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Iron (Fe) is a critical micronutrient for the growth and survival of most organisms, playing an important structural role in proteins and as an enzyme cofactor. Despite its abundance in nature, Fe is often not available to plants, particularly in alkaline soils, since it exists mostly in its oxidized state, Fe (III). To prevent chlorosis and poor development, plants have evolved strategies to acquire Fe from the rhizosphere. Non-grasses plants use a Strategy I, which involves a plasma membrane H+-ATPase to acidify the rhizosphere and solubilize Fe, a ferric reductase (FRO1) to reduce Fe (III) to Fe (II), and the Fe (II) transporter (IRT) for uptake across the plasma membrane. Grasses, on the other hand, employ a Strategy II, which includes the production of phytosiderophores (PS) to chelate Fe (III) and oligopeptide transporters YS1 or YS1-like to transport the PS-Fe chelates into root cells. The establishment of beneficial associations with soil microorganisms is another strategy evolved by plants to cope with Fe deficiency. Arbuscular mycorrhizal (AM) fungi, belonging to the subphylum Glomeromycotina, are among the most prominent microorganisms that contribute to plant nutrition. These fungi form a mutualistic symbiosis with most terrestrial plant species. They colonize biotrophically the root cortex and develop an extensive network of extraradical hyphae in the soil that overgrows the soil surrounding the roots. In return for the carbon compounds provided by the plants, AM fungi deliver to the plant the nutrients they take up beyond the nutrient depletion zones developed around the roots. It is well established that AM fungi can help plants to acquire low mobility nutrients, such as phosphorus, nitrogen, zinc, copper and Fe. Besides enhancing nutrient uptake to their host plants, AM fungi provide increased tolerance against biotic and abiotic stresses. AM fungi play a crucial role in modulating plant metal acquisition over a wide range of soil metal concentrations, as they increase plant metal acquisition in soils deficient in these elements but reduce metal uptake in contaminated soils. The importance of the AM symbiosis for plant development in both Fe-deficient and Fecontaminated soils has been established. However, little is known about the mechanisms of Fe transport and homeostasis in AM. Within this PhD thesis to get further insights into the mechanisms of Fe homeostasis in AM, we employed a multidisciplinary approach combining in silico, physiological and molecular tools. We used the model AM fungus Rhizophagus irregularis DAOM197198 v2.0 and A1, A4, A5, B3 and C2 v1.0, which can be easily grown in in vitro monoxenic cultures and in vivo whole plant bidimensional experimental systems. For the studies on the plant side we used Solanum lycopersicum, an economically important crop that has been used as a model plant for studying Fe homeostasis in Strategy I plants., El hierro (Fe) es un micronutriente esencial para el crecimiento y supervivencia de la mayoría de los organismos, ya que desempeñado un papel estructural clave en muchas proteínas y actúa como cofactor de múltiples enzimas. A pesar de que el Fe es uno de los elementos más abundantes en la naturaleza, a menudo no está disponible para las plantas debido a que se encuentra principalmente en su estado oxidado, Fe (III), lo que se agrava en los suelos alcalinos. Para evitar la clorosis férrica y las deficiencias de desarrollo asociadas a la limitación de Fe, las plantas han evolucionado una serie de estrategias que les permitan obtener eficientemente el Fe presente en la rizosfera. Las plantas no gramíneas utilizan la llamada “Estrategia I”, que consta de una H+- ATPasa de membrana plasmática que acidifica la rizósfera y solubiliza el Fe, de una reductasa férrica (FRO1) que reduce el Fe (III) a Fe (II) y de un transportador específico de Fe (II) (IRT) que para su absorción a través de la membrana plasmática. Por otro lado, las gramíneas utilizan la “Estrategia II”, que incluye la producción y exudación de fitosideróforos (PS) que quelan el Fe (III) y transportadores de oligopéptidos YS1 o similares que transportan los quelatos PS-Fe a las células de la raíz. Otra estrategia utilizada por las plantas para incrementar la absorción de hierro lo es el establecimiento de asociaciones beneficiosas con microorganismos del suelo. Los hongos micorrícicos arbusculares (MA), pertenecientes al subfilo Glomeromycotina, son unos de los principales microorganismos que contribuyen a la nutrición de las plantas. Estos hongos forman una simbiosis mutualista con la mayoría de las especies de plantas terrestres. Los hongos MA colonizan biotróficamente el córtex de la raíz, al tiempo que desarrollan una red extensa de hifas en el exterior de la raíz que alcanza y explora zonas del suelo no accesibles a la raíz. A cambio de los compuestos de carbono proporcionados por las plantas, los hongos suministran a la planta los nutrientes minerales que absorben más allá de las zonas de agotamiento de nutrientes desarrolladas alrededor de las raíces. Es ampliamente reconocido que los hongos MA ayudan a las plantas a obtener nutrientes de baja movilidad, como fósforo, nitrógeno, zinc, cobre y Fe. Además de mejorar la captación de nutrientes, los hongos MA incrementan la tolerancia de la planta hospedadora a estreses bióticos y abióticos. Los hongos MA desempeñan un papel crucial en la modulación de la adquisición de metales por parte de las plantas en un amplio rango de concentraciones de metales del suelo, ya que aumentan la adquisición de metales en suelos deficitarios en estos elementos, pero la reducen en suelos contaminados. Varios estudios han puesto de manifiesto que la simbiosis MA mejora el desarrollo de las plantas tanto en suelos deficientes como contaminados con Fe. Sin embargo, los conocimientos actuales sobre los mecanismos de transporte y homeostasis de Fe en MA son muy limitados., Tesis Univ. Granada., BES-2016-078463 Ministerio de Ciencia, Innovación y Universidades, Proyectos de Investigación del Plan Nacional AGL2015-67098 y RTI2018-098756- B-I00

RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2-9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2-9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.