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The market for microalgae is substantial and currently expanding. To meet this demand, it is necessary to demonstrate the feasibility of large-scale biomass production over long periods. This study aims to demonstrate the feasibility and sustainability of a semi-industrial process for the outdoor cultivation of the microalga Nannochloropsis gaditana in flat-panel reactors using flue gases on demand from a coal-fired power plant. To this end, four secondary objectives were defined: (i) To generate an accurate estimate of the annual average biomass production using this process; (ii) To determine how the production rate and the biochemical composition of the biomass vary with the weather conditions over the course of a year; (iii) To reduce operational costs by developing a new culture medium based on natural seawater enriched with agricultural fertilizers; and (iv) To increase the productivity yield by optimizing the air flow rate in outdoor flat-panel reactors. This research is relevant because it fills an information gap in the available literature regarding experimentation with outdoor microalgae culture, on a semi-industrial scale, using flue gases, for a period of 2 full years. Results showed that biomass production and growth rate range from 0.040 ± 0.022 g (dry weight) L−1 d−1 and 0.088 ± 0.044 d−1 to 0.078 ± 0.033 g dwt L−1 d−1 and 0.179 ± 0.079 d−1, respectively, depending on the environmental conditions. Protein and lipid content range from 34.4 ± 8.163 to 43.5 ± 13.250% dry weight and from 12.5 ± 2.243 to 14.4 ± 0.936% dwt, respectively. The culture was maintained during a complete cycle of two years, with the stated conditions, and demonstrated the technical feasibility and sustainability of the process.

During the last decades, proteomics approaches has become in a fundamental tool to unravel the proteins dynamic of biological processes, being an essential technique for basic and applied research [1-4]. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and MS/MS improvements has allowed the application of proteomic of non-model microorganisms, which have been deeply described as a novel source of metabolites [5-8]. Between them, Nannochloropsis gaditana that has been pointed out as an alternative source of biomolecules [9-13]. Recently, our research group has reported the first complete proteome analysis of this microalga [14], which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we select UCA01 protein from prohibitin family. The recombinant protein showed its antitumor activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data has been transformed into relevant biotechnological information. In summary, the framework of the European Union “Blue growth” politicies, the revalorization of microalgae biomass is crucial point. From Nannochloropsis gaditana has been developed a new tool against cancer, the protein named UCA01. This protein has selective effect inhibiting the growth of tumour cells, while does not show any effect over control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named “applied proteomics”. It is based in a novel bio-algorism, which is able to identified proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bio-algorism identified over 400 proteins with potential use; one of them was selected as UCA01, “in vitro” and its potential prove it against cancer. The potential applications from this approach are unpredictable.

Nannochloropsis gaditana is a non-flagellated microalgae that has been widely used for different purposes, mostly related with the industrial production of biofuels or aquiculture. However, in order to increase the economic viability of the obtained microalgae biomass from a production plant coupled to a coal power plant, a proteomic approach was initiated by using fresh and atomized microalgae samples, as the main used commercial forms. Above 51,000 high quality spectra were obtained per sample in the MS/MS analysis of whole proteome of N. gaditana, yielding above 7,500 peptides, leading the identification of 1,950 proteins, from the N. gaditana protein database, where 655 proteins were presented in all the replicates. The identified proteins were categorized according to gene ontology classification by molecular function and biological process. In this study, it has been described the first proteomic analysis of the microalgae N. gaditana under industrial conditions containing an important number of identified proteins. A significative presence of proteins with a potential role in different agri-food and biomedical applications was detected and studied being the core of future N. gaditana research to expand the current biotechnological applications of this microalga. SIGNIFICANCE OF THE STUDY: Three quarters of the planet earth correspond to seas and oceans, however its potential biotechnological use is still unknown. We described the first proteomic description of the microalgae N. gaditana under industrial conditions. Following the spirit of the EU initiatives of blue growth and the statements of circular economy, CO2 waste from a coal plant power has been transformed in a resource for microalgae biomass production, common product presentations were evaluated by proteomic, and its potential use of identified proteins in Agri-food and Biomedicine has been revealed.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein&ndash, protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named &ldquo, Applied Proteomics&rdquo, has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer&mdash, the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named &ldquo, applied proteomics&rdquo, It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses, one of them was selected as UCA01, &ldquo, in vitro&rdquo, and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

The ascomycete Botrytis cinerea is one of the most relevant plant pathogenic fungi, affecting fruits, flowers, and greenhouse-grown crops. The infection strategy used by the fungus comprises a magnificent set of tools to penetrate and overcome plant defenses. In this context, the plant-pathogen communication through membrane receptors and signal transduction cascades is essential to trigger specific routes and the final success of the infection. In previous reports, proteomics approaches to B. cinerea signal transduction cascades changes in response to different carbon source and plant-based elicitors have been performed. Analyzing the secretome, membranome, phosphoproteome, and the phosphomembranome. Moreover, phenotypic changes in fungal biology was analyzed, specifically toxin production. To obtain the whole picture of the process and reveal the network from a system biology approach, this proteomic information has been merged with the phenotypic characterization, to be analyzed using several bioinformatics algorithms (GO, STRING, MCODE) in order to unravel key points in the signal transduction regulation crucial to overcome plant defenses, as well as new virulence/pathogenicity factors that could be used as therapeutic targets in the control of the gray mold rot disease. A total of 1721 and 663 exclusive or overexpressed proteins were identified under glucose (GLU) and deproteinized tomato cell walls (TCW), summarizing all of the protein identifications under phenotypic characterized stages. Under GO analysis, there are more biological process and molecular functions described in GLU, highlighting the increase in signaling related categories. These results agree with the high number of total identified proteins in GLU, probably indicating a more varied and active metabolism of the fungus. When analyzing only GO annotations related with signal transduction, it was revealed that there were proteins related to TOR signaling, the phosphorelay signal transduction system, and inositol lipid-mediated signaling, only under GLU conditions. On the contrary, calcium-mediated signaling GO annotation is only present between the proteins identified under TCW conditions. To establish a potential relationship between expressed proteins, cluster analyses showed 41 and 14 clusters under GLU and TCW conditions, confirming an increase in biological activity in GLU, where we identified a larger number of clusters related to transcription, translation, and cell division, between others. From these analyses, clusters related to signal transduction and clusters related to mycotoxin production were found, which correlated with the phenotypic characterization. The identification of the proteins encompassed in each condition and signal transduction cascade would provide the research community with new information about the B. cinerea infection process and potential candidates of pathogenicity/virulence factors, overcoming plant defenses, and new therapeutic targets., The present research was made possible due to the funding received from Universidad de Cadiz. Project: development of new proteomic approaches to B. cinerea to detect rapid changes in signaling cascades responsible for triggering the first steps of phytopathogenic infective processesPROTEOCAS (ref. PR2020-002).

Knowledge at the molecular level of microalgae has obtained great scientific interest in recent years, because of their potential for biofuel production, as well as other highly valued molecules. Recent advances in the area of the genetic manipulation of microalgae open an entirely new field of possible applications for these organisms as biorefineries. This review represents a compendium that gathers the main milestones that have marked the area of genetic engineering in microalgae during the last decade, from classical techniques for the transformation and expression of transgenes, to the most avant‐garde techniques currently used for gene silencing and DNA edition, as well as a discussion about the future perspectives in this field., This research has been financed by A4HW project (RTC‐2016‐4860‐2) by the Spanish Ministry of Economy, Industry and Competitiveness, under the Collaboration Challenges programme. This aid is co‐financed by the European Union through FEDER funds, with the aim of promoting Technological Development, Innovation and Quality Research.

