灰葡萄孢产孢新基因的功能分析

灰葡萄孢是一种重要的植物病原真菌,其寄主范围广泛,能危害世界上230多种双子叶植物,常给农业生产造成重大的经济损失[1-3].由灰葡萄孢引起的灰霉病是目前我国温室蔬菜生产中最主要的病害之一,一般造成全年减产20％-25％,严重时达到40％以上[4].因此,研究该病菌的致病机理对该病防治具有重要意义,并且随着灰葡萄孢基因组测序的完成,灰葡萄孢已成为发育生物学、分子植物病理学研究的模式生物之一.     

Melanin in the extracellular matrix of germlings of Botrytis cinerea

Previous work on the composition of the extracellular matrix of germlings of the plant pathogenic fungus Botrytis cinerea demonstrated the presence of carbohydrate, protein, and simple lipids; which, together, comprised 50-60% of the dry weight. Here we show that most of the remaining mass of the extracellular matrix consists of a chemically inert dark pigment with the electron paramagnetic resonance characteristics of a melanin. Scanning electron micrographs of the purified pigment, and transmission electron micrographs of thin sections made using the pigment indicate that it has a filamentous structure. We conclude that melanin is an important component of the extracellular matrix of germlings of B. cinerea. This is the first report of a melanin present in the extracellular matrix of a plant pathogenic fungus.     

Lipid composition of the extracellular matrix of Botrytis cinerea germlings

Six simple lipid classes (mono-, di- and tri-acylglycerols, free fatty acids, free fatty alcohols and wax esters) were identified by TLC in the extracellular matrix of Botrytis cinerea germlings and the molecular components of each class were characterized using GC-MS. The relative amounts of fatty acids and fatty alcohols within each lipid class were determined by GC-FID. Over all the lipid classes, the most abundant saturated fatty acids were palmitic (ca. 30%) and stearic acid (ca. 22%). Palmitoleic and oleic acids made up ca. 21% and 24% (respectively) of the free fatty acids, while erucic (ca. 4.1%) and linoleic (ca. 3.6%) acids were the most abundant unsaturated fatty acids in the acylglycerides. The acylglycerides also contained almost 35% long chain fatty acids (C20:0 to C28:0). Six fatty acids were identified which had odd-numbered carbon chain lengths (C15:0, C17:0, C19:0, C21:0, C23:0 and C25:0). Of these, pentacosanoic acid made up almost 14% of the fatty acids in the acylglycerides. Three methyl-branched chain fatty acids, namely isopalmitic, isoheptadecanoic and anteisopalmitic, were identified in the ECM, all in small amounts. Of the fatty alcohols identified, only palmityl and stearyl alcohols were found in the free form (ca. 57% and 43%, respectively) but arachidyl alcohol (ca. 47%) and 1-octacosanol (ca. 30%) were the most abundant fatty alcohols found in the wax ester fraction.     

Evolutionary analysis of endopolygalacturonase-encoding genes of Botrytis cinerea

Sequence analysis of five of the six endopolygalacturonase-encoding genes ( Bcpg1 , Bcpg2 , Bcpg3 , Bcpg4 , Bcpg5 ) from 32 strains of Botrytis cinerea showed marked gene to gene differences in the amount of among-strains diversity. Bcpg4 was almost invariable in all strains; Bcpg3 and Bcpg5 showed a moderate variability, similar to that of non-pathogenicity-associated genes examined in other studies. Conversely, Bcpg1 and Bcpg2 were highly variable and were shown to be under positive selection based on the McDonald–Kreitman test and likelihood ratio test. The evolution of the five endopolygalacturonase genes is explained by their different ecophysiological role. Diversification and balancing selection, as detected in Bcpg1 and Bcpg2 , can be used by the pathogen to escape recognition by the host and delay plant reaction in the early phases of infection. The analysis of the polymorphisms and the location of the sites with high probability of being positively selected highlighted the relevance of variability of the BcPG1 and BcPG2 proteins at their C-terminal end. By contrast, the absence of variability in Bcpg4 suggests that the efficiency of the product of this gene is critical for B. cinerea growth in late phases of infection or during intraspecific competition, thus markedly affecting strain fitness.     

Genetic analysis and relationship to pathogenicity in Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. Ten strains from different locations collected on different years have been isolated and characterized by several methods (morphological, biochemical, genetical and molecular). Results showed that clear morphological differences exist between strains, and showing a relationship between the presence of sclerotia and pathogenicity. The conidial size and the nuclear number were highly variable between different strains. Pulsed-field gel electrophoresis showed a unique karyotype for each strain, highly polymorphic between strains and with a number of bands ranging from 4 to 8. An efficient transformation system has been achieved through the plasmid pAMPF21, containing the region AMA1 of Aspergillus nidulans. Lastly, from a genomic library the gdhA gene has been cloned. This gene produces an RNAm of 1.7 Kb and complements the deficiency on glutamate dehydrogenase activity of A. nidulans.     

