http://hdl.handle.net/10366/4028 Fri, 24 Apr 2026 07:21:21 GMT 2026-04-24T07:21:21Z http://hdl.handle.net/10366/170354 [ES] Las RASopatías asociadas a alteraciones en los genes NF1 y SPRED1 presentan una marcada heterogeneidad clínica y molecular, donde el principal obstáculo tras el diagnóstico genético es la interpretación funcional de las variantes identificadas. Con el objetivo de superar este desafío, la presente tesis doctoral ha creado un flujo de trabajo experimental multidisciplinar que combina el análisis de variantes en NF1 y SPRED1 reportadas en bases de datos públicas y de pacientes de la DIERCyL, el modelado estructural y de estabilidad proteica in silico, la señalización celular, proliferación, y morfometría en las líneas celulares HEK293T y NIH3T3. Los análisis de las bases de datos genéticas identificaron hotspots estructurales críticos en la proteína neurofibromina (CSRD, GRD y Armadillo1), mientras que las variantes en SPRED1 mostraron una distribución homogénea, sugiriendo mecanismos patogénicos distintos. En el caso de las variantes en NF1 y SPRED1 de los pacientes, el análisis energético reveló un espectro continuo en neurofibromina que va desde haploinsuficiencia hasta dominancia negativa en truncantes tardías, mientras que en SPRED1 predominó la pérdida de función. Los ensayos celulares in vitro, mostraron dinámicas de señalización celular diferentes según el tipo de variante analizada en los genes NF1 y SPRED1. En particular, la hiperactivación de ERK emergió como el evento molecular más determinante para la patogenicidad de las variantes. La coactivación de p38 identificó variantes de mayor severidad clínica, mientras que la relación inversa p- ERK/p-AKT(Ser473) reveló un circuito de retroalimentación negativa entre las rutas de señalización MAPK y PI3K/AKT. Los ensayos de proliferación celular y el análisis cuantitativo del tamaño celular revelaron fenotipos consistentes con el grado de desregulación de MAPK, proporcionando biomarcadores fenotípicos adicionales para diferenciar entre variantes patogénicas y benignas en ambos genes. La relevancia tisular de HEK293T (contextos epiteliales/neuroectodérmicos) y NIH3T3 (mesenquimales) permitió reproducir la heterogeneidad clínica observada en pacientes. Los resultados de esta tesis doctoral han permitido reclasificar variantes de significado incierto en ambos genes e identificar la hiperactivación de ERK como diana terapéutica prioritaria, apoyando el uso de inhibidores de MEK en contextos compatibles con esta desregulación. En conjunto, este trabajo establece un marco integral para la interpretación funcional de variantes en RASopatías y para el desarrollo de futuras estrategias de medicina personalizada.; [EN] RASopathies associated with alterations in the NF1 and SPRED1 genes exhibit marked clinical and molecular heterogeneity, where the main challenge after genetic diagnosis lies in the functional interpretation of the identified variants. To address this issue, this doctoral thesis established a multidisciplinary experimental workflow that integrates the analysis of NF1 and SPRED1 variants reported in public databases and in patients from the DIERCyL cohort, together with in silico structural and protein-stability modeling, cellular signaling assays, proliferation studies, and morphometric analyses in HEK293T and NIH3T3 cell lines. Database analyses identified critical structural hotspots in neurofibromin (CSRD, GRD, and Armadillo1), whereas SPRED1 variants showed a more homogeneous distribution, suggesting distinct pathogenic mechanisms. In patient-derived NF1 and SPRED1 variants, energy-based analyses revealed a continuous spectrum in neurofibromin, ranging from haploinsufficiency to dominant-negative effects in late truncating variants, while SPRED1 alterations predominantly showed loss-of-function behavior. The in vitro cellular assays revealed distinct signaling dynamics depending on the type of variant analyzed in NF1 and SPRED1. Notably, ERK hyperactivation emerged as the most determinant molecular event for pathogenicity. Co-activation of p38 identified variants associated with more severe clinical outcomes, whereas the inverse p-ERK/p- AKT(Ser473) relationship revealed a negative feedback circuit between the MAPK and PI3K/AKT pathways. Cell-proliferation assays and quantitative cell-size analyses uncovered phenotypes consistent with the degree of MAPK deregulation, providing additional phenotypic biomarkers to discriminate between pathogenic and benign variants in both genes. The tissue relevance of HEK293T (epithelial/neuroectodermal contexts) and NIH3T3 (mesenchymal) cells allowed us to recapitulate the clinical heterogeneity observed in patients. The results of this doctoral thesis enabled the reclassification of variants of uncertain significance in both genes and identified ERK hyperactivation as a priority therapeutic target, supporting the use of MEK inhibitors in contexts consistent with this dysregulation. Taken together, this work establishes a comprehensive framework for the functional interpretation of variants in RASopathies and for the development of future personalizedmedicine strategies. Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/10366/170354 2025-01-01T00:00:00Z http://hdl.handle.net/10366/169986 [EN]Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing Fri, 01 Jan 2021 00:00:00 GMT http://hdl.handle.net/10366/169986 2021-01-01T00:00:00Z http://hdl.handle.net/10366/169937 [EN]Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen Wed, 01 Dec 2021 00:00:00 GMT http://hdl.handle.net/10366/169937 2021-12-01T00:00:00Z http://hdl.handle.net/10366/169930 [EN]KP4 killer toxins are secreted proteins that inhibit cell growth and induce cell death in target organisms. In Fusarium graminearum, KP4-like (KP4L) proteins contribute to fungal virulence in wheat seedling rot and are expressed during Fusarium head blight development. However, fungal KP4L proteins are also hypothesized to support fungal antagonism by permeabilizing cell walls of competing fungi to enable penetration of toxic compounds. Here, we report the differential expression patterns of F. graminearum KP4L genes (Fgkp4l-1, -2, -3 and -4) in a competitive interaction, using Trichoderma gamsii as the antagonist. The results from dual cultures indicate that Fgkp4l-3 and Fgkp4l-4 could participate in the recognition at the distance of the antagonist, while all Fgkp4l genes were highly activated in the pathogen during the physical interaction of both fungi. Only Fgkp4l-4 was up-regulated during the interaction with T. gamsii in wheat spikes. This suggests the KP4L proteins could participate in supporting F. graminearum interspecific interactions, even in living plant tissues. The distribution of KP4L orthologous within the genus Fusarium revealed they are more represented in species with broad host-plant range than in host-specific species. Phylogeny inferred provides evidence that KP4L genes evolved through gene duplications, gene loss and sequence diversification in the genus Fusarium Fri, 16 Sep 2022 00:00:00 GMT http://hdl.handle.net/10366/169930 2022-09-16T00:00:00Z http://hdl.handle.net/10366/169920 [EN]Species of fungal genus Trichoderma are characterized by a versatile lifestyle, high adaptability to the changing environmental conditions and the ability to establish sophisticated interactions with other organisms. Due to their ability to antagonize plant pathogens and to elicit the plant defence responses against biotic/abiotic stresses, Trichoderma spp. are commonly used as commercially biopesticides and biofertilizers. The Trichoderma success in the rhizosphere is supported by a wide arsenal of specialised metabolites (SMs) providing morphological and physiological autoregulation, self-protection and facilitating fungal communication. This review aims to explore the roles of SMs in the biology of fungi, with special emphasis on the genus Trichoderma and on how divergence in the SMs genetic structure determine Trichoderma lifestyles. Trichoderma genomes are endowed with a high number of SMs biosynthetic genes, and understanding the genetic basis of their biosynthesis is crucial for determining the role of these metabolites in Trichoderma ecophysiology and for expanding their application in crop protection. Recent advances on the characterization of the Trichoderma SMs genetic inventory driven by computational biology are discussed Sat, 01 Jan 2022 00:00:00 GMT http://hdl.handle.net/10366/169920 2022-01-01T00:00:00Z http://hdl.handle.net/10366/169919 [EN]Trichoderma is a fungal genus comprising species used as biocontrol agents in crop plant protection and with high value for industry. The beneficial efects of these species are supported by the secondary metabolites they produce. Terpenoid compounds are key players in the interaction of Trichoderma spp. with the environment and with their fungal and plant hosts; however, most of the terpene synthase (TS) genes involved in their biosynthesis have yet not been characterized. Here, we combined comparative genomics of TSs of 21 strains belonging to 17 Trichoderma spp., and gene expression studies on TSs using T. gamsii T6085 as a model. An overview of the diversity within the TS-gene family and the regulation of TS genes