Study of phytopathogenesis regulation through chromatine modification

Chromatin is a structure formed by DNA and protein found in eukaryotic cells. This structure serves to package and condense the DNA. Histones are the main proteins forming part of this structure. The histones have aminoacidic tails which modifications are highly involved in transcriptional control of genetic programs that lead essentials processes like mitosis or more complex processes as can be cell differentiation. We have done a phylogenetic study and have found 3 genes which can encode the enzymes in charge of histone H3 methylation at its lysine resides 9 (KMT1) and 36 (KMT3 and KMT2H). The lysine 9 methylation in H3 is the main hallmark of constitutive heterochromatin (1,2) and the H3 lysine 36 methylation is generally associated with active transcription (3). We have deleted these putative methyltransferases in Ustilago maydis. While KMT1 deletion has not showed a significant virulence phenotype, both H3K36 methyltransferases mutants present infection defects. Interestingly, meanwhile KMT3 deletion shows a reduction in virulence, KMT2H deletion improve pathogenesis. In addition, by western blot analysis we have observed a reduction of H3K36 trimethylation in both mutants respect the wild type strain, indicating there are two H3K36 methyltransferases with opposite roles in infection. These observations are in agreement with a recent publication where the fungus Fusarium fujikuroi is methylating H3K36 in heterochromatin and euchromatin area through two different methyltransferases (4). We are currently studying the differences between these methyltrasnferases at the molecular and cellular level during the pathogenesis process

Study of PMT target specificity in Ustilago maydis

Ustilago maydis is a pathogenic fungi responsible for the corn smut fungus disease, which causes a significant loss in mayze production every year. The PMT is a family of well-conserved O-mannosylating proteins. In Ustilago maydis, the deletion of Pmt2 has been shown to be deleterious and the deletion of Pmt4 disrupts completely the infectious process; on the other hand, the deletion of Pmt1 doesn't manifest any phenotype. All PMT proteins have three domains, named PMT, MIR and 4TMC, and several transmembrane regions. We hipothesized that one of these domains of Pmt4 is responsable for the substrate specificity that confers the virulence phenotype in Ustilago maydis Pmt4 and, if this would be the case, we could develop an antifungal drug specific to this domain. To check this, we built three chimerical protein strains, chanching one domain in Pmt4 for the same domain of Pmt1 at a time, and measure the tumor formation in 3 independent experiments. We also built a full Pmt4 length strain as a positive control. The results indicate that either the protein requires all three Pmt4 domains to infect or the chimerical protein is not working properly.Furthermore, we also wanted to check what region gives Pmt2 its specificity for growth. In Schizosaccharomyces pombe, a ortholog of Pmt2, named Ogm2, present the same essencial-for-growth phenotype than in U. maydis. Since U. maydis doesn't have any repressable promoter, we built a S. pombe strain with the shut-off promoter nmt81 in the endougenous Ogm2 gene and will complement the phenotype with mutagenized plasmids containing the U. maydis Pmt2 and measure its viability.Finally, we also plan to build other three strains interchanching Pmt4 domains in the Pmt1 protein

Study of physico-chemical parameters in smallscale craft beer production

In this past decade, the craft beer market has been gaining importance from large national andinternational beer breweries, especially micro-breweries and brew pubs. The evidence of thiscompetition is the fact that many large companies’ advertisements remind the public that theirroots were originally a microbrewery (1,2).However, the smaller budget of microbreweries incomparison to large companies, requires greater efficiency because a big loss for a smallbusiness would be a significant cost which could affect the competitive power of the companyin the market.In our microbrewery we have been studying different physico-chemical parameters (pH anddensity between others), changing critical steps in the elaboration of the different craft beersand analysing which factors influence these parameters the most, with the objective of makingthe beer production much more efficient. At the same time, we are going to compare theseparameters between the different types of beers that we produce in the factory.Our results show that the pH is slightly more acidic in beers that use a lower proportion ofPilsen-type malts. In relation to density it seems to be the opposite, the higher of thisproportion of this type of malt, the higher the density. Furthermore, some correlation has beenobserved between the degree of milling and the density, with a higher degree of milling of thegrain the higher the density is in the wort which allows to use less malt for the same volume,something that can have beneficial economic repercussions.Another problem that we found in this kind of industries it's the contamination of the finalproduct. However, we have designed a plan for waste management that consists in all thecontaminated beer previously analysed, is distilled in an alembic that extracts the ethanol fromthis useless product, that could then be used to make different valuable alcoholic drinks (gins,vodkas, whiskies, liqueurs). The point of this waste management plan is to reduce the costs aswell as improve the efficiency. We observed that the higher the alcoholic strength of thesubstrate to be distilled the greater the alcoholic strength of the final product. At the same time,the higher the volume to distill the longer it takes to start producing the final product