© 2020 by the authors., Pyrocystis lunula is considered a model organism due to its bioluminescence capacity linked to circadian rhythms. The mechanisms underlying the bioluminescent phenomenon have been well characterized in dinoflagellates; however, there are still some aspects that remain an enigma. Such is the case of the presence and diversity of the luciferin-binding protein (LBP), as well as the synthesis process of luciferin. Here we carry out a review of the literature in relation to the molecular players responsible for bioluminescence in dinoflagellates, with particular interest in P. lunula. We also carried out a phylogenetic analysis of the conservation of protein sequence, structure and evolutionary pattern of these key players. The basic structure of the luciferase (LCF) is quite conserved among the sequences reported to date for dinoflagellate species, but not in the case of the LBP, which has proven to be more variable in terms of sequence and structure. In the case of luciferin, its synthesis has been shown to be complex process with more than one metabolic pathway involved. The glutathione S-transferase (GST) and the P630 or blue compound, seem to be involved in this process. In the same way, various hypotheses regarding the role of bioluminescence in dinoflagellates are exposed., This work was partly supported by the Ministry of Economy, Industry and Competitiveness of Spanish Government under the Collaboration Challenges program (A4HW project, RTC-2016-4860-2), and co-financed by the European Union through FEDER funds. C.F. was partly sponsored through scholarships granted by the International Relations Office of the University of Cadiz (ORI-UCA) in the frame of ERASMUS+ program; and the Ibero-American Postgraduate University Association (AUIP). B.C. was supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal) through grant IF/00197/2015.

Botrytis cinerea is a critically important phytopathogenic fungus, causing devastating crop losses; signal transduction cascades mediate the “dialogue” among the fungus, plant, and environment. Surface proteins play important roles as front-line receptors. We report the first description of the surfactome of a filamentous fungus. To obtain a complete view of these cascades during infection of B. cinerea, its surfactome has been described by optimization of the “shaving” process and LC–MS/MS at two different infection stages, and with both rapid and late responses to environmental changes. The best results were obtained using PBS buffer in the “shaving” protocol. The surfactome obtained comprises 1010 identified proteins. These have been categorized by gene ontology and protein–protein interactions to reveal new potential pathogenicity/virulence factors. From these data, the percentage of total proteins predicted for the genome of the fungus represented by proteins identified in this and other proteomics studies is calculated at 54%, a big increase over the previous 12%. The new data may be crucial for understanding better its biological activity and pathogenicity. Given its extensive exposure to plants and environmental conditions, the surfactome presents innumerable opportunities for interactions between the fungus and external elements, which should offer the best targets for fungicide development.

Botrytis cinerea is considered a model organism for the study of plant–pathogen interaction showing great genetic diversity and a high degree of morphological variability depending on environmental conditions. The use of new compounds and plant-based elicitors may trigger the expression of different B. cinerea genes, providing new sources of virulence factors. This work is focused on elucidating the phenotypic effect in B. cinerea of different carbon sources such as glucose, cellulose and tomato cell walls (TCW). Production of botrydial and dihydrobotrydial toxins was evaluated using thin-layer chromatography (TLC), proton nuclear magnetic resonance spectroscopy (1H-NMR) and mass spectrometry (UPLC-HRESIMS). Expression of the toxin biosynthesis gene BcBOT2 was followed using RT-qPCR. Results show an inhibition of the toxin biosynthesis pathway when TCW are present as a sole carbon source, suggesting that the toxin is only produced when rich molecules, like glucose, are available for fungal metabolism. That suggests a connection between gene expression of virulence factors and environmental conditions, where the silent genes can be induced by different culture conditions. Electronic supplementary material The online version of this article (10.1007/s00284-017-1399-3) contains supplementary material, which is available to authorized users.

