scholarly journals Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

2020 ◽  
Author(s):  
Liz M. Florez ◽  
Reiny W. A. Scheper ◽  
Brent M. Fisher ◽  
Paul W. Sutherland ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Time Course ◽  
Gene Expression Analysis ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Post Inoculation ◽  
In Planta ◽  
Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

scholarly journals Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0238157
Author(s):  
Liz M. Florez ◽  
Reiny W. A. Scheper ◽  
Brent M. Fisher ◽  
Paul W. Sutherland ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Time Course ◽  
Gene Expression Analysis ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Post Inoculation ◽  
In Planta ◽  
Regulation Of Expression

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

scholarly journals Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0226168 ◽  
Author(s):  
Li Li ◽  
Kangyu Wang ◽  
Mingzhu Zhao ◽  
Shaokun Li ◽  
Yue Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Hairy Roots ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Qrt Pcr

scholarly journals Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0160637 ◽  
Author(s):  
Sarena Che Omar ◽  
Michael A. Bentley ◽  
Giulia Morieri ◽  
Gail M. Preston ◽  
Sarah J. Gurr
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Magnaporthe Oryzae ◽  
Reference Genes ◽  
Rice Blast ◽  
Gene Expression Analysis ◽  
Rice Blast Fungus ◽  
Qrt Pcr ◽  
Blast Fungus

scholarly journals Validation of reference genes for expression analysis in a murine trauma model combining traumatic brain injury and femoral fracture

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ellen Otto ◽  
Paul Köhli ◽  
Jessika Appelt ◽  
Stefanie Menzel ◽  
Melanie Fuchs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Reference Gene ◽  
Expression Analysis ◽  
Bone Fracture ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Tissue Specific ◽  
Qrt Pcr

Abstract Systemic and local posttraumatic responses are often monitored on mRNA expression level using quantitative real-time PCR (qRT-PCR), which requires normalisation to adjust for confounding sources of variability. Normalisation requests reference (housekeeping) genes stable throughout time and divergent experimental conditions in the tissue of interest, which are crucial for a reliable and reproducible gene expression analysis. Although previous animal studies analysed reference genes following isolated trauma, this multiple-trauma gene expression analysis provides a notable study analysing reference genes in primarily affected (i.e. bone/fracture callus and hypothalamus) and secondarily affected organs (i.e. white adipose tissue, liver, muscle and spleen), following experimental long bone fracture and traumatic brain injury. We considered tissue-specific and commonly used top-ranked reference candidates from different functional groups that were evaluated applying the established expression stability analysis tools NormFinder, GeNorm, BestKeeper and RefFinder. In conclusion, reference gene expression in primary organs is highly time point as well as tissue-specific, and therefore requires careful evaluation for qRT-PCR analysis. Furthermore, the general application of Ppia, particularly in combination with a second reference gene, is strongly recommended for the analysis of systemic effects in the case of indirect trauma affecting secondary organs through local and systemic pathophysiological responses.

scholarly journals Expression analysis of defense-related genes in cucumber (Cucumis sativus L.) against Phytophthora melonis

2020 ◽  
Author(s):  
Lida Hashemi ◽  
Ahmad Reza Golparvar ◽  
Mehdi Nasr Esfahani ◽  
Maryam Golabadi
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Molecular Mechanisms ◽  
Crown Rot ◽  
Economic Losses ◽  
Post Inoculation ◽  
Expression Ratio ◽  
Phytophthora Melonis ◽  
Maximum Expression

AbstractPhytophthora melonis is the causal agent of damping-off or crown rot, one of the most destructive cucumber diseases that causes severe economic losses in Iran and some other parts of the world. Despite intense research efforts made in the past years, no permanent cure currently exists for this disease. With the aim to understand the molecular mechanisms of defense against P. melonis, root collars and leaves of four cucumber genotypes consisting of resistant Ramezz; moderately resistant Baby and very susceptible Mini 6-23 and Extrem, were monitored for quantitative gene expression analysis of five antifungal and/or anti-oomycete genes (CsWRKY20, CsLecRK6.1, PR3, PR1-1a and LOX1) at three points after inoculation with P. melonis. The gene expression analysis indicated that P. melonis strongly enhanced the expression of these genes after inoculation in both leaves and root collars. Further, not only the transcript levels of these genes were significantly higher in the resistant and moderately resistance genotypes, but also the time point of the highest relative expression ratio for the five genes was different in the four cucumber genotypes. CsWRKY20 and PR3 showed the maximum expression in Ramezz at 48 hours post inoculation (hpi) while CsLecRK6.1, and LOX1 showed the highest expression at 72 hpi. In addition, PR1-1a showed the maximum expression in the Baby at 72 hpi. Root collars responded faster than leaves and some responses were more strongly up-regulated in root collars than in leaves. The genes found to be involved in disease resistance in two different organs of cucumber after pathogen infection. The results suggest that increased expression of these genes led to activation of defense pathways and could be responsible for a reduced P. melonis colonization capacity in Ramezz and Baby. Overall, this work represents a valuable resource for future functional genomics studies to unravel the molecular mechanisms of C. sativus- P. melonis interaction.

scholarly journals Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2331 ◽  
Author(s):  
Qianqian Zhang ◽  
Wei Liu ◽  
Yingli Cai ◽  
A-Feng Lan ◽  
Yinbing Bian
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Expression Analysis ◽  
Internal Control ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Stable Gene ◽  
Experimental Conditions ◽  
The Stability ◽  
Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

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