Caracterización y mejora de levaduras de flor aisladas en la Denominación de Origen Montilla-Moriles

Programa de Doctorado en Biotecnología y Tecnología QuímicaLos vinos finos se someten a un original proceso de crianza biológica que los caracteriza, y que ocurre bajo una gruesa capa de levaduras que se desarrolla en la superficie del vino denominada velo de flor. El objetivo principal de este trabajo ha sido analizar las distintas poblaciones de levaduras que constituyen este velo de flor, responsables de la maduración biológica, en diferentes bodegas de la Denominación de Origen Montilla-Moriles. Se han analizado cinco bodegas de la zona, donde hemos estudiado 9 botas de cada una de ellas, para completar un total de 1700 muestras. Las muestras tomadas de cada bodega se han sometido a técnicas de biología molecular (RFLPmit, análisis de microsatélites y CHEF) para su identificación en función de los patrones obtenidos. Se han identificado cinco cepas diferentes, denominadas A, B. C. D y E, todas ellas pertenecientes a S. cerevisiae raza levaduras de flor. De estas, es la cepa A la que domina en la Denominación de Origen con un porcentaje del 79%, siendo la mayoritaria en cuatro de las cinco bodegas estudiadas. Las cinco cepas identificadas se han sometido a una caracterización que incluye un estudio de crecimiento en distintos medios, velocidad de formación de flor, competencia entre levaduras a diferentes concentraciones de alcohol y temperatura, y microvinificación, entre otras. Una vez caracterizadas se ha procedido a la mejora de estas levaduras tratando de reunir en una levadura características destacables de ambos parentales, mediante la fusión de protoplastos de las mismas o rare mating, asi como intentando intercambiar la mitocondria entre las distintas cepas de flor aisladas. La frecuencia de éxito en cualquier caso ha sido baja. Dado que la principal característica de estas levaduras es la formación de un biofilm sobre la superficie del vino, hemos realizado diferentes abordajes proteómicos para determinar cuáles son las proteínas que pueden estar implicadas en este proceso. Para esto último hemos utilizado el hongo U. maydis, que presenta una gran capacidad de formar biofilm sobre superficie líquida, al igual que lo hacen las levaduras de flor. En estos estudios hemos hallado tres proteínas posiblemente implicadas en la formación de biofilm, y de forma paralela en la patogénesis de dicho hongo. Esto último vincula ambos procesos y abre una vía de estudio sobre su posible relación.Universidad Pablo de Olavide. Centro de Estudios de Postgrad

Caracterización y mejora de levaduras de flor aisladas en la Denominación de Origen Montilla-Moriles

Memoria presentada por la licenciada Miriam Marín Menguiano para optar al grado de Doctora por la Universidad Pablo de Olavide, de Sevilla.-- Excepto si se señala otra cosa, la licencia del ítem se describe como Atribución-NoComercial-SinDerivadas 3.0 EspañaLos vinos finos se someten a un original proceso de crianza biológica que los caracteriza, y que ocurre bajo una gruesa capa de levaduras que se desarrolla en la superficie del vino denominada velo de flor. El objetivo principal de este trabajo ha sido analizar las distintas poblaciones de levaduras que constituyen este velo de flor, responsables de la maduración biológica, en diferentes bodegas de la Denominación de Origen Montilla-Moriles. Se han analizado cinco bodegas de la zona, donde hemos estudiado 9 botas de cada una de ellas, para completar un total de 1700 muestras. Las muestras tomadas de cada bodega se han sometido a técnicas de biología molecular (RFLPmit, análisis de microsatélites y CHEF) para su identificación en función de los patrones obtenidos. Se han identificado cinco cepas diferentes, denominadas A, B. C. D y E, todas ellas pertenecientes a S. cerevisiae raza levaduras de flor. De estas, es la cepa A la que domina en la Denominación de Origen con un porcentaje del 79%, siendo la mayoritaria en cuatro de las cinco bodegas estudiadas. Las cinco cepas identificadas se han sometido a una caracterización que incluye un estudio de crecimiento en distintos medios, velocidad de formación de flor, competencia entre levaduras a diferentes concentraciones de alcohol y temperatura, y microvinificación, entre otras. Una vez caracterizadas se ha procedido a la mejora de estas levaduras tratando de reunir en una levadura características destacables de ambos parentales, mediante la fusión de protoplastos de las mismas o rare mating, asi como intentando intercambiar la mitocondria entre las distintas cepas de flor aisladas. La frecuencia de éxito en cualquier caso ha sido baja. Dado que la principal característica de estas levaduras es la formación de un biofilm sobre la superficie del vino, hemos realizado diferentes abordajes proteómicos para determinar cuáles son las proteínas que pueden estar implicadas en este proceso. Para esto último hemos utilizado el hongo U. maydis, que presenta una gran capacidad de formar biofilm sobre superficie líquida, al igual que lo hacen las levaduras de flor. En estos estudios hemos hallado tres proteínas posiblemente implicadas en la formación de biofilm, y de forma paralela en la patogénesis de dicho hongo. Esto último vincula ambos procesos y abre una vía de estudio sobre su posible relación.Peer Reviewe

Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.1,2 The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals.3-11 Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues.12 Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless.13-18 Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. We have used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs).19 We report that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, we provide evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche

Identification of O-mannosylated Virulence Factors in Ustilago maydis

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis

mastermind regulates niche ageing independently of the Notch pathway in the Drosophila ovary