Protein phosphorylation and membrane proteins play an important role in the infection of plants by phytopathogenic fungi, given their involvement in signal transduction cascades. Botrytis cinerea is a well-studied necrotrophic fungus taken as a model organism in fungal plant pathology, given its broad host range and adverse economic impact. To elucidate relevant events during infection, several proteomics analyses have been performed in B. cinerea, but they cover only 10% of the total proteins predicted in the genome database of this fungus. To increase coverage, we analysed by LC-MS/MS the first-reported overlapped proteome in phytopathogenic fungi, the "phosphomembranome" of B. cinerea, combining the two most important signal transduction subproteomes. Of the 1112 membrane-associated phosphoproteins identified, 64 and 243 were classified as exclusively identified or overexpressed under glucose and deproteinized tomato cell wall conditions, respectively. Seven proteins were found under both conditions, but these presented a specific phosphorylation pattern, so they were considered as exclusively identified or overexpressed proteins. From bioinformatics analysis, those differences in the membrane-associated phosphoproteins composition were associated with various processes, including pyruvate metabolism, unfolded protein response, oxidative stress response, autophagy and cell death. Our results suggest these proteins play a significant role in the B. cinerea pathogenic cycle. This research has been financed by the Spanish Government under project AGL2015-65684-C2-2-R supported by the Spanish Ministry of Economy and Competitiveness. Eva Liñeiro was supported by an FPI grant from the University of Cádiz (2010-152). The CRG/UPF Proteomics Unit is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013–2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012-0208, and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595).

Worldwide, 20–25% of all harvested fruit and vegetables are lost annually in the field and throughout the postharvest supply chain due to rotting by fungal pathogens. Most postharvest pathogens exhibit necrotrophic or saprotrophic lifestyles, resulting in decomposition of the host tissues and loss of marketable commodities. Necrotrophic fungi can readily infect ripe fruit leading to the rapid establishment of disease symptoms. However, these pathogens generally fail to infect unripe fruit or remain quiescent until host conditions stimulate a successful infection. Previous research on infections of fruit has mainly been focused on the host’s genetic and physicochemical factors that inhibit or promote disease. Here, we investigated if fruit pathogens can modify their own infection strategies in response to the ripening stage of the host. To test this hypothesis, we profiled global gene expression of three fungal pathogens that display necrotrophic behavior—Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer—during interactions with unripe and ripe tomato fruit. We assembled and functionally annotated the transcriptomes of F. acuminatum and R. stolonifer as no genomic resources were available. Then, we conducted differential gene expression analysis to compare each pathogen during inoculations versus in vitro conditions. Through characterizing patterns of overrepresented pathogenicity and virulence functions (e.g., phytotoxin production, cell wall degradation, and proteolysis) among the differentially expressed genes, we were able to determine shared strategies among the three fungi during infections of compatible (ripe) and incompatible (unripe) fruit tissues. Though each pathogen’s strategy differed in the details, interactions with unripe fruit were commonly characterized by an emphasis on the degradation of cell wall components, particularly pectin, while colonization of ripe fruit featured more heavily redox processes, proteolysis, metabolism of simple sugars, and chitin biosynthesis. Furthermore, we determined that the three fungi were unable to infect fruit from the non-ripening (nor) tomato mutant, confirming that to cause disease, these pathogens require the host tissues to undergo specific ripening processes. By enabling a better understanding of fungal necrotrophic infection strategies, we move closer to generating accurate models of fruit diseases and the development of early detection tools and effective management strategies.

Pyrocystis lunula (Schutt) is a photoautotrophic dinoflagellate without armored form, frequently found in marine environments. Today, there are several biotechnological applications derived from the bioluminescent system of this species. From a post-genomic perspective, in order to have a starting point for studying the proteome of P. lunula, an “omics” approach (transcriptomics-proteomics) was assessed using fresh microalgae samples. A total of 80,874,825 raw reads were generated (11,292,087,505 bp; 55.82% GC) by mRNA sequencing. Very high-quality sequences were assembled into 414,295 contigs (219,203,407 bp; 55.38% GC) using Trinity software, generating a comprehensive reference transcriptome for this species. Then, a P. lunula proteome was inferred and further employed for its analysis on this species. A total of 17,461 peptides were identified, yielding 3182 protein identification hits, including 175 novel proteins. The identified proteins were further categorized according to functional description and gene ontology classification. Significance: The major contribution of the present work is making available a reference transcriptome and proteome of P. lunula, that is now accessible for the research community, and a functional description of the 3182 proteins inferred from the transcriptome, including 175 novel proteins, which have already been deposited in the ProteomeXchange and NCBI SRA databases, respectively. In addition to this, a series of important factors related to the bioluminescent system and the regulation of gene expression, were identified and described., This work was supported by the Ministry of Economy, Industry and Competitiveness of Spanish Government under the Collaboration Challenges program [RTC-2016-4860-2, A4HW project, 2016], and co-financed by the European Union through FEDER Funds.

