Molecular mechanisms of Strawberry Plant Defence against colletotrichum acutatum

This thesis is focused on strawberry molecular studies aimed by the strong economic impact and social staple that represents this crop. With an annual production of 500000 tons and an economic weigh of 650 million €, Spain is the third producing country in world (FAOSTAT Agriculture Data [http://faostat.fao.org/]). Important losses in strawberry yields occur due to diseases and pests. Although resistant cultivars are a priority of most strawberry breeding programs, completely resistant cultivars have not yet been reported, relying pathogen control on the excessive use of chemical products (mostly environmental contaminants). Despite of the immediate necessity on developing new strategies to improve resistance in this crop plant, molecular knowledge is still scarce and most components and mechanisms of the strawberry defense network remain unknown and poorly understood. The main aim of this work is to get molecular clues about how plant immunity is activated in strawberry to face pathogen attack. Understanding the molecular interplay between strawberry plant and microbes will successful contribute to identify candidate genes useful for developing biotechnological strategies and help breeding to increase resistance against specific pathogens. This work has been structured in eight chapters which contributed to mount and exploit new technical platforms to subsequently uncover the strawberry defense response at molecular level. Chapter I is an intense and comprehensive compendium of all available information on strawberry immunity, and contribute to the field by discussing, updating and compiling research focused on the molecular aspects and events of the strawberry defense mechanisms against pathogens, concluding that major progress in the physiology, genetics and molecular biology of strawberry, is still needed to fully uncover the logic of its elaborate plant innate immune system. In this context, the use of high-throughput technologies will provide large amount of molecular information related with defense response in strawberry. However, the lack on commercially-available tools focused on crop plants such as strawberry, aimed us to produce our private platforms to be exploited in strawberry-based studies, as it is described in Chapter II. Thus, the generation of a ESTs collection enriched on defense-related genes, which allow the identification of new components of strawberry immunity, and provides information on those biological...Esta tesis está enfocada en estudios moleculares de la planta de fresa animada por el importante impacto económico y estabilizador social que representa este cultivo. Con una producción anual de 500000 toneladas y una relevancia económica de 650 millones de euros, España es el tercer país productor en el mundo (FAOSTAT Agriculture Data [http://faostat.fao.org/]). La producción de fresa sufre importantes pérdidas como consecuencia de las enfermedades y las plagas. Aunque la obtención de cultivares resistentes son una prioridad para la mayoría de los programas de mejora, aún no se han obtenido cultivares completamente resistentes, por lo que el control de los patógenos ha recaído en el uso excesivo de productos químicos (mayoritariamente contaminantes ambientales). Aún con la urgente necesidad de desarrollar nuevas estrategias para mejorar la resistencia en este cultivo, el conocimiento a nivel molecular de la mayoría de los componentes y mecanismos de la respuesta de defensa de la planta de fresa permanecen desconocidos y difícilmente entendibles. El objetivo principal de este trabajo consiste en la obtención de pistas a nivel molecular a cerca de cómo la inmunidad de la planta de fresa es activada para enfrentarse al ataque de los patógenos. Conocer la interacción molecular entre la planta de fresa y los microbios contribuirá eficazmente a la identificación de genes candidatos útiles en el desarrollo de estrategias biotecnológicas y ayudará en los programas de mejora para incrementar la resistencia contra patógenos específicos. Este trabajo se ha estructurado en ocho capítulos que contribuyen en la tarea de montar y explotar nuevas plataformas técnicas para descubrir secuencialmente, a un nivel molecular, los distintos pasos en la respuesta de defensa desplegados por la planta de fresa. El Capítulo I es un amplio e intenso compendio de toda la información disponible sobre la inmunidad de la fresa, y contribuye a este campo con la discusión, actualización y compilación de toda la investigación enfocada en los aspectos moleculares de los mecanismos de la defensa de esta planta. Este capítulo concluye que aún es necesario un importante esfuerzo en los estudios de la fisiología, la genética y la biología molecular en fresa, para llegar a discernir por completo la lógica de su muy elaborado sistema de inmunidad innata. En este contexto, el uso de tecnologías de gran escala proporcionará una gran cantidad de información molecular relacionada con la respuesta de defensa en..