Proper stem cell activity in tissues ensures the correct balance between proliferation and differentiation, thus allowing tissue homeostasis and repair. The Drosophila ovary develops well-defined niches that contain on average 2–4 germline stem cells (GSCs), whose maintenance depends on systemic signals and local factors. A known player in the decline of tissue homeostasis is ageing, which correlates with the waning of resident stem cell populations. In Drosophila, ovaries from old females contain fewer GSCs than those from young flies. We isolated niche cells of aged ovaries, performed a transcriptomic analysis and identified mastermind (mam) as a factor for Drosophila ovarian niche functionality during ageing. We show that mam is upregulated in aged niche cells and that we can induce premature GSC loss by overexpressing mam in otherwise young niche cells. High mam levels in niche cells induce reduced Hedgehog amounts, a decrease in cadherin levels and a likely increase in reactive oxygen species, three scenarios known to provoke GSC loss. Mam is a canonical co-activator of the Notch pathway in many Drosophila tissues. However, we present evidence to support a Notch-independent role for mam in the ovarian germline niche.This work was funded by the Spanish State Agency for Research (MCUI/AEI; grant nos. BFU2015-65372-P, PGC2018-097115-B-I00 to A.G.-R. and MDM-2016-0687) and by the European Regional Development Fund (http://ec.europa.eu/regional_policy/en/funding/erdf/).Peer reviewe

N-glycosylation of the protein disulfide isomerase Pdi1 ensures full Ustilago maydis virulence

© 2019 Marín-Menguiano et al.Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.MMM was supported by P09- AGR-5241 Junta de Andalucía. IMS was awarded by BES-2014-069149 MINECO AEI/FEDER, UE, Spain. This work was supported by Junta de Andalucía P09- AGR-5241 grant (https://www.juntadeandalucia.es/organismos/economiaconocimientoempresasyuniversidad.html), Spanish Government BIO2013–48858-P and BIO2016-80180-P grants from MINECO AEI/FEDER, UE to JII and Ramon y Cajal program, RyC-2016-19659 to AFA (http://www.mineco.gob.es/portal/site/mineco/)

Population analysis of biofilm yeasts during fino sherry wine aging in the Montilla-Moriles D.O. region

Fino is the most popular sherry wine produced in southern Spain. Fino is matured by biological aging under a yeast biofilm constituted of Saccharomyces cerevisiae yeasts. Although different S. cerevisiae strains can be identified in such biofilms, their diversity and contribution to wine character have been poorly studied. In this work, we analyse the flor yeast population in five different wineries from the Montilla-Moriles D.O. (Denominación de Origen) in southern Spain. Yeasts present in wines of different ages were identified using two different culture-dependent molecular techniques. From 2000 individual yeast isolates, five different strains were identified with one of them dominating in four out of the five wineries analysed, and representing 76% of all the yeast isolates collected. Surprisingly, this strain is similar to the predominant strain isolated twenty years ago in Jerez D.O. wines, suggesting that this yeast is particularly able to adapt to such a stressful environment. Fino wine produced with pure cultures of three of the isolated strains resulted in different levels of acetaldehyde. Because acetaldehyde levels are a distinctive characteristic of fino wines and an indicator of fino aging, the use of molecular techniques for yeast identification and management of yeast populations may be of interest for fino wine producers looking to control one of the main features of this wine.This work was supported by Junta de Andalucía Grant AGR 05241. MMM was supported by a fellowship from the Junta de Andalucía AGR 05241.Peer Reviewe

Visualization and Quantification of Drosophila Larval Ovaries

The morphogenesis of the ovarian germline stem cell (GSC) niche during larval stages in Drosophila provides the initial cellular and molecular basis for female gamete production in the adult. During larval instars, the Drosophila female gonad matures gradually from a round structure enclosing primordial germ cells (PGCs) and somatic cells into a functional organ containing GSC populations in their niches that later in adult stages support oogenesis. In this chapter, we describe a technique for dissecting, staining, and analyzing gonads from female Drosophila larvae and early pupae, offering the possibility of a direct view of the morphogenesis of an ovarian niche

Endoplasmic reticulum glucosidases and protein quality control factors cooperate to establish biotrophy in Ustilago maydis

Secreted fungal effectors mediate plant-fungus pathogenic interactions. These proteins are typically N-glycosylated, a common posttranslational modification affecting their location and function. N-glycosylation consists of the addition, and subsequent maturation, of an oligosaccharide core in the endoplasmic reticulum (ER) and Golgi apparatus. In this article, we show that two enzymes catalyzing specific stages of this pathway in maize smut (Ustilago maydis), glucosidase I (Gls1) and glucosidase II β-subunit (Gas2), are essential for its pathogenic interaction with maize (Zea mays). Gls1 is required for the initial stages of infection following appressorium penetration, and Gas2 is required for efficient fungal spreading inside infected tissues. While U. maydis Δgls1 cells induce strong plant defense responses, Δgas2 hyphae are able to repress them, showing that slight differences in the N-glycoprotein processing can determine the extent of plant-fungus interactions. Interestingly, the calnexin protein, a central element of the ER quality control system for N-glycoproteins in eukaryotic cells, is essential for avoiding plant defense responses in cells with defective N-glycoproteins processing. Thus, N-glycoprotein maturation and this conserved checkpoint appear to play an important role in the establishment of an initial biotrophic state with the plant, which allows subsequent colonization. © 2013 American Society of Plant Biologists. All rights reserved.This work was supported by Ministerio de Ciencia e Innovación Grant BIO2010-16787. A.F.-A. and A.E.-V. were supported by fellowships from Ministerio de Ciencia e Innovación. Centro Andaluz de Biología del Desarrollo is institutionally supported by Consejo Superior de Investigaciones Científicas, Universidad Pablo de Olavide, and Junta de AndalucíaPeer Reviewe

N-glycosylation of the protein disulfide isomerase Pdi1 ensures full Ustilago maydis virulence.

Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis