Raquel González-Fernández

Proteomics of plant pathogenic fungi.

Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

Contribution of Proteomics to the Study of Plant Pathogenic Fungi

Phytopathogenic fungi are one of the most damaging plant parasitic organisms, and can cause serious diseases and important yield losses in crops. The study of the biology of these microorganisms and the interaction with their hosts has experienced great advances in recent years due to the development of moderm, holistic and high-throughput -omic techniques, together with the increasing number of genome sequencing projects and the development of mutants and reverse genetics tools. We highlight among these -omic techniques the importance of proteomics, which has become a relevant tool in plant-fungus pathosystem research. Proteomics intends to identify gene products with a key role in pathogenicity and virulence. These studies would help in the search of key protein targets and in the development of agrochemicals, which may open new ways for crop disease diagnosis and protection. In this review, we made an overview on the contribution of proteomics to the knowledge of life cycle, infection mechanisms, and virulence of the plant pathogenic fungi. Data from current, innovative literature, according to both methodological and experimental systems, were summarized and discussed. Specific sections were devoted to the most studied fungal phytopathogens: Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium graminearum.

Proteomic analysis of mycelium and secretome of different Botrytis cinerea wild-type strains ☆ ☆☆

UNLABELLED The necrotrophic fungus Botrytis cinerea is a very damaging phytopathogen of wide host range and environmental persistence. It is difficult to control because of its genetic versatility, expressed in the many phenotypical differences among isolates. The genomes of the B. cinerea B05.10 and T4 strains have been recently sequenced, becoming a model system for necrotrophic pathogens, and thus opening new alternatives for functional genomics analysis. In this work, the mycelium and secreted proteome of six wild-type strains with different host range, and grown in liquid minimal medium, have been analyzed by using complementary gel-based (1-DE and 2-DE) and gel-free/label-free (nUPLC-MS(E)) approaches. We found differences in the protein profiles among strains belonging to both the mycelium and the secretome. A total of 47 and 51 variable proteins were identified in the mycelium and the secretome, respectively. Some of them, such as malate dehydrogenase or peptidyl-prolyl cis-trans isomerase from the mycelium, and endopolygalacturonase, aspartic protease or cerato-platanin protein from the secretome have been reported as virulence factors, which are involved in host-tissue invasion, pathogenicity or fungal development. BIOLOGICAL SIGNIFICANCE The necrotrophic fungus Botrytis cinerea is an important phytopathogen of wide host range and environmental persistence, causing substantial economic losses worldwide. In this work, the mycelium and secreted proteome of six B. cinerea wild-type strains with different host range have been analyzed by using complementary gel-based and gel-free/label-free approaches. Fungal genetic versatility was confirmed at the proteome level for both mycelium proteome and secreted proteins. A high number of hypothetical proteins with conserved domains related to toxin compounds or to unknown functions were identified, having qualitative differences among strains. The identification of hypothetical proteins suggests that the B. cinerea strains differ mostly in processes involved in adaptation to a particular environment or a growth condition, rather than in essential metabolic reactions. Proteomics can help in the identification of variable proteins related to the infection and colonization of host plant tissues, as well as of virulence and aggressiveness factors among different B. cinerea wild-type strains. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Physiological and Proteomic Analyses of Drought Stress Response in Holm Oak Provenances

Responses to drought stress by water withholding have been studied in 1 year old Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) seedlings from seven provenances from Andalusia (southern Spain). Several physiological parameters, including predawn xylem water potentials and relative water content in soil, roots, and leaves as well as maximum quantum efficiency and yield of PSII were evaluated for 28 days in both irrigated and nonirrigated seedlings. The leaf proteome map of the two provenances that show the extreme responses (Seville, GSE, is the most susceptible, while Almerı́a, SSA, is the least susceptible) was obtained. Statistically significant variable spots among provenances and treatments were subjected to MALDI-TOF/TOF-MS/MS analysis for protein identification. In response to drought stress, ~12.4% of the reproducible spots varied significantly depending on the treatment and the population. These variable proteins were mainly chloroplastic and belonged to the metabolism and defense/stress functional categories. The 2-DE protein profile of nonirrigated seedlings was similar in both provenances. Physiological and proteomics data were generally in good agreement. The general trend was a decrease in protein abundance upon water withholding in both provenances, mainly in those involved in ATP synthesis and photosynthesis. This decrease, moreover, was most marked in the most susceptible population compared with the less susceptible one.

Application of Label-Free Shotgun nUPLC–MSE and 2-DE Approaches in the Study of Botrytis cinerea Mycelium

The phytopathogenic fungus Botrytis cinerea infects more than different 200 plant species and causes substantial losses in numerous crops. The B05.10 and T4 wild-type strain genomes have been recently sequenced, becoming a model system for necrotrophic pathogens, as well as opening up new alternatives in functional genomics, such as proteomics. We analyzed B. cinerea mycelium from these two wild-type strains, introducing label-free shotgun nUPLC-MS(E) methodology to complement the 2-DE-MS-based approach. We assessed the label-free nUPLC-MS(E) methodology for protein identification and quantification using five mycelium protein dilutions. A total of 225 and 170 protein species were identified by nUPLC-MS(E) in the B05.10 and T4 strains, respectively. Moreover, 129 protein species were quantified in both strains. Significant differences in protein abundance were found in 15 more abundant and 16 less abundant protein species in the B05.10 strain compared to the T4 strain. Twenty-nine qualitative and 15 significant quantitative differences were found using 2-DE. The label-free nUPLC-MS(E) was a reliable, reproducible and sensitive method for protein identification and quantification to study the B. cinerea mycelial proteome. Results obtained by gel-based and gel-free complementary approaches allow a deeper characterization of this fungus, as well as the identification of potential virulence factors.