Phosphorylation is one of the main post-translational modification (PTM) involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW). A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099). Further interpretation and discussion of these data are provided in our research article entitled “Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors” (Liñeiro et al., 2016) [1]. This research has been financed by the Spanish Government under Project DGICYTAGL2012-39798-C02-01/02 (www.micinn.es/portal/site/MICINN/). Eva Liñeiro was supported by a FPI grant from the University of Cádiz (2010-152). The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of Instituto de Salud Carlos III (ISCIII) and it is also supported by the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’ (SEV-2012-0208), and by the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (Project 2014SGR678).

The phytopathogen Botrytis cinerea is a ubiquitous fungus with a high capacity to adapt its metabolism to different hosts and environmental conditions in order to deploy a variety of virulence and pathogenicity factors and develop a successful plant infection. Here we report the first comparative phosphoproteomic study of B. cinerea, aimed to analyze the phosphoprotein composition of the fungus and its changes under different phenotypical conditions induced by two different carbon sources as plant based elicitors: glucose and deproteinized tomato cell wall (TCW). A total of 2854 and 2269 different phosphosites (2883 and 1137 phosphopeptides) were identified in glucose and TCW respectively, which map to 1338 phosphoproteins in glucose and 733 in TCW. Out of the identified phosphoproteins, 173 were exclusively found when glucose was the only carbon source and 11 when the carbon source was TCW. Differences in the pattern of phosphorylation-sites were also detected according to the carbon source. Gene ontology classification of the identified phosphoproteins showed that most of the characteristic proteins of the different carbon sources were related to signalling and transmembrane transport, thus highlighting the importance of these processes in the fungal adaptation to the surrounding conditions. Biological significance The characterization of the B. cinerea phosphoproteome under different induction conditions reported here is the first comparative phosphoproteomic approach in this model phytopathogenic fungus. The identified phosphopeptides contribute to expand the map of known phosphoproteins in this pathogen and the observed changes according to the used carbon source contribute to understand the adaptation of the fungus to the environment changes. This knowledge improves the understanding of the adaptation mechanism, defines the role of the phosphoproteins involved in this process, and enables the advance in the design of novel strategies against the fungi.

Botrytis cinerea is one of the most relevant plant pathogenic fungi. The first step during its infection process is the germination of the conidia. Here, we report on the first proteome analysis during the germination of B. cinerea conidia, where 204 spots showed significant differences in their accumulation between ungerminated and germinated conidia by two-dimensional polyacrylamide gel electrophoresis and qPCR. The identified proteins were grouped by gene ontology revealing that the infective tools are mainly preformed inside the ungerminated conidia allowing a quick fungal development at the early stages of conidial germination. From 118 identified spots, several virulence factors have been identified while proteins, such as mannitol-1-phosphate dehydrogenase, 6,7-dimethyl-8-ribityllumazine synthase or uracil phosphoribosyltransferase, have been disclosed as a new potential virulence factors in botrytis whose role in pathogenicity needs to be studied to gain new insights about the role of these proteins as therapeutic targets and virulence factors.

In the current context of global warming and fossil fuel depletion, the aim of the EU Blue Growth concept is to support sustainable growth in the marine and maritime sectors. A crucial issue under this initiative is the valorization of microalgae biomass. The production of sugar and polyalcohols from microalgae Nannochloropsi s gaditana , Arthrospira platensis, and Pyrocystis lunula has been studied in the context of this initiative. Using fresh and/or atomized samples, several extraction methods were assayed. In brief, two types of lysis (mechanical and ultrasonic), variations in temperature and treatments times, and treatments with HCl and maceration. The obtained mono-saccharides, di-saccharides and polyalcohols were analyzed by HPLC. Best obtained results were for saccharose with the HCl treatment and mannitol with maceration treatment extracted from N. gaditana biomass (160250 and 72148 mg/L respectively). These results may open new routes for the commercial exploitation of microalgae biomass as a completely novel raw material for the food, drinks and pharmaceuticals manufacturing industries.

