Caracterización funcional de genes reguladores del proceso de desarrollo, maduración y senescencia del fruto de fresa

“Caracterización funcional de genes de fresa (Fragaria × ananassa) relacionados con la maduración identificados a través de una plataforma de microarrays de oligos hecha a medida” A lo largo del desarrollo de esta Tesis se han estudiado los cambios transcriptómicos que ocurren en el receptáculo del fruto de fresa (Fragaria × ananassa) durante su desarrollo y maduración usando una plataforma de microarrays de oligos. Este análisis permitió seleccionar varios genes de interés biotecnológico potencialmente implicados en el proceso de maduración, jugando papeles significativos en diversos procesos fisiológicos tales como producción hormonal, regulación de compuestos volátiles y tamaño/forma celular que contribuyen en la modulación de las propiedades organolépticas finales del fruto. Uno de los genes seleccionados (FaNCED1) mostraba homología de secuencia significativa con genes de plantas superiores que codifican 9-cis-dioxigenasas de epoxicarotenoides (NCEDs), descritas como enzimas claves en la producción de ácido abscísico (ABA). Estas enzimas han sido descritas como la última enzima que actúa en el interior de los plastidios en la ruta de biosíntesis de ABA, catalizando el corte del precursor C40-xantofilas (9-cis-neoxantina y 9-cis-violaxantina) para producir C25 epoxiapo- aldehido y xantosina que es liberada al citosol donde finalmente da lugar a ABA. Se ha propuesto que en frutos no-climatéricos como fresa, uva y cítricos, ABA juega un importante papel en la regulación de la maduración de los mismos, mucho más que el llevado a cabo por el etileno. En este trabajo, se ha observado una clara correlación entre el incremento de la expresión génica de FaNCED1 y el contenido de ABA a lo largo de la maduración de la fresa. Además, se ha encontrado que la expresión de FaNCED1 está regulada negativamente por las auxinas sintetizadas en los aquenios. Por otro lado, se llevaron a cabo ensayos de actividad enzimática de la proteína recombinante FaNCED1 derivada de la secuencia completa de ADN copia de FaNCED1. La enzima recombinante mostró actividad solamente en presencia de precursores en configuración cis localizados un paso corriente arriba en la ruta de biosíntesis de ABA, permitiendo identificar, al menos, uno de los dos productos de la...“Functional characterization of strawberry (Fragaria × ananassa) ripening-related genes identified throughout a custom-made oligo-based microarray platform” Along the development of this thesis, I have studied the transcriptomic changes that occur in the receptacle of the strawberry fruit (Fragaria × ananassa) during development and ripening using an oligo microarray platform. This analysis allowed selecting several target genes with biotechnological importance potentially involved in the process of fruit ripening, playing significant roles in various physiological processes such as hormone production, regulation of volatile compounds and cell size/shape that contribute to modulate the final organoleptic properties of the fruit. One of the selected genes (FaNCED1) showed significant homology of sequence with genes of higher plants encoding 9-cis-epoxycarotenoid dioxigenases (NCEDs), key enzymes in the production of abscisic acid (ABA). These enzymes are described as the last enzyme that acts into the plastids in the ABA biosynthetic pathway, catalyzing the cleavage of the precursor C40-xanthophylls (9-cis-neoxanthin and 9-cis-violaxanthin) to produce C25 epoxy apo-aldehyde and xanthoxin that is released into the cytosol and finally gives rise to ABA. It has been proposed that, in non-climacteric fruits such as strawberry, grape and citrus; ABA plays an important role in regulating the maturation of the fruit, much more than that undertaken by ethylene. In this work, we have observed a clear correlation between the increase in FaNCED1 gene expression and the increase of ABA content along the strawberry maturation. Furthermore, it has been found that its expression is negatively regulated by auxins synthesized in the achenes. On the other hand, I assessed the enzymatic activity of the recombinant protein FaNCED1 derived from the full-length FaNCED1 cDNA. The recombinant enzyme showed activity only in the presence of precursors in the cis configuration located one step upstream in ABA biosynthesis pathway, allowing identify, at least, one of the two products. Therefore, these results suggest that the FaNCED1 protein is related to the production of ABA..

β-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis

The plant hormone salicylic acid (SA) is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) and NON RECOGNITION OF BTH-4 (NRB4) are required for the response to SA in Arabidopsis (Arabidopsis thaliana). Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, βCA1 and βCA2, are β-carbonic anhydrase family proteins. Since double mutant βca1 βca2 plants did not show any obvious phenotype, we investigated other βCAs and found that NRB4 also interacts with βCA3 and βCA4. Moreover, several βCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between βCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4- βCA1-NPR1. The quintuple mutant βca1 βca2 βca3 βca4 βca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.Facultad de Ciencias Exacta

Cross-Resistance to Abiraterone and Enzalutamide in Castration Resistance Prostate Cancer Cellular Models Is Mediated by AR Transcriptional Reactivation

Androgen deprivation therapy (ADT) and novel hormonal agents (NHAs) (Abiraterone and Enzalutamide) are the goal standard for metastatic prostate cancer (PCa) treatment. Although ADT is initially effective, a subsequent castration resistance status (CRPC) is commonly developed. The expression of androgen receptor (AR) alternative splicing isoforms (AR-V7 and AR-V9) has been associated to CRPC. However, resistance mechanisms to novel NHAs are not yet well understood. Androgen-dependent PCa cell lines were used to generate resistant models to ADT only or in combination with Abiraterone and/or Enzalutamide (concomitant models). Functional and genetic analyses were performed for each resistance model by real-time cell monitoring assays, flow cytometry and RT-qPCR. In androgen-dependent PCa cells, the administration of Abiraterone and/or Enzalutamide as first-line treatment involved a critical inhibition of AR activity associated with a significant cell growth inhibition. Genetic analyses on ADT-resistant PCa cell lines showed that the CRPC phenotype was accompanied by overexpression of AR full-length and AR target genes, but not necessarily AR-V7 and/or AR-V9 isoforms. These ADT resistant cell lines showed higher proliferation rates, migration and invasion abilities. Importantly, ADT resistance induced cross-resistance to Abiraterone and/or Enzalutamide. Similarly, concomitant models possessed an elevated expression of AR full-length and proliferation rates and acquired cross-resistance to its alternative NHA as second-line treatment.Instituto de Salud Carlos III PI17/00989European Regional Development Fund "A way to build Europe"Ramon y Cajal - Ministry of Economy and Competitiveness RYC-2015-18382Ministry of Education, Culture and Sport FPU14/05461University of Granad

FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.This work was funded by the Spanish Ministerio de Ciencia e Innovacion (AGL2014-55784-C2-2-R and AGL2017-86531-C2-2-R). FJMR is supported by a ‘Margarita Salas’ post-doctoral fellowship (UCOR02MS) from the University of Cordoba (Requalification of the Spanish university system) from the Ministry of Universities financed by the European Union (NexGenerationEU). FJMH is supported by a ‘Juan de la Cierva-Incorporacion’ fellowship (IJC2020- 045526-I), funded by MCIN/AEI/10.13039/501100011033 and the European Union ‘NextGenerationEU’/PRTR. AR-F and SA are on the European Union’s Horizon 2020 Research and Innovation Program, Project PlantaSYST (SGA-CSA No. 739582 under FPA No. 664620). The authors thank Dr. Gema Garc ıa from the Microscopy Unit of UCAIB-IMIBIC for technical help with the microscope. Funding for open access charge: University of Cordoba/CBUA.S

Prevalence and Associated Factors of Low Bone Mineral Density in the Femoral Neck and Total Hip in Axial Spondyloarthritis: Data from the CASTRO Cohort

Studies on osteoporosis in axial spondyloarthritis (axSpA) have focused on the lumbar segment, and few studies have assessed bone mineral density (BMD) in the hip and femoral neck in these patients. The aim of this study was to evaluate the prevalence of low BMD and osteopenia in the total hip or femoral neck and the factors associated with these conditions in axSpA patients. This was a single-centre, observational, cross-sectional study among consecutive patients with axSpA according to the ASAS criteria from the CASTRO registry. All patients underwent total hip and femoral neck DXA BMD measurements. Low BMD was defined as a Z-score less than −1, and osteopenia was defined as a T-score less than −1. Multivariate logistic and generalised linear regressions were used to evaluate factors independently associated with low BMD and osteopenia in the hip or femoral neck and those associated with variability in BMD, respectively. A total of 117 patients were included, among which 30.8% were female and the mean age was 45 years. A total of 36.0% of patients had low BMD (28.1% in the total hip and 27.4% in the femoral neck), and 56.0% of patients had osteopenia (44.7% in the total hip and 53.8% in the femoral neck). A multivariate logistic regression showed that age, radiographic sacroiliitis and ASAS-HI were independently associated with low BMD in the total hip or femoral neck. Factors that were independently associated with osteopenia were Body Mass Index, disease duration, radiographic sacroiliitis and ASAS-HI. In conclusion, 36% of the patients with axSpA had low BMD in the total hip or femoral neck. A younger age and radiographic sacroiliitis were the most important factors associated with decreased BMD

A Defense Pathway Linking Plasma Membrane and Chloroplasts and Co-opted by Pathogens

Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon activation of plant defense, interfering with the chloroplast-dependent anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defenses following an association with membranes, which relies on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defenses from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defense exists in plants and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses

A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an antiviral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus can inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homolog, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signaling components have been coopted to play multiple functions in plants

A plant virus causes symptoms through the deployment of a host-mimicking protein domain to attract the insect vector.

During compatible plant-virus interactions, viruses can interfere with the normal developmental program of their hosts, leading to the appearance of phenotypes that we usually identify as ‘’symptoms of infection’’ (leaf curling and yellowing, stunting, dwarfism, necrosis). Despite their relevance, the molecular mechanisms underlying symptom induction and their biological meaning, if any, remain poorly understood. By using tomato yellow leaf curl virus (TYLCV, Geminivirus) as model, we have isolated C4 as the main protein responsible for the induction of TYLCV-associated symptoms in tomato. C4, by mimicking a host protein domain, the Conserved C-termini in LAZY1 protein family (CCL) domain, physically interacts with the RCC1-like domain-containing plant proteins (RLDs). By interacting with the RLDs through the CCL-like domain, C4 displaces one endogenous interactor, LAZY (LZY), interfering with RLD functions in processes such as auxin signaling and endomembrane trafficking, which correlates with the manifestation of symptoms. Surprisingly, we observed that appearance of C4-mediated symptoms in tomato plants plays no major role in viral replication nor movement, but they serve as attractants for the insect vector, the whitefly Bemisia tabaci, which preferentially feeds on tomato plants exhibiting strong symptoms of viral infection. These results suggest that, during plant-virus co-evolution, symptoms may have appeared as a strategy to promote viral transmission by the insect vector, at least in some specific plant-virus-vector pathosystems.Work in RLD’s lab is partially funded by the Excellence Strategy of the German Federal and State Governments, the ERC-COG GemOmics (101044142), the DeutscheForschungsgemeinschaft (DFG, German Research foundation) (project numbers LO 2314/1-1 and SBF 1101/3, C08), and a Royal Society Newton Advance grant (NA140481 – NAF\R2\180857). EA is the recipient of a Marie Skłodowska-Curie Grant from the European Union’s Horizon 2020 Research and Innovation Program (Grant 896910-GeminiDECODER). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech