scholarly journals Validation of RT-qPCR approaches to monitor Pseudomonas syringae gene expression during infection and exposure to pattern-triggered immunity

2017 ◽  
Author(s):  
Amy Smith ◽  
Amelia H. Lovelace ◽  
Brian H. Kvitko
Keyword(s):  
Gene Expression ◽  
Pseudomonas Syringae ◽  
Marker Genes ◽  
Amplification Efficiency ◽  
Relative Expression ◽  
Expression Of Genes ◽  
Bacterial Rna ◽  
Transcriptional Changes ◽  
Total Tissue

AbstractPseudomonas syringae pv. tomato DC3000 (DC3000) is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a RT-qPCR assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P.syringae-specific16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of Type III secretion system, coronatine synthesis genes and flagellar marker genes.

scholarly journals Validation of RT-qPCR Approaches to Monitor Pseudomonas syringae Gene Expression During Infection and Exposure to Pattern-Triggered Immunity

2018 ◽  
Vol 31 (4) ◽  
pp. 410-419 ◽  
Author(s):  
Amy Smith ◽  
Amelia H. Lovelace ◽  
Brian H. Kvitko
Keyword(s):  
Gene Expression ◽  
Pseudomonas Syringae ◽  
Marker Genes ◽  
Amplification Efficiency ◽  
Relative Expression ◽  
Expression Of Genes ◽  
Bacterial Rna ◽  
Transcriptional Changes ◽  
Total Tissue

Pseudomonas syringae pv. tomato DC3000 is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a quantitative reverse transcription-polymerase chain reaction assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P. syringae–specific 16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of type III secretion system, coronatine synthesis genes, and flagellar marker genes.

Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1661-1671
Author(s):  
Klaus Maleck ◽  
Urs Neuenschwander ◽  
Rebecca M Cade ◽  
Robert A Dietrich ◽  
Jeffery L Dangl ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Constitutive Expression ◽  
Firefly Luciferase ◽  
Marker Genes ◽  
Arabidopsis Mutants ◽  
Isolation And Characterization ◽  
Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

scholarly journals Coordinated multitissue transcriptional and plasma metabonomic profiles following acute caloric restriction in mice

2006 ◽  
Vol 27 (3) ◽  
pp. 187-200 ◽  
Author(s):  
Colin Selman ◽  
Nicola D. Kerrison ◽  
Anisha Cooray ◽  
Matthew D. W. Piper ◽  
Steven J. Lingard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Caloric Restriction ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Proton Nuclear Magnetic Resonance ◽  
Cellular Transport ◽  
Expression Of Genes ◽  
Transcriptional Changes

Caloric restriction (CR) increases healthy life span in a range of organisms. The underlying mechanisms are not understood but appear to include changes in gene expression, protein function, and metabolism. Recent studies demonstrate that acute CR alters mortality rates within days in flies. Multitissue transcriptional changes and concomitant metabolic responses to acute CR have not been described. We generated whole genome RNA transcript profiles in liver, skeletal muscle, colon, and hypothalamus and simultaneously measured plasma metabolites using proton nuclear magnetic resonance in mice subjected to acute CR. Liver and muscle showed increased gene expressions associated with fatty acid metabolism and a reduction in those involved in hepatic lipid biosynthesis. Glucogenic amino acids increased in plasma, and gene expression for hepatic gluconeogenesis was enhanced. Increased expression of genes for hormone-mediated signaling and decreased expression of genes involved in protein binding and development occurred in hypothalamus. Cell proliferation genes were decreased and cellular transport genes increased in colon. Acute CR captured many, but not all, hepatic transcriptional changes of long-term CR. Our findings demonstrate a clear transcriptional response across multiple tissues during acute CR, with congruent plasma metabolite changes. Liver and muscle switched gene expression away from energetically expensive biosynthetic processes toward energy conservation and utilization processes, including fatty acid metabolism and gluconeogenesis. Both muscle and colon switched gene expression away from cellular proliferation. Mice undergoing acute CR rapidly adopt many transcriptional and metabolic changes of long-term CR, suggesting that the beneficial effects of CR may require only a short-term reduction in caloric intake.

scholarly journals Relative expression of mRNAs related to cavitation process in bovine embryos produced in vivo and in vitro

2011 ◽  
Vol 40 (1) ◽  
pp. 124-128
Author(s):  
Sabine Wohlres-Viana ◽  
Mariana Cortes Boite ◽  
João Henrique Moreira Viana ◽  
Marco Antonio Machado ◽  
Luiz Sérgio de Almeida Camargo
Keyword(s):  
Culture System ◽  
Rna Extraction ◽  
Endogenous Control ◽  
Bovine Embryos ◽  
Relative Expression ◽  
Gapdh Gene ◽  
Expression Of Genes ◽  
In Vitro Culture System

