Identification of suitable reference genes for real-time RT-PCR normalization in the grapevine-downy mildew pathosystem.

Due to its reproducibility and sensitivity, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become the method of choice for quantifying gene expression. However, the accuracy of RT-qPCR is prone to bias if proper precautions are not taken, e.g. starting with intact, non-degraded RNA, considering the PCR efficiency and using the right reference gene(s) for normalization. It has been reported that some of the well-known reference genes are differentially regulated under certain experimental conditions suggesting that there is no gene that could be used as a universal reference. This paper aims at selecting the most suitable reference gene(s) out of six putative genes to be used as normalizer(s) for quantification of gene expression in the grapevine-downy mildew interaction as well as upon induced resistance with chemical elicitors. Moreover, the paper aims at determining the optimal number of reference genes to be used in normalization, since it has been emphasized in the literature that using multiple reference genes increases accuracy. Two different software tools, geNorm and Normfinder, were used to identify the most stable reference genes in grapevine under the aforementioned conditions. The importance of the choice of adequate reference genes is highlighted by studying chitinase expression.

Methodical approaches to determine the rate of radial muscle displacement using tensiomyography: A scoping review and new reporting guideline.

Tensiomyography is a non-invasive method to assess skeletal muscle contractile properties from the stimulated radial displacement. Many studies have used the rate of displacement (Vc) as an indirect measure of muscle contraction velocity. However, no standardised methodical approach exists to measure displacement and determine Vc. This review aimed to provide an overview of concepts to determine Vc and measurement protocols to foster the development of a standardised methodical approach. This review followed the Preferred Reporting Items for Systematic Reviews and meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guideline. Systematic searches were performed within five electronic databases and additional sources. The included 62 studies reported 10 different concepts to determine Vc, which we summarised in three groups. The determination concepts differed mainly regarding time intervals during the contraction phase considered and criteria used to define these intervals. Essential information on the equipment and raters, measurement setup, electrical stimulation procedure, and data analysis were frequently not reported. In conclusion, no consensus on how to determine Vc existed. Incomplete reporting of measurement protocols hindered study comparison, which obstructs developing a standardised approach. Therefore, we propose a new guideline for reporting measurement protocols, which covers the 1) equipment and rater, 2) measurement setup, including positioning of the subject, sensor and electrodes, 3) electrical stimulation, including initial stimulation amplitude, increment, and endpoint, and 4) data analysis, including selection criteria and number of analysed signals and a definition of derived parameters.

Expression analysis of genes induced in barley after chemical activation reveals distinct disease resistance pathways.

Abstract Salicylic acid (SA) and its synthetic mimics 2,6-dichloroisonicotinic acid (DCINA) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), protect barley systemically against powdery mildew (Blumeria graminis f.sp. hordei, Bgh) infection by strengthening plant defence mechanisms that result in effective papillae and host cell death. Here, we describe the differential expression of a number of newly identified barley chemically induced (BCI) genes encoding a lipoxygenase (BCI-1), a thionin (BCI-2), an acid phosphatase (BCI-3), a Ca(2+)-binding EF-hand protein (BCI-4), a serine proteinase inhibitor (BCI-7), a fatty acid desaturase (BCI-8) and several further proteins with as yet unknown function. Compared with SA, the chemicals DCINA and BTH were more potent inducers of both gene expression and resistance. Homologues of four BCI genes were detected in wheat and were also differentially regulated upon chemical activation of disease resistance. Except for BCI-4 and BCI-5 (unknown function), the genes were also induced by exogenous application of jasmonates, whereas treatments that raise endogenous jasmonates as well as wounding were less effective. The fact that BCI genes were not expressed during incompatible barley-Bgh interactions governed by gene-for-gene relationships suggests the presence of separate pathways leading to powdery mildew resistance.

Absence of the outer membrane phospholipase A suppresses the temperature-sensitive phenotype of Escherichia coli degP mutants and induces the Cpx and sigma(E) extracytoplasmic stress responses.

DegP is a periplasmic protease that is a member of both the sigma(E) and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42 degrees C. [U-(14)C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, beta-lactamase, and beta-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42 degrees C, displayed increased viability at 44 degrees C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and sigma(E) regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and sigma(E) regulons rather than by inactivating the phospholipase per se.