Searching for gene clusters related to virulence by coding sequence conservation

Motivation: Due to the increasing world population, the need to improve food production is growing. This can be helped byfighting the pathogens which affect the main crops as maize, wheat, barley and sugar cane. Among those, biotrophic parasitessuch as smut fungi can be found. To study how those microorganisms infect their host, the model system Ustilago maydis canbe used.U. maydis secretes protein effectors to infect its host, and at least 25% of them are known to be grouped in 13 different geneclusters. In addition to these characterized clusters, 7 new clusters have been described in the bibliography but notexperimentally tested. The aim of this work is to find out new clusters with features similar to the known ones (controls), mainlylow conservation, which can affect the infection process.Methods: To achieve this goal, candidate gene clusters were initially discovered based on coding sequence conservation viathe computational tool AnABlast [1], which highlitghted genomic coding region with conservation signal similar to the initialcontrols. Then, the candidates were functionally annotated using the tool Sma3s_v2 [2]. To select the best candidates, aprincipal component analysis (PCA) was done using the following factors, which were trained with the controls: sequenceconservation obtained by a similarity search by Blast against close organisms (Ensembl fungi phylogeny), expression dataduring infection, and signal peptide presence (SignalP and TargetP), usually present in effectors.Currently, a laboratory experiment has been began to elucidate if the chosen candidates affect the pathogenity, deleting themby homologous recombination.Results: We have been able to identify 49 new clusters by comparing their coding signal with those already known. After thesubsequent analysis three of them, and one from the bibliography have been chosen to be tested in laboratory to elucidatetheir virulence phenotype (swelling and tumors).In the PCA our best candidate is located among the clusters previously described as pathogenic, showing genes beingsecreted with high levels of expressionConclusions: In brief, we propose that putative cluster of virulence sequences could be found by the presented strategy. So,it could constitute a new silico approach to find out specific genes involved in different biological processes such as inffection

Mutagenesis in the control of virulence in Ustilago maydis

Ustilago maydis is a pathogenic fungus that infects maize plant resulting in stunted plant growth and in tumors, leading to high economic losses every year. Most of the virulence related genes are clustered together in specific regions of the genome (1). As most plant fungal pathogens, U. maydis develops many morphological changes in order to ensure proper infection, which imply a rigorous regulation. The control of genetic programs involved in developmental changes has been typically shown to be regulated by chromatin modifying factors. The principal mechanism by which eukaryotic cells repress large chromosomic regions is through the modification of histones resulting in the formation of a transcriptionally inactive chromatin called heterochromatin. Our main aim is to know if Ustilago maydis owns heterochromatin too and, if through it, this fungus is able to control the expression or repression of genes related of virulence

Chromatin regulation of virulence gene clusters in Ustilago maydis.

Pathogenic fungi possess chromatin modification mechanisms that are involved in their virulence and interaction with the host 1. This modification usually occurs as a consequence of the presence or absence of certain molecular marks on histones, such as methylations or acetylations. It allows the transition between their two conformational states, euchromatin and heterochromatin, associated with gene transcriptional activation and gene silencing, respectively 2,3. The maize fungus Ustilago maydis has become a model organism for the study of plant pathogenesis due to its ease of handling and the multiple available genetic and cellular tools 4. This pathogen uses a large number of factors and pathways that regulate gene expression at each stage of the infective process, but little is known about the role of chromatin in that 1. Many genes involved in that process are clustered in gene regions and are activated at specific times 4,5. Therefore, we should think of an active role of chromatin in this regulation. However, the absence of typical heterochromatin-associated marks and regulators in U. maydis has so far been observed 1. In this laboratory, we have sought other likely regulators of this process and found that the histone methyltransferase Ash1 affects the virulence activity through the silencing of gene clusters involved in this process. In this project we try to find out if this is the only regulator of heterochromatin in U. maydis. For this purpose, different strains with antibiotic resistance markers in silenced regions controlled by Ash1 were developed, allowing antibiotic resistance when these regions are derepressed. Spontaneous resistant mutants were obtained from these strains and Ash1 was consequently sequenced to determine if the silencing was affected by a mutation in Ash1. Surprisingly, the methyltransferase gene was not mutated, suggesting the presence of other regulators. In addition, we performed a ChIP-qPCR to see if the epigenetic mark associated with Ash1 was still present. We observed a decrease in this mark in one of the spontaneous mutants in different virulence regions, which also suggests the presence of other regulators, such as some demethylases. Subsequently, in order to discover whether the decrease in this mark is maintained over time, we have studied its stability in successive generations. We noticed antibiotic-resistant colonies trended towards a decrease which might imply a reacquisition of the epigenetic mark