is provided. We identified 15 groups of TSs, and the presence of clade-specific enzymes revealed a variety of terpenoid chemotypes evolved to cover diferent ecological demands. We propose that functional diferentiation of gene family members is the driver for the high number of TS genes found in the genomes of Trichoderma. Expression studies provide a picture in which diferent TS genes are regulated in many ways, which is a strong indication of diferent biological functions. Thu, 01 Oct 2020 00:00:00 GMT http://hdl.handle.net/10366/169919 2020-10-01T00:00:00Z http://hdl.handle.net/10366/169918 [EN]Fungal pathogens are the main factors responsible for the most severe diseases affecting plants, leading to significant reduction in yield and crop quality and causing enormous economic losses worldwide. It is estimated that around 30% of the emerging diseases are caused by fungi thus requiring new strategies to improve their management. Biological control approach, frequently referred to the use of non-pathogenic microbial antagonists or products derived from their metabolism, represents a valid and promising alternative under a more ecological perspective to reduce the activities and to control populations of target pathogens. However, although the use of antagonists belonging to species different from that of the pathogen has been successfully reported, the use of competitors belonging to the same species of the pathogen is not widespread. A biocontrol strategy based on competition for space and nutrients and/or the induction of plant defenses against virulent pathogens performed by attenuated or avirulent pathogens could, therefore, be considered a valid alternative. Fri, 15 Feb 2019 00:00:00 GMT http://hdl.handle.net/10366/169918 2019-02-15T00:00:00Z http://hdl.handle.net/10366/169917 [EN]Fusarium graminearum está entre los principales agentes causales del tizón de la espiga por Fusarium (FHB) o escabiosis en trigo y otros cereales, enfermedad presente en todo el mundo y producida por un complejo de especies de Fusarium. Además de causar pérdidas económicas importantes en rendimiento y calidad de los cultivos, F. graminearum representa una amenaza grave para la salud animal y humana. En este trabajo presentamos la primera secuencia genómica preliminar (borrador) del genoma completo de la cepa micotoxigénica Fusarium graminearum ITEM 124, proporcionando además información útil para estudios de genómica comparativa. Thu, 09 Nov 2017 00:00:00 GMT http://hdl.handle.net/10366/169917 2017-11-09T00:00:00Z http://hdl.handle.net/10366/169884 [EN]The gene Bcin04g03490 has been shown to control development and pathogenicity in the plant necrotroph Botrytis cinerea. We have identified the orthologous gene in two formae speciales of the Fusarium oxysporum species complex; it maps to chromosome 7 in the core genome of the lycopersici forma specialis. It encodes a protein with two distinct domains, a GAL4-like Zn(II)2Cys6 binuclear cluster DNA-binding domain and an acetyltransferase domain, as previously shown in B. cinerea. Null mutants were generated in both formae speciales (phaseoli and lycopersici), and the phenotypes exhibited during in culture growth and plant colonization were analyzed. The results obtained demonstrate that this virulence factor is required for complete colonization of the plant host stem vascular tissue and full virulence in F. oxysporum but not for the initial stages of root colonization. In addition, null mutants showed a severe alteration in the formation of aerial mycelia and a drastic reduction of sporulation. These observations are consistent with a master role of FoG4MAT in the gene regulation of different processes crucial for normal development and full disease induction in the plant host. Tue, 01 Jul 2025 00:00:00 GMT http://hdl.handle.net/10366/169884 2025-07-01T00:00:00Z http://hdl.handle.net/10366/169877 [EN]In planta gene expression analysis and GFP-based confocal microscopy are two powerful techniques that may be coupled to assess the extent and dynamics of plant colonization by a fungal pathogen. Here we describe methods to prepare common bean plants for inoculation with a highly virulent strain of Fusarium oxysporum f. sp. phaseoli, quantify the extent of colonization by RT-qPCR, and visualize the colonized tissues by confocal microscopy. Sat, 01 Jan 2022 00:00:00 GMT http://hdl.handle.net/10366/169877 2022-01-01T00:00:00Z http://hdl.handle.net/10366/169874 [EN]Natural populations provide valuable information and resources for addressing the genetic characterization of biological systems. Botrytis cinerea is a necrotrophic fungus that exhibits