Colletotrichum acutatum is an important phytopathogenic fungus causing anthracnose in commercially important fruit crops, such as strawberry. The conidia produced by the fungus are survival structures which play a key role in host infection and fungal propagation. Despite its relevance to the fungal life cycle, conidial biology has not been extensively investigated. Here, we provide the first proteomic description of the conidial germination in C. acutatum by comparing the proteomic profiles of ungerminated and germinated conidia. Using two-dimensional electrophoresis combined with MALDI-TOF/TOF mass spectrometry, we have identified 365 proteins in 354 spots, which represent 245 unique proteins, including some proteins with key functions in pathogenesis. All these proteins have been classified according to their molecular function and their involvement in biological processes, including cellular energy production, oxidative metabolism, stress, fatty acid synthesis, protein synthesis, and folding. This report constitutes the first comprehensive study of protein expression during the early stage of the C. acutatum conidial germination. It advances our understanding of the molecular mechanisms involved in the conidial germination process, and provides a useful basis for the further characterization of proteins involved in fungal biology and fungus life cycles.

Phosphorylation is one of the main post-translational modification (PTM) involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW). A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099). Further interpretation and discussion of these data are provided in our research article entitled “Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors” (Liñeiro et al., 2016) [1].

Recent work has clearly shown the capacity of proteomics-based methodologies to establish the roles played by specific proteins in different biological processes. Beyond the study of genes, it has been established that proteins are the relevant set to be analyzed in research aiming to solve specific biological questions. Proteomics approaches can be categorized according to three different methodologies; gel-based, mainly two-dimension gel electrophoresis (2-DE); gel free, based on liquid chromatography-mass spectrometry (LC-MS); and quantitative proteomics, by isobaric markers. Most of these methodologies have been applied to studies of the proteome of Botrytis cinerea. Since the publication of the first proteomics report on Botrytis, technological advances have accelerated the identification of global protein content. Clearly, the publication of the B. cinerea genome has been of tremendous value to the proteomics research community; this has supported the accurate identification, through MS, of this fungus’ peptides. This landmark event has greatly facilitated the development of proteomics studies exploring the biology of the fungus; to date, mainly mycelium samples have been used. Only a few reports have aimed at the study of fractions of the total proteome, and all of these are focused on the secretome. The role of several particular proteins related to fungal pathogenicity, metabolism, biology, etc. has been elucidated, but the number of Botrytis proteins found, as a proportion of the total proteins predicted from the genome, remains below 10 %. There is much work to be done.

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.

Pathogenic fungi in plants are now one of the most serious causes of crop losses in Europe. Phytopathogenic fungi are able to infect any tissue at any stage of plant growth. Most significantly, these organisms are able to maintain their infective capacity between seasons because sources of inoculum can remain present in soils and plant debris. The value of producers' crop losses and the costs of fungicide treatments are extremely relevant economic data. The complex cycle of infection is directed by a set of genes/proteins known as pathogenicity or virulence factors. Proteomics is one of the techniques that is currently attracting much interest in the study of these organisms. At present, there is a significant increase in the number of research projects in which proteomics approaches are being used to understand the complex interactions between plants and their pathogens. This trend confirms the usefulness of these techniques for studying the biology of these pathogens, their pathogenicity or virulence factors, and for exploring new proteins that could be used as therapeutic targets. However, there are still problems to be resolved in these approaches. There is also a continuous process of improvement in many crucial stages of these proteomic studies of fungal plant pathogens - in experimental designs, in protein extraction procedures, in the separation and identification of proteins, in the analysis of results of proteome studies, and more. This review will describe the different approaches being taken in proteomics studies of fungal plant pathogens. It will highlight the solutions being adopted to overcome the complications arising from the nature of the specific biological samples. Currently, there are no fungicides on the market that have been developed from molecular biology studies. Consequently the knowledge gained from past and present proteomic studies is still to be used in the design of fungicides, although the authors are optimistic in this respect. © 2010 Bentham Science Publishers Ltd.