A Comprehensive Study of the WRKY Transcription Factor Family in Strawberry

WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry

Proteomic analysis of goat milk kefir: profiling the fermentation-time dependent protein digestion and identification of potential peptides with biological activity

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date

Ion Torrent and lllumina, two complementary RNA-seq platforms for constructing the holm oak (Quercus ilex) transcriptome

Transcriptome analysis is widely used in plant biology research to explore gene expression across a large variety of biological contexts such as those related to environmental stress and plant-pathogen interaction. Currently, next generation sequencing platforms are used to obtain a high amount of raw data to build the transcriptome of any plant. Here, we compare Illumina and Ion Torrent sequencing platforms for the construction and analysis of the holm oak (Quercus ilex) transcriptome. Genomic analysis of this forest tree species is a major challenge considering its recalcitrant character and the absence of previous molecular studies. In this study, Quercus ilex raw sequencing reads were obtained from Illumina and Ion Torrent and assembled by three different algorithms, MIRA, RAY and TRINITY. A hybrid transcriptome combining both sequencing technologies was also obtained in this study. The RAY-hybrid assembly generated the most complete transcriptome (1,116 complete sequences of which 1,085 were single copy) with a E90N50 of 1,122 bp. The MIRAIllumina and TRINITY-Ion Torrent assemblies annotated the highest number of total transcripts (62,628 and 74,058 respectively). MIRA-Ion Torrent showed the highest number of shared sequences (84.8%) with the oak transcriptome. All the assembled transcripts from the hybrid transcriptome were annotated with gene ontology grouping them in terms of biological processes, molecular functions and cellular components. In addition, an in silico proteomic analysis was carried out using the translated assemblies as databases. Those from Ion Torrent showed more proteins compared to the Illumina and hybrid assemblies. This new generated transcriptome represents a valuable tool to conduct differential gene expression studies in response to biotic and abiotic stresses and to assist and validate the ongoing Q. ilex whole genome sequencing

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics

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-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 "Applied Proteomics" 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-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 "applied proteomics". 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, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined

The Strawberry FaWRKY1 Transcription Factor Negatively Regulates Resistance to Colletotrichum acutatum in Fruit Upon Infection

Strawberry (Fragaria ×ananassa) is a major food crop worldwide, due to the flavor, aroma and health benefits of the fruit, but its productivity and quality are seriously limited by a large variety of phytopathogens, including Colletotrichum spp. So far, key factors regulating strawberry immune response remain unknown. The FaWRKY1 gene has been previously proposed as an important element mediating defense responses in strawberry to Colletotrichum acutatum. To get further insight into the functional role that FaWRKY1 plays in the defense mechanism, Agrobacterium-mediated transient transformation was used both to silence and overexpress the FaWRKY1 gene in strawberry fruits (Fragaria ×ananassa cv. Primoris), which were later analyzed upon C. acutatum inoculation. Susceptibility tests were performed after pathogen infection comparing the severity of disease between the two agroinfiltrated opposite halves of the same fruit, one half bearing a construct either for FaWRKY1 overexpression or RNAi-mediated silencing and the other half bearing the empty vector, as control. The severity of tissue damage was monitored and found to be visibly reduced at five days after pathogen inoculation in the fruit half where FaWRKY1 was transiently silenced compared to that of the opposite control half and statistical analysis corroborated a significant reduction in disease susceptibility. Contrarily, a similar level of susceptibility was found when FaWRKY1 overexpression and control fruit samples, was compared. These results unravel a negative regulatory role of FaWRKY1 in resistance to the phytopathogenic fungus C. acutatum in strawberry fruit and contrast with the previous role described for this gene in Arabidopsis as positive regulator of resistance against the bacteria Pseudomonas syringae. Based on previous results, a tentative working model for WRKY75 like genes after pathogen infection is proposed and the expression pattern of potential downstream FaWRKY1 target genes was also analyzed in strawberry fruit upon C. acutatum infection. Our results highlight that FaWRKY1 might display different function according to species, plant tissue and/or type of pathogen and underline the intricate FaWRKY1 responsive defense regulatory mechanism taking place in strawberry against this important crop pathogen

Consens d’escales i eines per a la valoració multidimensional de les persones a Catalunya

Model d'atenció integral; Valoració multidimensional; DiagnòsticModelo de atención integral; Valoración multidimensional; DiagnósticoComprehensive care model; Multidimensional valuation; DiagnosisActualment no existeix a Catalunya un consens sobre quin hauria de ser el mínim comú denominador d’escales i eines d’elecció per a la valoració de les diferents dimensions de les persones grans amb multimorbiditat, fragilitat, cronicitat complexa o avançada. Aquest escenari fa evident la necessitat de disposar d’escales i eines consensuades i compartides que facilitin l’aproximació multidimensional a aquestes persones de forma objectiva i pragmàtica, amb l’objectiu d'ajudar els professionals en aquest procés d’individualització de l’atenció. És per aquest motiu que, impulsat per la Direcció General de Planificació i Recerca en Salut i per l’oficina eSalut, s’ha promogut i desenvolupat aquest consens i es vol assegurar que la proposta pugui ser suportada en l’entorn digital

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.This work was supported by Junta de Andalucía, Spain [Proyectos de Excelencia P07-AGR-02482/P12-AGR-2174, and grants to Grupo-BIO278].Peer reviewedPeer Reviewe

Comparative proteomic analysis of Arabidopsis wild-type, mutants, and Fa WRKY1 transgenic plant to characterize the function fo strawberry (Fragaria X ananassa) Fa WRKY1 protein and its Arabidopsis homolog, at WRKY75 two positive regulators of resistance

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