complex responses to light. Physiological analysis of B. cinerea populations from vineyards in Castilla y León (Spain) allowed for the identification of isolate Bc116. This field isolate shows a reduced pathogenicity that is conditioned by the light regime. Light also delays germination and accentuates the negative effect it exerts on the vegetative growth of B. cinerea. Bc116 also displays a marked hyperconidiation phenotype and a characteristic sclerotia production pattern. Genetic analysis demonstrates that the alternative phenotypes regarding pathogenicity, conidiation, and pattern of sclerotia production co-segregate in the progeny of crosses between isolate Bc116 and a wild-type field isolate, Bc448, showing that they are under the control of a single locus. By applying a strategy based on bulked segregant analysis, the mutation in Bc116 has been mapped to a 200 kb region on Chr14 and the analysis of this region identifies a 2 kb deletion affecting the bcltf1 gene, encoding the B. cinerea Light Responsive Transcription Factor 1 described in the reference isolate B05.10. Transformation of Bc116 with the B05.10 bcltf1 allele restored the wild-type phenotypes, providing functional evidence that the natural mutant Bc116 is altered in gene bcltf1. This study offers additional information, derived from the analysis of the genetic background of a natural mutant, on the physiological processes regulated by BcLTF1 and supports the key role of this TF in the pathogenicity and photobiology of B. cinerea. Tue, 08 Apr 2025 00:00:00 GMT http://hdl.handle.net/10366/169874 2025-04-08T00:00:00Z http://hdl.handle.net/10366/169868 [EN]Botrytis cinerea, a necrotrophic fungus responsible for grey rot disease, causes substantial economic losses. However, recent studies have discovered distinct non-sporogenic and non-infective isolates of this species, such as the B459 field strain. Examination of these isolates is particularly intriguing in the context of the development of methodological applications that could be useful in the biocontrol of this phytopathogenic species. This investigation revealed that B459 exhibited a reduced growth rate yet displayed enhanced resilience to stressors like oxidative stress agents, SDS, ethanol, and PhITC. Notably, its ability to generate reactive oxygen species (ROS) and acidic compounds, crucial in plant–pathogen interactions, was impacted. Bio-targeted toxin identification assays and the metabolomic study of extracts obtained from fermentations at seven and fourteen days revealed that this strain does not biosynthesize botrydial and derivatives neither botcinin family toxins. Furthermore, its inability to infect tomato fruits, grape, and gerbera petals coincides with its lack of toxin production under culture conditions typically adapted for reference strain B05.10. Mon, 01 Jan 2024 00:00:00 GMT http://hdl.handle.net/10366/169868 2024-01-01T00:00:00Z http://hdl.handle.net/10366/169865 [EN]Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants. Fri, 12 Nov 2021 00:00:00 GMT http://hdl.handle.net/10366/169865 2021-11-12T00:00:00Z http://hdl.handle.net/10366/169842 [ES] El uso indiscriminado de fertilizantes y pesticidas químicos en los sistemas agrícolas ha provocado una degradación progresiva de la calidad del suelo, alteraciones en la estructura y funcionalidad de las comunidades microbianas, así como la contaminación de otros ecosistemas. Frente a esta problemática ambiental, las bacterias promotoras del crecimiento vegetal (PGPB) se presentan como una alternativa biotecnológica sostenible, con el potencial de mantener o incluso mejorar el rendimiento de los cultivos sin comprometer la salud del ecosistema. El potencial de las PGPB se basa en diversos mecanismos, entre los que se destacan la colonización eficiente de tejidos vegetales en uno o varios hospedadores, la capacidad de facilitar la disponibilidad de nutrientes esenciales y la modulación de los niveles hormonales en las plantas. A través de estos mecanismos, dichas bacterias pueden influir de manera determinante en procesos fisiológicos clave, tales como la modificación de la arquitectura del sistema radicular, lo que favorece la adaptación del hospedador a su entorno, potencia interacciones sinérgicas con otros microorganismos beneficiosos y promueve una mayor resistencia frente a condiciones adversas como sequía o la salinidad. Las cepas bacterianas que presentan estas características se consideran candidatas para el desarrollo de bioinoculantes, susceptibles de ser formulados, producidos a escala industrial, distribuidos y aplicados en sistemas agrícolas. No obstante, los buenos resultados de estos bioinoculantes in vitro no garantizan la efectividad del producto bajo condiciones reales. Por tanto, durante el proceso de formulación es indispensable evaluar aspectos críticos como la estabilidad del producto, su vida útil y, fundamentalmente, su eficacia bajo condiciones no controladas. En este contexto, la cepa Pseudomonas sp. CDVBN10, endófito de raíces de colza, ha demostrado tener potencial como PGPB. Análisis genómicos y bioquímicos han evidenciado su capacidad para producir sideróforos, solubilizar fosfatos, sintetizar ácido indol-3-acético (AIA), presentar actividad ACC desaminasa y formar biofilm en la superficie radicular. Ensayos funcionales adicionales confirmaron su eficacia para promover el crecimiento de colza y cultivos hortícolas como espinaca y cilantro (Jiménez-Gómez, 2020; Jiménez-Gómez et al., 2020). La cepa CDVBN10-B deriva de la cepa CDVBN10, pero cuenta con una deleción genómica de origen desconocido que fue descubierta tras finalizar la presente tesis doctoral. No obstante, nuestros estudios han demostrado que esta cepa sigue manifestando las citadas propiedades. El objetivo general de la presente tesis doctoral es profundizar en la caracterización funcional de la cepa Pseudomonas sp. CDVBN10-B como un bioinoculante de amplio espectro. Para ello, se plantea la caracterización exhaustiva de su potencial como PGPB en cultivos de interés agronómico, la evaluación de los mecanismos moleculares implicados en su interacción con la planta, la remodelación de la arquitectura radicular y la inducción de resistencia sistémica, así como el diseño de un bioinoculante efectivo para su aplicación en condiciones de campo.; [EN] The indiscriminate use of chemical fertilizers and pesticides in agricultural systems has led to progressive soil degradation, disruptions in the structure and functionality of microbial communities, and contamination of surrounding ecosystems. In response to this environmental challenge, plant growth-promoting bacteria (PGPB) have emerged as a sustainable biotechnological alternative with the potential to maintain or even enhance crop yields without compromising ecosystem health. The potential of PGPB relies on several mechanisms, including efficient colonization of plant tissues in one or more hosts, enhanced availability of essential nutrients, and modulation of plant hormone levels. Through these mechanisms, PGPB can significantly influence key physiological processes such as the modification of root system architecture, thereby improving host adaptation to environmental conditions, facilitating synergistic interactions with other beneficial microorganisms, and enhancing tolerance to abiotic stresses such as drought and salinity. Bacterial strains exhibiting these traits are considered suitable candidates for the development of bioinoculants that can be formulated, produced at an industrial scale, distributed, and applied in agricultural systems. However, successful in vitro performance does not necessarily guarantee product effectiveness under field conditions. Thus, formulation development must include the assessment of critical factors such as product stability, shelf life, and, above all, efficacy under non-controlled environments. In this context, Pseudomonas sp. CDVBN10, an endophyte isolated from rapeseed roots, has demonstrated potential as PGPB. Genomic and biochemical analyses revealed its ability to produce siderophores, solubilize phosphate, synthesize indole-3-acetic acid (IAA), exhibit ACC deaminase activity, and form biofilms on root surfaces. Additional functional assays confirmed its capacity to promote the growth of rapeseed and horticultural crops such as spinach and coriander (Jiménez-Gómez, 2020; Jiménez-Gómez et al., 2020). CDVBN10-B, a derivative of strain CDVBN10, was later found to harbor a genomic deletion of unknown origin, identified upon completion of this doctoral thesis. Nevertheless, our studies have shown that this strain retains the aforementioned properties. The overall objective of this doctoral thesis is to conduct an in-depth functional characterization of Pseudomonas sp. CDVBN10-B as a broad-spectrum bioinoculant. This includes a comprehensive evaluation of its potential as PGPB in crops of agronomic interest, investigation of the molecular mechanisms involved in its interaction with host plants, its role in remodeling root architecture and inducing systemic resistance, and the development of an effective bioinoculant suitable for field application. Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/10366/169842 2025-01-01T00:00:00Z http://hdl.handle.net/10366/169778 [ES] Colletotrichum es un género de hongos filamentosos fitopatógenos que causan la enfermedad de la antracnosis. Colletotrichum graminicola es el agente causal de la antracnosis del maíz (Zea mays), una enfermedad de gran impacto económico cuya incidencia está en aumento en distintas regiones del mundo y cuyos principales síntomas son el tizón foliar y la pudrición del tallo. Su éxito infeccioso depende en gran medida de la acción de proteínas efectoras, muchas de las cuales permanecen poco caracterizadas. Esta Tesis Doctoral se centró en dos efectores nucleares clave que son parálogos entre sí: CgEP4 y CgEP1 (Colletorichum graminicola Effector Protein 4 and 1). El estudio de CgEP4 reveló que se trata de un efector con localización nuclear altamente conservado, originado por una duplicación ancestral, cuya función resulta indispensable para la virulencia. Este gen está altamente conservado entre aislados de campo de C. graminicola de distintos continentes, lo que subraya su importancia biológica. A nivel experimental, se comprobó que CgEP4 se expresa de manera específica durante las fases iniciales de la infección y se transloca al núcleo de las células de maíz. Los mutantes de deleción mostraron una notable reducción en la capacidad de penetración y colonización, así como una activación fuerte de defensas basales en el hospedador, lo que indica que este efector cumple un papel esencial en la supresión de respuestas inmunitarias tempranas. Además, los ensayos in vitro revelaron que su ausencia altera procesos fúngicos fundamentales como el crecimiento, la esporulación y la tolerancia al estrés oxidativo y salino, lo que apunta a una doble función de CgEP4: actuar sobre el hospedador y, al mismo tiempo, regular aspectos internos de la fisiología del hongo. El análisis de CgEP1 se centró en la influencia de las repeticiones en tándem que presenta en su secuencia sobre la virulencia. A partir de un conjunto de aislados naturales se identificaron variantes alélicas que diferían en el número de repeticiones intragénicas (entre 5 y 8), lo que permitió establecer una relación directa entre la arquitectura del gen y la virulencia. Experimentos de complementación funcional demostraron que se requieren al menos siete repeticiones para mantener la virulencia, mientras que alelos con menos repeticiones pierden eficacia infecciosa y se comportan como mutantes nulos. Modelados estructurales realizados con AlphaFold2 confirmaron que la pérdida de repeticiones compromete la estabilidad tridimensional de la proteína, explicando la pérdida de función. A nivel transcriptómico, se comprobó que CgEP1 no solo suprime genes de defensa en las primeras etapas de la infección, sino que también reprograma el metabolismo del maíz en fases posteriores, favoreciendo la colonización. De forma paralela, en el propio hongo regula la expresión de genes asociados a la biosíntesis proteica, al metabolismo energético y a enzimas de degradación de la pared vegetal, reforzando su papel como efector multifuncional capaz de coordinar tanto la respuesta del hospedador como la del patógeno. En conjunto, esta tesis doctoral demuestra que CgEP4 y CgEP1 son efectores nucleares fundamentales para la virulencia de C. graminicola. Ambos casos ilustran la complejidad de las interacciones planta-patógeno y aportan nuevas perspectivas para el diseño de estrategias de control frente a la antracnosis del maíz. Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/10366/169778 2025-01-01T00:00:00Z http://hdl.handle.net/10366/169695 [EN]Guanine nucleotide exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to physical and chemical stimuli and ultimately regulate numerous cellular responses such as polarized growth, morphogenesis, and movement. The GEF proteins are characterized by a Dbl-homology (DH) domain that contacts the Rho GTPases, to catalyzing nucleotide exchange, and an associated Pleckstrin homology (PH) domain, which fine-tunes the exchange process by a variety of mechanisms related to the binding of phosphoinositides. Most GEFs are divergent in regions outside the DH/PH module and contain additional protein-protein or lipid-protein interaction domains that presumably dictate unique cellular functions. Fission yeast Rho1-GEFs act as a link between growth processes and the cell cycle machinery. In this chapter, we focus on the recent leaps in our understanding of how Rho1-GEFs control interphase and cytokinesis in fission yeast. Furthermore, we will go beyond mitosis and highlight the unexpected roles of Rho1-GEFs in the DNA damage response. Mon, 01 Jan 2018 00:00:00 GMT http://hdl.handle.net/10366/169695 2018-01-01T00:00:00Z