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.

Colletotrichum acutatum is a major plant pathogen which infects a broad range of host plants. Extensive research has been carried out on C. acutatum populations affecting various hosts in different geographical locations, showing a considerable genotypic and phenotypic diversity. Anthracnose, caused by Colletotrichum spp., is the major disease of cultivated strawberry, Fragaria x ananassa. In the present study, the phylogenetic relationships within a worldwide sample of fifty-two C. acutatum isolates collected from different strawberry cultivars have been established, by using ITS sequence analyses. Twenty-nine isolates clustered in the molecular group A2, in which seventeen out of eighteen Spanish isolates were included; this may indicate that the group A2 is the key group in Spain. The molecular polymorphism among C. acutatum isolates was determined by southern-blot hybridisation using a telomeric DNA probe. Results indicated that the minimum number of estimated chromosomes ranges between six and nine. The molecular characterisation of C. acutatum isolates was completed using the Pulsed-Field Gel Electrophoresis (PFGE) technique that resolved from six to nine chromosomal bands, this number being coincident with the number of chromosomes obtained by telomeric fingerprinting. The minimum total genome size was estimated to range from 29 to 36 Mb. Comparison of karyotypes patterns and southern-blot analysis demonstrated a high level of molecular polymorphism among C. acutatum isolates from different origins.

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.

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month (P

Petroleum hydrocarbon contamination of harbor sediments from shipping activity, fuel oil spills, and runoffs are becoming a great concern because of the toxicity and recalcitrance of many of the fuel components. Polycyclic aromatic hydrocarbons (PAHs) are of most concern due to their toxicity, low volatility, resistance to degradation, and high affinity for sediments. Microorganisms, especially bacteria, play an important role in the biodegradation of these hydrocarbons. The objective of the present study was to characterize and isolate PAH-(naphthalene) degrading bacteria in the coastal sediments of Cadiz (SW Spain), since this area is mostly polluted by PAH occurrence. A total of 16 naphthalene-utilizing bacteria were isolated from these sites. Introduction of bacteria isolated from contaminated sediments into mineral medium contributed to the increased rate of hydrocarbon utilization. The bacterial isolates obtained from these sites are very potent in utilizing naphthalene and crude oil. It would be interesting to assess if the selected naphthalene-degrading isolates may degrade other compounds of similar structure. Hence these isolates could be very helpful in bioremediating the PAH-contaminated sites. Further pursue on this work might represent eco-friendly solution for oil contamination on sea surface and coastal area.

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.

Phytopathogenic fungi are organisms responsible for several plant diseases in different crops around the world, causing very important economic losses to the farmers. Fungi have a complicated life cycle, normally with asexual and sexual reproduction that involves the formation of different reproductive structures. Moreover, during plant disease, fungi produce different components that are essential to complete the infection process (enzymes, toxins, etc) and are named "pathogenicity factors". Two dimensional gel electrophoresis (2-DE) has been widely used to study the proteome of a good number of microorganisms. However, few of them have been carried out to study the proteome of phytopathogenic fungi, mainly due to the difficulty of obtaining fungal protein extracts and/or the lack of available fungal protein databases. This review shows the strategies that can be addressed to overcome those problems and how different approaches have resulted to be useful in (i) obtaining the proteomic maps of several phytopathogenic fungi, (ii) determining fungal proteins involved in the formation of infective structures and (iii) studying the production of extracellular proteins or pathogenicity factors in phytopathogenic fungi. Fungal proteomic is still at an early stage, but recent reports show that it is a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

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).

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D-DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC-MS was performed. The proteomic and "aromatomic" results are discussed in relation to the oenological properties of S. bayanus var. uvarum.

