Jesús Manuel Cantoral

2-DE proteomic approach to the Botrytis cinerea secretome induced with different carbon sources and plant-based elicitors

Botrytis cinerea is a phytopathogenic fungus infecting a number of crops (tomatoes, grapes and strawberries), which has been adopted as a model system in molecular phytopathology. B. cinerea uses a wide variety of infection strategies, which are mediated by a set of genes/proteins called pathogenicity/virulence factors. Many of these factors have been described as secreted proteins, and thus the study of this sub‐proteome, the secretome, under changing circumstances can help us to understand the roles of these factors, possibly revealing new loci for the fight against the pathogen. A 2‐DE, MALDI TOF/TOF‐based approach has been developed to establish the proteins secreted to culture media supplemented with different carbon sources and plant‐based elicitors (in this study: glucose, cellulose, starch, pectin and tomato cell walls). Secreted proteins were obtained from the culture media by deoxycholate‐trichloroacetic acid/phenol extraction, and 76 spots were identified, yielding 95 positive hits that correspond to 56 unique proteins, including several known virulence factors (i.e. pectin methyl esterases, xylanases and proteases). The observed increases in secretion of proteins with established virulence‐related functions indicate that this in vitro‐induction/proteome‐mining approach is a promising strategy for discovering new pathogenicity factors and dissecting infection mechanisms in a discrete fashion.

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

The phytotoxic activity of some metabolites of Botrytis cinerea

Abstract A fungus-free culture filtrate from a static culture of Botrytis cinerea reproduced the symptoms of the ‘grey mould’ disease on tobacco leaves. This aspect of the phytoxicity of B. cinerea could not be attributed to enzyme action. Two metabolites, botrydial and dihydrobotrydial, isolated from the culture filtrate appeared to be responsible for the phytotoxic effect.

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.

Development of Proteomics-Based Fungicides: New Strategies for Environmentally Friendly Control of Fungal Plant Diseases

Proteomics has become one of the most relevant high-throughput technologies. Several approaches have been used for studying, for example, tumor development, biomarker discovery, or microbiology. In this “post-genomic” era, the relevance of these studies has been highlighted as the phenotypes determined by the proteins and not by the genotypes encoding them that is responsible for the final phenotypes. One of the most interesting outcomes of these technologies is the design of new drugs, due to the discovery of new disease factors that may be candidates for new therapeutic targets. To our knowledge, no commercial fungicides have been developed from targeted molecular research, this review will shed some light on future prospects. We will summarize previous research efforts and discuss future innovations, focused on the fight against one of the main agents causing a devastating crops disease, fungal phytopathogens.

Structure-activity relationships of new phytotoxic metabolites with the botryane skeleton from Botrytis cinerea

Abstract The fungal antibiotic botrydial (1) and related compounds constitute an important group of metabolites whose biological activity was not previously known in depth. The isolation, in addition to known compounds, of three new epimer metabolites with the botryane structure has allowed us to study the structure-activity relationships. The results suggest that, in addition to the presence of the dialdehyde functionality, the antibiotic, phytotoxic and cytostatic activities shown by some of these compounds are strongly correlated with the stereochemistry of the C-1/C-8 dialdehyde moieties. The relative configuration (S) of the C-1 substituent seems to play a critical role in the binding of the substrate to the chemoreceptor.

Genomic characterization and selection of wine yeast to conduct industrial fermentations of a white wine produced in a SW Spain winery

Aims:  To analyse the diversity of wild yeast in spontaneous fermentations of a white wine and to select the most suitable autochthonous starter yeasts. The selected yeasts would be used for inoculation of industrial fermentations in several years.

Metabolites from a shake culture of Botrytis cinerea

Abstract Four new metabolites, 10-oxo-dihydrobotrydial, 4β-acetoxy-9β-10β-15α-trihydroxyprobotrydial, β - O -methyldihydrobotrydialone and α - O -methyldihydrobotrydialone, were isolated from a shake culture of Botrytis cenerea . The second compound, a tricyclic sesquiterpene, is a key biosynthetic intermediate and sheds light on the last steps of the biosynthesis of botrydial derivatives. A higher oxidation level was observed in the metabolites isolated. The structures were elucidated by extensive NMR investigations of the natural compounds and their derivatives.

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.

The biodegradation of the phytotoxic metabolite botrydial by its parent organism, Botrytis cinerea

The inhibition of the growth of Botrytis cinerea has been found to be directly proportional to the concentration of its metabolite, botrydial (1). The fungus transforms botrydial (1) to the less active phytotoxins dihydrobotrydial (2), botryenedial (3), and secobotrytrienediol (4). Two main biodegradative pathways of botrydial (1) and the interconversion of the botryane toxins excreted by B. cinerea are proposed.