New strategies to find chromatin silencers in the pathogenic fungus Ustilago maydis

Ustilago maydis is a smut fungus that infects maize, causing tumors, stunted growth and consequently reduced yields leadingto economic losses [2]. A key aspect of the pathogenic development of U.maydis is the action of effectors, which are secretedvirulence factors with principal roles in plant defense suppression and host's metabolism alterations. Many genes encodingeffector proteins are grouped in silenced clusters in the genome highly induced during infection. It has been shown thatintroduction of resistance marker genes with high expression promoters in these clusters de-repress the surrounding region ofthe insertion point [3]. Consequently, it is suggested that these clusters are subjected to chromatin silencing. However, U. maydislacks the canonical factors involved in chromatin silencing. The main purpose of this project is to find regulators that control thesilencing state of these regions. To achieve this goal we are going to perform a screening in a U.maydis strain harboring anantibiotic resistant marker gene inside a silenced cluster. In order to do this strain, we decided to introduce in one of theseclusters an antibiotic marker that will be controlled by an endogenous promoter followed of a different resistance marker genewith a high expressed promoter that will disturb the silencing of the region of insertion. Once we obtained this strain, we restoredsilencing by removing the high expressed gene, which is flanked by two direct repeat sequences, expressing the flippaserecombinase [1]. We are currently performing the first steps of the mutagenesis assay using the recently generated strain

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery

International audienceAbstract Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is not well understood. Here we demonstrate that Schizosaccharomyces pombe Lem2, an NE protein, regulates nuclear-exosome-mediated RNA degradation. Lem2 deletion causes accumulation of RNA precursors and meiotic transcripts and de-localization of an engineered exosome substrate from the nuclear periphery. Lem2 does not directly bind RNA but instead interacts with the exosome-targeting MTREC complex and its human homolog PAXT to promote RNA recruitment. This pathway acts largely independently of nuclear bodies where exosome factors assemble. Nutrient availability modulates Lem2 regulation of meiotic transcripts, implying that this pathway is environmentally responsive. Our work reveals that multiple spatially distinct degradation pathways exist. Among these, Lem2 coordinates RNA surveillance of meiotic transcripts and non-coding RNAs by recruiting exosome co-factors to the nuclear periphery

La gestión de la responsabilidad social en las universidades Iberoamericanas

La presente aportación recoge el trabajo de 47 especialistas de 14 países iberoamericanos que aportan sus estudios y valoraciones sobre la temática. Se proporciona así una visión amplia del desarrollo de esta en los distintos países, proporcionando para cada uno de ellos referencias normativas, la realidad en sus universidades, algún ejemplo relevante y algunas reflexiones y propuestas para la mejora. Se cumple así y una vez más el compromiso social de la Red de Apoyo a la Gestión Educativa (RedAGE) de emitir un informe anual sobre aspectos relevantes de la organización y gestión de la educación. La temática y estructura del informe fue debatido y aprobado en la reunión anual celebrada en Barcelona el 9 de abril de 2019, donde también se analizaron algunas de las problemáticas y retos que la vinculación con el entorno supone para los administradores y gestores de las universidades. Las diferentes aportaciones tratan de reflejar la realidad en los distintos países desde los redactores de los informes nacionales. El propósito no es tanto el de identificar todas las realizaciones existentes como el de conocer aportaciones significativas y tener una valoración cualificada de la realidad y perspectivas en la temática abordada. Esperemos se haya cumplido el propósito mencionado y el resultado sea de interés para los especialistas e interesados