Phytophthora spp. is one of the phytopathogenic Oomycete responsible for many important crop losses. Relevant species are P. infestans (causing potato late blight) and P. capsici (causing blight in pepper). In recent years, the use of conventional fungicides has favoured the appearance of different resistant strains. This study analyses the effect of various compounds on these two Phytophthora species. Those compounds were designed on the basis of known structures of natural compounds to obtain a rational control of these fungal-like species. All the analysed products showed a fungistatic activity against both strains, one of them reduced mycelial growth by over 46% at 100 p.p.m.

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

Southwestern Spain is one of the main areas for strawberry culture in the world. Large losses are due to grey mould caused by Botrytis cinerea, a plant pathogen that affects a wide variety of crops. Resistance to synthetic fungicides is one of the more important problems that hamper control of grey mould on strawberry. We have characterized the relative response to benzimidazole (benomyl and carbendazaim) and dicarboximide (vinclozolin) among 36 isolates of B. cinerea obtained from different plots in six counties of the province of Huelva during a 2-year period. Three phenotypes with resistance to benzimidazole were detected: (i) BenR1 having phenotype resistant (R) to benomyl and high-resistant (HR) to carbendazim; (ii) BenR2 with phenotype HR to both benomyl and carbendazim; and (iii) BenR3 with phenotype HR to benomyl and R to carbendazim. Strains were either resistant or sensitive to the dicarboximide vinclozolin (DicR or DicS, respectively), but high-resistance to this fungicide (DicHR strains) was n...

Molecular characterization of wine yeast population during spontaneous fermentation in biodynamic wines from Ribera del Duero D.O. located at northern plateau of Spain has been carried out during two consecutive years. A total of 829 yeast strains were isolated from the samples and characterized by electrophoretic karyotype. The results show the presence of three population of yeast differentiated by their electrophoretic karyotypes, (1) non-Saccharomyces yeast dominant in the initial phase of the fermentations (NS); (2) Saccharomyces bayanus var uvarum detected mainly mid-way through the fermentation process at 20–25 °C; and (3) Saccharomyces cerevisiae which remained dominant until the end of the fermentation. This is the first study showing the population dynamic of S. bayanus var. uvarum in red wines produced in Ribera del Duero that could represent an important source of autochthonous wine yeasts with novel oenological properties.

Fungi is an extensive group of eukaryotic microorganisms, generally they are microscopic and usually filamentous. It is estimated that there are between 70,000 and 1.5 millions species of fungi, most of them are being discovering and describing (Agrios, 2005). Most of the known hundred thousand fungal species are strictly saprophytic, living on decomposing dead organic matter. About fifty species cause disease in human, and more than ten thousand species can cause disease in one (obligate parasite) or many kinds of plants (nonobligate parasites) (Fernandez-Acero et al., 2007a).

which were introduced in the laboratory for genetics and molecular studies in about 1935. There is, however, a second type of yeast comprising those used in industrial processes, for example, in brewing, baking and winemaking. Wine yeast and its properties have been known to humans for as long as civilizations have existed, and the earliest evidence of this yeast has been dated to Neolithic times (Mortimer, 2000). Most wine yeast strains are diploid and have a low frequency of sporulation. Another important characteristic of wine yeasts, and those used in other industries, is their highly polymorphic chromosomes: their genetic constitution is affected by the frequent and extensive mutation they undergo. These effects include (i) aneuploidy, (ii) polyploidy, (iii) amplification and deletion of chromosomal region or single gene, and (iv) the presence of hybrid chromosomes. The chromosomal polymorphism obtained by applying the technique known as pulsed field gel electrophoresis (PFGE) has been used to characterize and to classify strains that belong to the same species. In the wine industry, knowledge of the yeast species responsible for the alcoholic fermentation is important because these yeasts with their metabolism contribute significantly to the organoleptic characteristics of the finished wine (Fleet, 2008). The diverse range of yeasts associated with the vinification process can be classified in two groups. The first group is formed principally by the genera

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.