytoplasm and then it was completely degraded. Collectively, our results show that anti A-PCD activity of BcBir1 occurs in the cytoplasm, the C' end mediates regulation of steady state level of BcBir1 in the nucleus, and the N' end mediates anti A-PCD activity as well as fast degradation of BcBir1 in the cytoplasm.

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.

Modifications of fungal membrane proteins profile under pathogenicity induction: A proteomic analysis of Botrytis cinerea membranome.

Botrytis cinerea is a model fungus for the study of phytopathogenicity that exhibits a wide arsenal of tools to infect plant tissues. Most of these factors are related to signal transduction cascades, in which membrane proteins play a key role as a bridge between environment and intracellular molecular processes. This work describes the first description of the membranome of Botrytis under different pathogenicity conditions induced by different plant-based elicitors: glucose and tomato cell wall (TCW). A discovery proteomics analysis of membrane proteins was carried out by mass spectrometry. A total of 2794 proteins were successfully identified, 46% of them were classified as membrane proteins based on the presence of transmembrane regions and lipidation. Further analyses showed significant differences in the membranome composition depending on the available carbon source: 804 proteins were exclusively identified when the fungus was cultured with glucose as a sole carbon source, and 251 proteins were exclusively identified with TCW. Besides, among the 1737 common proteins, a subset of 898 proteins presented clear differences in their abundance. GO enrichment and clustering interaction analysis revealed changes in the composition of membranome with increase of signalling function in glucose conditions and carbohydrate degradation process in TCW conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD003099 (http://proteomecentral.proteomexchange.org/dataset/PXD003099).

Effect of calcium on cell-wall degrading enzymes of Botrytis cinerea.

Effective anti-Botrytis strategies leading to reduce pesticides on strawberries are examined to provide the protection that is harmless to humans, higher animals and plants. Calcium treatments significantly inhibited the spore germination and mycelial growth of B. cinerea. The intracellular polygalacturonase and CMCase showed low activities in B. cinerea cultivated by medium containing calcium. On the other hand, calcium-stimulated ß-glucosidases production occurred. Our findings suggest that the calcium treatments keep CMCase activity low and cause low activities of cell-wall degrading enzymes of B. cinerea in the late stage of growth.

BcGs1, a glycoprotein from Botrytis cinerea, elicits defence response and improves disease resistance in host plants.

In this study, a necrosis-inducing protein was purified from the culture filtrate of the necrotrophic fungus Botrytis cinerea BC-98 strain. Secreted proteins were collected and fractionated by liquid chromatography. The fraction with the highest necrosis-inducing activity was further purified. A glycoprotein named BcGs1 was identified by 2D electrophoresis and mass spectrometry. The BcGs1 protein consisted of 672 amino acids with a theoretical molecular weight of 70.487 kDa. Functional domain analysis indicated that BcGs1 was a glucan 1,4-alpha-glucosidase, a cell wall-degrading enzyme, with a Glyco_hydro_15 domain and a CBM20_glucoamylase domain. The BcGs1 protein caused necrotic lesions that mimicked a typical hypersensitive response and H2O2 production in tomato and tobacco leaves. BcGs1-treated plants exhibited resistance to B. cinerea, Pseudomonas syringae pv. tomato DC3000 and tobacco mosaic virus in systemic leaves. In addition, BcGs1 triggered elevation of the transcript levels of the defence-related genes PR-1a, TPK1b and Prosystemin. This is the first report of a Botrytis glucan 1,4-alpha-glucosidase triggering host plant immunity as an elicitor. These results lay a foundation for further study of the comprehensive interaction between plants and necrotrophic fungi.

Biological control of the grapevine diseases 'grey mold' and 'powdery mildew' by Bacillus B27 and B29 strains.

Uncinula necator and Botrytis cinerea are the most destructive pathogens of the grapevine in Tunisia and elsewhere. We used two strains of Bacillus subtilis group, B27 and B29 to control powdery mildew and the grey mold disease of the grapevine. Green house experiments showed that B29 and B27 strains of the bacteria efficiently reduced the severity of powdery mildew up to 50% and 60%, respectively. Further, they decreased Botrytis cinerea development on grape leaf by 77% and 99%, respectively. The mode of action has been shown to be chitinolytic. These two bacteria showed significant production of total proteins discharged into the culture medium. Determination of some chitinolytic enzymes revealed the involvement of N-acetyl glucosaminidase (Nagase), the chitin-1,4-chitobiosidase (Biase) and endochitinase in degrading the mycelium of B. cinerea.

Characterization of endophytic Bacillus strains from tomato plants (Lycopersicon esculentum) displaying antifungal activity against Botrytis cinerea Pers.

