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..

Proteomic study of the membrane components of signalling cascades of Botrytis cinerea controlled by phosphorylation

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 cycl

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

Biomethanisation of sewage sludge: Sonication pretreatment and monitoring of microbial communities

The improvement of mesophilic biomethanisation of recalcitrant sewage sludge derived from urban wastewater treatment through the application of a sonication pretreatment was evaluated in parallel in two pilot-scale anaerobic digesters (two biological replicates: reactors RA and RB). The valorisation process was monitored through a novel and holistic approach that related the biomethanisation yield, and its main batch operational parameters, with the abundance of archaeal and bacterial communities in the anaerobic inocula. Sonication allowed achieving a methane yield coefficient derived from sewage sludge of 240 ± 20 mLSTPCH4/g VS (volatile solids) at the load range of 0.8–4.0 g VS/L in both reactors. The process was more stable in reactor B, with a wider range of loads being allowed (up to 5.29 g VS/L). Monitoring the presence of Archaea in the mixing liquor revealed a variation in their abundance throughout the process which was directly related to the availability of organic matter and pH. Advanced metagenomic analysis showed the phylogenetic and functional diversity of the complex microbiome involved. While Bacteria were widely distributed in 35 phyla, Archaea fitted in only two. Euryarchaeota was the majoritarian archaeal phylum (99.5 %) and its more abundant families are linked to methanogenic metabolism. Functional analysis revealed several relevant metabolic pathways that followed similar trends in both reactors. “Methane metabolism” clearly diminished at the end of the process in concordance with the exhaust of methane generation, while “ABC transporters” or “two-component systems”, involved in bacterial survival to changing environments, followed the opposite pattern. This integrated approach could help to increase the methanogenic valorisation of sewage sludge

FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum

Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to control diseases that has been found so far. Alternatively, genetic manipulation, employing plant key genes involved in defense, such as members of the NPR-like gene family, has been successful in many crops to improve resistance. The identification and use of the endogenous counterpart genes in the plant of interest (as it is the case of strawberry) is desirable as it would increase the favorable outcome and requires prior knowledge of their defense-related function. Using RNAi technology in strawberry, transient silencing of Fragaria ananassa NPR3 members in fruit significantly reduced tissue damage after Colletotrichum acutatum infection, whereas the ectopic expression of either FaNPR3.1 or FaNPR3.2 did not have an apparent effect. Furthermore, the ectopic expression of FaNPR3.2 in Arabidopsis thaliana double-mutant npr3npr4 reverted the disease resistance phenotype to Pseudomonas syringe to wild-type levels. Therefore, the results revealed that members of the strawberry FaNPR3 clade negatively regulate the defense response to pathogens, as do their Arabidopsis AtNPR3/AtNPR4 orthologs. Also, evidence was found showing that FaNPR3 members act in strawberry (F. ananassa) as positive regulators of WRKY genes, FaWRKY19 and FaWRKY24; additionally, in Arabidopsis, FaNPR3.2 negatively regulates its orthologous genes AtNPR3/AtNPR4. We report for the first time the functional characterization of FaNPR3 members in F. ananassa, which provides a relevant molecular basis for the improvement of resistance in this species through new breeding technologies

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

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population