Unraveling the in vitro secretome of the phytopathogen Botrytis cinerea to understand the interaction with its hosts

Botrytis cinerea is a necrotrophic fungus with high adaptability to different environments and hosts. It secretes a large number of extracellular proteins, which favor plant tissue penetration and colonization, thus contributing to virulence. Secretomics is a proteomics sub-discipline which study the secreted proteins and their secretion mechanisms, so-called secretome. By using proteomics as experimental approach, many secreted proteins by B. cinerea have been identified from in vitro experiments, and belonging to different functional categories: (i) cell wall-degrading enzymes such as pectinesterases and endo-polygalacturonases; (ii) proteases involved in host protein degradation such as an aspartic protease; (iii) proteins related to the oxidative burst such as glyoxal oxidase; (iv) proteins which may induce the plant hypersensitive response such as a cerato-platanin domain-containing protein; and (v) proteins related to production and secretion of toxins such as malate dehydrogenase. In this mini-review, we made an overview of the proteomics contribution to the study and knowledge of the B. cinerea extracellular secreted proteins based on our current work carried out from in vitro experiments, and recent published papers both in vitro and in planta studies on this fungi. We hypothesize on the putative functions of these secreted proteins, and their connection to the biology of the B. cinerea interaction with its hosts.

Metabolite and proteome changes during the ripening of Syrah and Cabernet Sauvignon grape varieties cultured in a nontraditional wine region in Brazil

UNLABELLED Grapevines are an important fruit crop from economic and cultural point of views in many countries, including Brazil, where the practice of vitiviniculture is being developed in different regions. We compared the anthocyanin concentration, the main organic acids and sugars, and the proteome profiles during berry ripening of Syrah and Cabernet Sauvignon grapes from two distinct geographical sources in São Paulo State. The proteome was mapped by two-dimensional gel electrophoresis and differentially abundant proteins during the ripening process were subjected to MALDI-TOF/TOF-MS analysis. An increase in sugar concentration and in anthocyanin content was observed, as well a decrease in the tartaric and malic acid concentration. A total of 128 spots varied with geographical origin, grape variety, and ripening stage, with 108 being identified. The identified proteins resulted in 80 gene products. A multivariate analysis of protein abundance clustered the samples according to grape variety, geographical origin, and stage of ripening, and showed the possibility of using proteomics to characterize three variables: variety, area where grown, and the ripening process. The changes observed during the ripening process corresponded to enzymes involved in sugar and organic acid metabolism. These results are in accordance with the metabolic profile reported for the process. BIOLOGICAL SIGNIFICANCE Given the importance of discriminating grapes, thus making the adulteration of wines more difficult, in this paper we showed the possibility of differentiating varieties of grapes, geographical area of cultivation and stage of ripening by combining the results of differentially abundant protein determinations and multivariate analysis.

Changes in the Proteome of Functional and Regressing Corpus Luteum During Pregnancy and Lactation in the Rat

Abstract The corpus luteum (CL) is an exquisitely regulated transitory endocrine gland necessary for the onset and maintenance of pregnancy in mammals. Most of the data on the mechanisms of CL differentiation at the molecular level come from genomic studies, but direct protein data are scarce. Here we have undertaken a differential expression proteomic approach to identify, in an unbiased way, those proteins whose levels change significantly in the rat CL as it evolves from functionality during pregnancy to regression after parturition. Moreover, we have compared the regressing CL with the newly formed functional CL that coexist during lactation under the same endocrine environment. We have defined a “proteomic signature” of CL functionality, which is constituted by a set of 24 proteins with a few differences between pregnancy and lactation. Most of these markers are new and are involved in microtubule assembly, retinoic acid transport, and Raf kinase signaling cascade; 10 are enzymes that define a ketogenic metabolic landscape, demonstrating, for the first time, the prevalence of de novo cholesterol synthesis in luteal cells. The “proteomic signature of regression,” on the other hand, is composed of nine proteins, one of which is 20alpha-hydroxysteroid dehydrogenase and two, ferritin and gamma-actin, are new. The discovery of unpredictable new actors in the differentiation process of CL reported here will contribute to new hypotheses that explain the complex female reproductive function at the protein level. It will also open new doors to research on each identified protein by relating them to cellular differentiation.

Evaluation of antagonist capacity of bacillales strains isolated from vermicompost leachate on phytopatogenic fungi

The Bacillales order has been reported to exhibit an antagonistic activity toward some phytopathogens. In addition, several studies have mentioned the presence of some species of this order in the vermicompost leachate. In the present study , the antagonistic effect of five Bacillales strains on the mycelial growth of Phytophthora capsici , Fusarium oxysporum, Alternaria solani and Rhizopus sp. was evaluated. Five bacterial strains were isolated from vermicompost leachate and identified by their morphological and biochemical characteristics. Subsequently these strains and a standard Bacillus subtilis strain were used to observe the competition against four fungi strains by determining the percentage inhibition of mycelial growth. Significant differences in percentage inhibition were founded among strains. The greatest antagonistic capacity was observed in Bacillus licheniformis and B. badius . Results showed that vermicompost leachate containing Bacillales strains could be used as a biocontrol agent of various pathogenic fungi, runing to a more effective and environmentally-friendly product to control those phytopathogens.