Eighty endophytic bacteria were isolated from healthy tissues of roots, stems, leaves and fruits of tomato plants (Lycopersicon esculentum). Four strains, named BL1, BT5, BR8 and BF11 were selected for their antagonism against Botrytis cinerea, a phytopathogenic fungus responsible of gray mold in several important crops, with growth inhibitory activity ranging from 27 to 53%. Morphological, biochemical, and molecular parameters as 16S rDNA sequencing demonstrated that the selected bacterial strains were related to Bacillus species which are known to produce and secrete a lot of lipopeptides with strong inhibitory effect against pathogen mycelial growth. Electrospray mass spectrometry analysis showed that these strains produced heterogeneous mixture of antibiotics belonging to fengycin and surfactin for BL1 and BT5, to iturin and surfactin for BR8, to bacillomycin D, fengycin and surfactin for BF11. Furthermore, these bacteria exhibited biocontrol potential by reducing the disease severity when tested on detached leaflets. Based on their antifungal activity against Botrytis cinerea, these strains could be used for biological control of plant diseases.

Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures.

This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. These enzymes contain four Cu atoms per molecule organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymatic reaction and is determined by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-dimensional structures of the wild-type enzyme and the L499M mutant were determined by X-ray crystallography at 1.7 Šresolution. Crystals suitable for X-ray analysis could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were determined: E0 = 720 and 580 mV for the wild-type enzyme and the mutant, respectively. Since the structures of the wild-type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.

Identification of glycoproteins secreted by wild-type Botrytis cinerea and by protein O-mannosyltransferase mutants.

BACKGROUND: Botrytis cinerea secretes a high number of proteins that are predicted to have numerous O-glycosylation sites, frequently grouped in highly O-glycosylated regions, and analysis of mutants affected in O-glycosylation has shown, in B. cinerea and in other phytopathogenic fungi, that this process is important for fungal biology and virulence. RESULTS: We report here the purification of glycoproteins from the culture medium, for a wild-type strain of B. cinerea and for three mutants affected in the first step of O-glycosylation, and the identification of components in the purified protein samples. Overall, 158 proteins were identified belonging to a wide diversity of protein families, which possess Ser/Thr-rich regions (presumably highly O-glycosylated) twice as frequently as the whole secretome. Surprisingly, proteins predicted to be highly O-glycosylated tend to be more abundant in the secretomes of the mutants affected in O-glycosylation than in the wild type, possibly because a correct glycosylation of these proteins helps keep them in the cell wall or extracellular matrix. Overexpression of three proteins predicted to be O-glycosylated in various degrees allowed to confirm the presence of mannose α1-2 and/or α1-3 bonds, but no mannose α1-6 bonds, and resulted in an enhanced activity of the culture medium to elicit plant defenses. CONCLUSIONS: Glycosylation of secretory proteins is very prevalent in B. cinerea and affects members of diverse protein families. O-glycosylated proteins play a role in the elicitation of plant defenses.

Exploring mutasynthesis to increase structural diversity in the synthesis of highly oxygenated polyketide lactones.

The enantioselective synthesis of (2R,3R,4E,8E)-3-hydroxy-2,4,8-trimethyldeca-4,8-dienolide (5) by ring-closing metathesis is described. This compound is an analogue of 3,4-dihydroxy-2,4,6,8-tetramethyldec-8-enolide (4) which is a rare 11-membered lactone produced by the fungus, Botrytis cinerea. Mutasynthetic studies with compound 5 using two mutants of B. cinerea led to the isolation of four new highly oxygenated 11-membered lactones (11-14) in which compound 5 has been stereoselectively epoxidized and hydroxylated at sites that were not easily accessible by classical synthetic chemistry.

Lipoxygenase activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.).

In this study we investigated the influence of biotic elicitor (phytopathogenic fungus Botrytis cinerea) and abiotic elicitors (methyljasmonate [MJ] and salicylic acid [SA]) on lipoxygenase (LOX) activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.). We have observed different time effects of elicitors (10, 24, 48 and 72 h) on LOX activity and production of sanguinarine in in vitro cultures. All elicitors used in the experiments evidently increased the LOX activity and sanguinarine production in contrast to control samples. The highest LOX activities were determined in samples elicitated by MJ after 48 h and 72 h and the lowest LOX activities (in contrast to control samples) were detected after biotic elicitation by Botrytis cinerea. These activities showed about 50% lower level against the activities after MJ elicitation. The maximal amount of sanguinarine was observed after 48 h in MJ treated cultures (429.91 mg/g DCW) in comparision with control samples. Although all elicitors affect the sanguinarine production, effect of SA and biotic elicitor on sanguinarine accumulation in in vitrocultures was not so significant than after MJ elicitation.