Generation and analysis of expressed sequence tags from Botrytis cinerea

Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen.     

The Botrytis cinerea early secretome

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2‐D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC‐MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.     

Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.     

Oxidation of Alcohols by Botrytis cinerea

Crude cell-free preparations of Botrytis cinerea were found to oxidize straight-chain primary alcohols (except methanol), aromatic primary alcohols, and unsaturated primary alcohols. The resulting products were the corresponding aldehydes and an equal molar quantity of hydrogen peroxide.     

Bioconversion of alpha-Damascone by Botrytis cinerea

Bioconversion of alpha-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-alpha-damascone, cis- and trans-3-hydroxy-alpha-damascone, gamma-damascenone, 3-oxo-8, 9-dihydro-alpha-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-alpha-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-alpha-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.     

Bioconversion of citronellol by Botrytis cinerea

Summary Bioconversion of citronellol 1 was studied with four strains of Botrytis cinerea. Using grape must predominant transformation of 1 to 2,6-dimethyl-1,8-octandiol 2 and (E)-2,6-dimethyl-2-octen-1,8-diol 3 was observed. In minor amounts 2,6-dimethyl-2,8-octandiol 4, two p-menthan-3,8-diol isomers 5a, 5b, (Z)-2,6-dimethyl-2-octen-1,8-diol 6, isopulegol 7, 2-methyl-2-hepten-6-one-1-ol 8 and 2-methyl--butyrolactone 9 were found. Using a small amount of grape must in a synthetic medium (1:700) the bioconversion products 2, 4, 5a and 5b were absent, but additionally 2-methyl-2-hepten-6-one 10, 2-methyl-2-hepten-6-ol 11 and citronellic acid 12 were detected. The results obtained were strongly dependent on the strains used; one strain did not show any metabolic activity against 1. The bioconversion products were identified by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e. on-line — mass spectrometry (HRGC-MS) and — Fourier transform infrared spectroscopy (HRGC-FTIR).     

Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation

The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. Moreover, in the last few years, B. cinerea has been adopted as an important model system in molecular phytopathology. In spite of these contributions, the molecular basis of the infection cycle remains unclear. Proteomic approaches have revealed significant information about the infective cycle of several pathogens, including B. cinerea. The main aim of this study is to make available a proteomic database containing a significant number of identified proteins from B. cinerea. In brief, three independent B. cinerea cultures supplemented with carboxymethylcellulose were used, and the extracted proteins were independently separated by 2‐D PAGE to obtain the proteome map from B. cinerea. Two hundred and sixty‐seven spots were selected for MALDI TOF/TOF MS analysis, resulting in 303 positive identifications, mostly representing unannotated proteins. Identified proteins were then classified into categories using the PANTHER classification system ( www.pantherdb.org ), showing the relevance of protein metabolism and modification process and oxidoreductase activity. Since cellulose is one of the major components of the plant cell wall, many of the identified proteins may have a crucial role in the pathogenicity process. In brief, this proteomic map of B. cinerea will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design.     

Adhesion of germlings of Botrytis cinerea.

Adhesion of conidia and germlings of the facultative plant parasite Botrytis cinerea occurs in two distinct stages. The first stage, which occurs immediately upon hydration of conidia and is characterized by relatively weak adhesive forces, appears to involve hydrophobic interactions (R. P. Doss, S. W. Potter, G. A. Chastagner, and J. K. Christian, Appl. Environ. Microbiol. 59:1786-1791, 1993). The second stage of adhesion, delayed adhesion, occurs after viable conidia have been incubated for several hours under conditions that promote germination. At this time, the germlings attach strongly to either hydrophobic or hydrophilic substrata. Delayed adhesion involves secretion of an ensheating film that remains attached to the substratum upon physical removal of the germlings. This fungal sheath, which can be visualized by using interference-contrast light microscopy, scanning electron microscopy, or atomic force microscopy, is 25 to 60 nm thick in the region immediately adjacent to the germ tubes. Germlings are resistant to removal by boiling or by treatment with a number of hydrolytic enzymes, 2.0 M periodic acid, or 1.0 M sulfuric acid. They are readily removed by brief exposure to 1.25 N NaOH. A base-soluble material that adheres to culture flask walls in short-term liquid cultures of B. cinerea is composed of glucose (about 30%), galactosamine (about 3%), and protein (30 to 44%).     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.