The objectives of this work were to identify and to evaluate possible differences on gene expression of aquaporins and Na/K-ATPases transcripts between embryos in vivo and in vitro produced. For each group, 15 blastocysts distributed in three pools were used for RNA extraction followed by amplification and reverse transcription. The resulting cDNAs were submitted to Real-Time PCR, using the GAPDH gene as endogenous control. It was not possible to identify AQP1 transcripts. Relative expression of AQP3 (1.33 ± 0.78) and AQP11 (2.00 ± 1.42) were not different in blastocysts in vitro and in vivo produced. Na/K-ATPase α1 gene (2.25 ± 1.07) was overregulated whereas Na/K-ATPase β2 transcripts 0.40 ± 0.30) did not differ among blastocysts produced in vitro from those produced in vivo. Transcripts for gene AQP1 are not present in bovine blastocysts. In vitro culture system does not alter expression of genes AQP3, AQP11 and Na/K-ATPase β2 genes, however, it affects expression of Na/K-ATPase α1.

Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function

2000 ◽  
Vol 113 (11) ◽  
pp. 1841-1849 ◽  
Author(s):  
T. Misteli
Keyword(s):  
Gene Expression ◽  
Cell Nucleus ◽  
Cell Biology ◽  
Nuclear Architecture ◽  
Molecular Techniques ◽  
Mrna Splicing ◽  
Cellular Organization ◽  
Control Of Gene Expression ◽  
Expression Of Genes

Gene expression is a fundamental cellular process. The basic mechanisms involved in expression of genes have been characterized at the molecular level. A major challenge is now to uncover how transcription, RNA processing and RNA export are organized within the cell nucleus, how these processes are coordinated with each other and how nuclear architecture influences gene expression and regulation. A significant contribution has come from cell biological approaches, which combine molecular techniques with microscopy methods. These studies have revealed that the mammalian cell nucleus is a complex but highly organized organelle, which contains numerous subcompartments. I discuss here how two essential nuclear processes - transcription and pre-mRNA splicing - are spatially organized and coordinated in vivo, and how this organization might contribute to the control of gene expression. The dynamic nature of nuclear proteins and compartments indicates a high degree of plasticity in the cellular organization of nuclear functions. The cellular organization of transcription and splicing suggest that the morphology of nuclear compartments is largely determined by the activities of the nucleus.

scholarly journals Nrg1 Is a Transcriptional Repressor for Glucose Repression of STA1 Gene Expression inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (3) ◽  
pp. 2044-2050 ◽  
Author(s):  
Seok Hee Park ◽  
Sang Seok Koh ◽  
Jae Hwan Chun ◽  
Hye Jin Hwang ◽  
Hyen Sam Kang
Keyword(s):  
Gene Expression ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Transcriptional Repressor ◽  
Dependent Manner ◽  
Expression Of Genes ◽  
Dnase I Footprinting ◽  
Genes Encoding

ABSTRACT Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. TheNRG1 gene encodes a 25-kDa C2H2zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of theNRG1 gene causes a fivefold increase in the level of theSTA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in bothssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.

scholarly journals Defects in Rhizobial Cyclic Glucan and Lipopolysaccharide Synthesis Alter Legume Gene Expression During Nodule Development

2008 ◽  
Vol 21 (1) ◽  
pp. 50-60 ◽  
Author(s):  
Alejandra L. D'Antuono ◽  
Thomas Ott ◽  
Lene Krusell ◽  
Vera Voroshilova ◽  
Rodolfo A. Ugalde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mutant Strain ◽  
Early Recognition ◽  
Lotus Japonicus ◽  
Cdna Array ◽  
Nodule Development ◽  
Marker Genes ◽  
Wild Type ◽  
Mesorhizobium Loti ◽  
Expression Of Genes

cDNA array technology was used to compare transcriptome profiles of Lotus japonicus roots inoculated with a Mesorhizobium loti wild-type and two mutant strains affected in cyclic β(1-2) glucan synthesis (cgs) and in lipopolysaccharide synthesis (lpsβ2). Expression of genes associated with the development of a fully functional nodule was significantly affected in plants inoculated with the cgs mutant. Array results also revealed that induction of marker genes for nodule development was delayed when plants were inoculated with the lpsβ2 mutant. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to quantify gene expression of a subset of genes involved in plant defense response, redox metabolism, or genes that encode for nodulins. The majority of the genes analyzed in this study were more highly expressed in roots inoculated with the wild type compared with those inoculated with the cgs mutant strain. Some of the genes exhibited a transient increase in transcript levels during intermediate steps of normal nodule development while others displayed induced expression during the final steps of nodule development. Ineffective nodules induced by the glucan mutant showed higher expression of phenylalanine ammonia lyase than wild-type nodules. Differences in expression pattern of genes involved in early recognition and signaling were observed in plants inoculated with the M. loti mutant strain affected in the synthesis of cyclic glucan.

