The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J.Fil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ramírez, Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Abd El Haliem, Ahmed. Wageningen University; Países BajosFil: Vossen, Jack H.. Wageningen University; Países BajosFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Joosten, Matthieu H. A. J.. Wageningen University; Países BajosFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Primordial fluctuations from nonlinear couplings

We study the spectrum of primordial fluctuations in theories where the inflaton field is coupled to massless fields and/or to itself. Conformally invariant theories generically predict a scale invariant spectrum. Scales entering the theory through infrared divergences cause logarithmic corrections to the spectrum, tiltilng it towards the blue. We discuss in some detail whether these fluctuations are quantum or classical in nature.Comment: 12 pages, Revtex, we added an appendix clarifying our assumptions about the initial conditions at the beggining of inflatio

Dyonic BIon black hole in string inspired model

We construct static and spherically symmetric particle-like and black hole solutions with magnetic and/or electric charge in the Einstein-Born-Infeld-dilaton-axion system, which is a generalization of the Einstein-Maxwell-dilaton-axion (EMDA) system and of the Einstein-Born-Infeld (EBI) system. They have remarkable properties which are not seen for the corresponding solutions in the EMDA and the EBI system.Comment: 13 pages, 15 figures, Final version in PR

Improving genetic diagnosis in Mendelian disease with transcriptome sequencing

Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI–like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy

Muscle contraction upon nerve stimulation relies on excitation–contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR

Phosphatidylinositol 4-phosphate is associated to extracellular lipoproteic fractions and is detected in tomato apoplastic fluids

We have recently detected phosphatidylinositol-4-phosphate (PI4P) in the extracellular medium of tomato cell suspensions. Extracellular PI4P was shown to trigger the activation of defence responses induced by the fungal elicitor xylanase. In this study, by applying a differential centrifugation technique, we found that extracellular PI4P is associated with fractions composed of diverse phospholipids and proteins, which were pelleted from the extracellular medium of tomato cell suspensions grown under basal conditions. Using mass spectrometry, we identified the proteins present in these pelleted fractions. Most of these proteins have previously been characterised as having a role in defence responses. Next, we evaluated whether PI4P could also be detected in an entire plant system. For this, apoplastic fluids of tomato plants grown under basal conditions were analysed using a lipid overlay assay. Interestingly, PI4P could be detected in intercellular fluids obtained from tomato leaflets and xylem sap of tomato plants. By employing electrospray ionisation tandem mass spectrometry (ESI-MS/MS), other phospholipids were also found in intercellular fluids of tomato plants. These had a markedly different profile from the phospholipid pattern identified in entire leaflets. Based on these results, the potential role of extracellular phospholipids in plant intercellular communication is discussed.Fil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Dekker, H. L.. University Of Amsterdam; Países BajosFil: Rep, M.. University Of Amsterdam; Países BajosFil: Munnik, T.. University Of Amsterdam; Países BajosFil: Testerink, C.. University Of Amsterdam; Países BajosFil: de la Canal, L. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentin

Silencing of the tomato phosphatidylinositol‐phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol‐phospholipase C (PI‐PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase‐induced expression of defense‐related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked‐down the expression of SlPLC2 by virus‐induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)‐defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non‐silenced infected plants, while transcripts of the jasmonic acid (JA)‐defense gene markers Proteinase Inhibitor I and II (SlPI‐I and SlPI‐II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Guzzo, Maria Carla. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Abd El Haliem, Ahmed M. Wageningen University. Laboratory of Phytopathology; HolandaFil: Joosten, Matthieu H. A. J. Wageningen University. Laboratory of Phytopathology; HolandaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models

International audienceMyotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets