miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Regulación de genes de resistencia de la familia TIR-NBS-LRR mediada por miRNA/phasiRNA durante la interacción con P. syringae

Durante un estrés biótico, las plantas modulan la expresión de una batería de genes involucrados en la respuesta de defensa, proceso donde recientemente se ha determinado el papel esencial que desempeña el silenciamiento génico. El silenciamiento génico es un mecanismo de regulación de la expresión génica, donde destacan como principales moléculas efectoras los pequeños RNAs (sRNAs). En plantas, estos sRNAs, son clasificados en pequeños RNAs interferentes (siRNAs) o microRNAs (miRNAs), presentando tamaños similares (20-24 nt) pero difiriendo en su biogénesis y modo de acción. Los miRNAs son pequeños RNAs de cadena sencilla que actúan regulando negativamente la expresión de genes, mediante su unión al complejo RISC (Rna Induced Silencing Complex) y en una forma dependiente de secuencia. En nuestro laboratorio, mediante el análisis de datos transcriptómicos, y el uso de herramientas bioinformáticas, identificamos un miRNA* de 22 nt como potencial regulador de la expresión de genes de resistencia (“R”) del tipo TIR-NBS-LRR. Posteriormente hemos validado dicha regulación y caracterizado los patrones de expresión tanto del Pri-miRNA como de un gen “R” regulado por este, en diferentes tejidos y estadios del desarrollo, así como durante la interacción con P. syringae. Por otro lado, hemos generado plantas transgénicas que presentan niveles alterados del miRNA* (incremento y reducción) y hemos observado que muestran fenotipos alterados de PTI y una mayor/menor colonización de P. syringae. Finalmente hemos identificado la producción de sRNAs (phasiRNAs) a partir del gen de resistencia, en una forma dependiente de miRNA*-RDR6-DCL4, pudiendo estos sRNAs secundarios regular otros transcritos de la misma familia de genes de resistencia.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Proteomic analysis of sera from rheumatoid arthritis B6 WT and CD38KO mice by using proteominer fractionation and two-dimensional difference gel electrophoresis (DIGE)

Comunicaciones a congreso

Extracellular vesicles from pristane-treated CD38-deficient mice express an antiinflammatory neutrophil protein signature, which reflects the mild lupus severity elicited in these mice

In CD38-deficient (Cd38-/-) mice intraperitoneal injection of pristane induces a lupus-like disease, which is milder than that induced in WT mice, showing significant differences in the inflammatory and autoimmune processes triggered by pristane. Extracellular vesicles (EV) are present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin and/or tissue pathological situation. The aim of this study was to analyze the protein composition, protein abundance, and functional clustering of EV released by peritoneal exudate cells (PECs) in the pristane experimental lupus model, to identify predictive or diagnostic biomarkers that might discriminate the autoimmune process in lupus from inflammatory reactions and/or normal physiological processes. In this study, thanks to an extensive proteomic analysis and powerful bioinformatics software, distinct EV subtypes were identified in the peritoneal exudates of pristane-treated mice: 1) small EV enriched in the tetraspanin CD63 and CD9, which are likely of exosomal origin; 2) small EV enriched in CD47 and CD9, which are also enriched in plasma-membrane, membrane-associated proteins, with an ectosomal origin; 3) small EV enriched in keratins, ECM proteins, complement/coagulation proteins, fibrin clot formation proteins, and endopetidase inhibitor proteins. This enrichment may have an inflammation-mediated mesothelial-tomesenchymal transition origin, representing a protein corona on the surface of peritoneal exudate EV; 4) HDL-enriched lipoprotein particles. Quantitative proteomic analysis allowed us to identify an anti-inflammatory, Annexin A1- enriched pro-resolving, neutrophil protein signature, which was more prominent in EV from pristane-treated Cd38-/- mice, and quantitative differences in the protein cargo of the ECM-enriched EV from Cd38-/- vs WT mice. These differences are likely to be related with the distinct inflammatory outcome shown by Cd38-/- vs WT mice in response to pristane treatment. Our results demonstrate the power of a hypothesis-free and data-driven approach to transform the heterogeneity of the peritoneal exudate EV from pristanetreated mice in valuable information about the relative proportion of different EV in a given sample and to identify potential protein markers specific for the different small EV subtypes, in particular those proteins defining EV involved in the resolution phase of chronic inflammation.Proyecto del plan estatal, Ministerio de Ciencia e Innovacion PT13/0001/011CSIC PT17/0019/0010 PID2020-119567RB-I0

Genetic Analysis of the Individual Contribution to Virulence of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1PtoDC3000 to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection

Antibodies Against β2-Glycoprotein I Complexed With an Oxidised Lipoprotein Relate to Intima Thickening of Carotid Arteries in Primary Antiphospholipid Syndrome

To explore whether antibodies against β2-glycoprotein I (β2GPI) complexed to 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) and to oxidised low-density lipoproteins (oxLDL) relate to paraoxonase activity (PONa) and/or intima media thickness (IMT) of carotid arteries in primary antiphospholipid syndrome (PAPS). As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL) without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: β2GPI−oxLDL complexes, IgG anti-β2GPI−oxLig-1, IgG anti-β2GPI−oxLDL antibodies (ELISA), PONa, (para-nitrophenol method), IMT of common carotid (CC) artery, carotid bifurcation (B), internal carotid (IC) by high resolution sonography. β2GPI−oxLDL complex was highest in the control group (p < 0.01), whereas, IgG anti-β2GPI−oxLig1 and IgG anti-β2GPI−oxLDL were highest in PAPS (p < 0.0001). In healthy controls, β2GPI−oxLDL complexes positively correlated to IMT of the IC (p = 0.007) and negatively to PONa after correction for age (p < 0.03). PONa inversely correlated with age (p = 0.008). In PAPS, IgG anti-2GPI−oxLig-1 independently predicted PONa (p = 0.02) and IMT of B (p = 0.003), CC, (p = 0.03) and of IC (p = 0.04). In PAPS, PONa inversely correlated to the IMT of B, CC and IC (p = 0.01, 0.02 and 0.003, respectively). IgG anti-2GPI−oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity

Mice deficient in CD38 develop an attenuated form of collagen type II-induced arthritis

CD38, a type II transmembrane glycoprotein expressed in many cells of the immune system, is involved in cell signaling, migration and differentiation. Studies in CD38 deficient mice (CD38 KO mice) indicate that this molecule controls inflammatory immune responses, although its involvement in these responses depends on the disease model analyzed. Here, we explored the role of CD38 in the control of autoimmune responses using chicken collagen type II (col II) immunized C57BL/6-CD38 KO mice as a model of collagen-induced arthritis (CIA). We demonstrate that CD38 KO mice develop an attenuated CIA that is accompanied by a limited joint induction of IL-1β and IL-6 expression, by the lack of induction of IFNγ expression in the joints and by a reduction in the percentages of invariant NKT (iNKT) cells in the spleen. Immunized CD38 KO mice produce high levels of circulating IgG1 and low of IgG2a anti-col II antibodies in association with reduced percentages of Th1 cells in the draining lymph nodes. Altogether, our results show that CD38 participates in the pathogenesis of CIA controlling the number of iNKT cells and promoting Th1 inflammatory responses

Downscaling Climate Change Impacts, Socio-Economic Implications and Alternative Adaptation Pathways for Islands and Outermost Regions

This book provides a comprehensive overview of the future scenarios of climate change and management concerns associated with climate change impacts on the blue economy of European islands and outermost regions. The publication collects major findings of the SOCLIMPACT project’s research outcomes, aiming to raise social awareness among policy-makers and industry about climate change consequences at local level, and provide knowledge-based information to support policy design, from local to national level. This comprehensive book will also assist students, scholars and practitioners to understand, conceptualize and effectively and responsibly manage climate change information and applied research. This book provides invaluable material for Blue Growth Management, theory and application, at all levels. This first edition includes up-to-date data, statistics, references, case material and figures of the 12 islands case studies. ¨Downscaling climate change impacts, socio-economic implications and alternative adaptation pathways for Islands and Outermost Regions¨ is a must-read book, given the accessible style and breadth and depth with which the topic is dealt. The book is an up-to-date synthesis of key knowledge on this area, written by a multidisciplinary group of experts on climate and economic modelling, and policy design