scholarly journals Developmental Anatomy Ontology of Zebrafish: an Integrative semantic framework

2007 ◽  
Vol 4 (3) ◽  
pp. 64-76 ◽  
Author(s):  
Mounia Belmamoune ◽  
Fons J. Verbeek
Keyword(s):  
Gene Expression ◽  
Information Systems ◽  
Heterogeneous Databases ◽  
Marker Genes ◽  
Learning Approaches ◽  
Anatomy Ontology ◽  
Developmental Anatomy ◽  
Zebrafish Development ◽  
Semantic Framework ◽  
Expression Of Genes

Summary Integration of information is quintessential to make use of the wealth of bioinformatics resources. One aspect of integration is to make databases interoperable through well annotated information. With new databases one strives to store complementary information and such results in collections of heterogeneous information systems. Concepts in these databases need to be connected and ontologies typically provide a common terminology to share information among different resources.Our focus of research is the zebrafish and we have developed several information systems in which ontologies are crucial. Pivot is an ontology describing the developmental anatomy, referred to as the Developmental Anatomy Ontolgoy of Zebrafish (DAOZ). The anatomical and temporal concepts are provided by the Zebrafish Information Network (ZFIN) and proven within the research community. We have constructed a 3D digital atlas of zebrafish development based on histology; the atlas is series of volumetric models; in each instance, every volume element is assigned to an anatomical term. Complementing the atlas we developed an information system with 3D patterns of gene expression in zebrafish development based on marker genes. The spatial and temporal annotations to these 3D images are drawn from the ontology that we have designed. In its design the DAOZ ontology is structured as a Directed Acyclic Graph (DAG). Such is required to find unique concept paths and prevent self referencing.As we need to address the ontology in a direct manner, the DAG structure is transferred to a database. The database is used in the integration of our databases that share concepts at different levels of aggregation. In order to make sure that sufficient levels of aggregation for applications in mind are present, the original vocabulary was enriched with more relations and concepts. Both databases can now be addressed with the same unique terms and co-occurrence and co-expression of genes can be readily extracted from the databases. Integration can be further extended to the ZFIN resource and also by including ontologies that relate to gene/gene expression (e.g. Gene Ontology). In this manner, interoperable information retrieval from heterogeneous databases can be realized. This greatly facilitates processing complex information and retrieving relations in the data through machine learning approaches.

scholarly journals Effect of Ambient pH on Growth, Pathogenicity, and Patulin Production of Penicillium expansum

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 550
Author(s):  
Carelle Kouasseu Jimdjio ◽  
Huali Xue ◽  
Yang Bi ◽  
Mina Nan ◽  
Lan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Soft Rot ◽  
Penicillium Expansum ◽  
Expression Of Genes ◽  
Transmission Electron ◽  
Expression Studies ◽  
Gene Expression Studies

Penicillium expansum is an important postharvest pathogen of pomaceous fruit and a causal agent of blue mold or soft rot. In this study, we investigated the effect of ambient pH on growth, ultrastructure alteration, and pathogenicity of P. expansum, as well as accumulation of patulin and expression of genes involved in patulin biosynthesis. Under different pH, the fungus was routinely cultured and collected for growth, pathogenicity, patulin production, and gene expression studies using transmission electron microscopy, apple inoculation, HPLC, and RT-qPCR methods. Different ambient pH had significant impact on expression of genes and growth factors involved in patulin biosynthesis. Under same range of pH, gene expression profile, growth factors, and patulin accumulation (in vivo and in vitro) all showed similar changing trends. A well-developed cell was observed in addition to upregulation of genes at pH between pH 5.0 and 7.0, while the opposite was observed when pH was too basic (8.5) or too acid (2.5). Additionally, ambient pH had direct or indirect influence on expression of PecreaA, PelaeA, and PepacC. These findings will help in understanding the effect of ambient pH on growth, pathogenicity, and patulin production and support the development of successful methods for combating P. expansum infection on apple fruits.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5223-5232 ◽  
Author(s):  
Naoshi Obara ◽  
Norio Suzuki ◽  
Kibom Kim ◽  
Toshiro Nagasawa ◽  
Shigehiko Imagawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Marker Genes ◽  
Central Vein ◽  
Specific Expression ◽  
Neuronal Marker ◽  
Nucleotide Mutation ◽  
Gata Box

Abstract In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type–specific expression of the Epo gene.

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