Validation of Melampsora larici-populina reference genes for in planta RT-quantitative PCR expression profiling during time-course infection of poplar leaves

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.

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

10.1101/2020.08.12.247601 ◽

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.

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Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2006.10.007 ◽

2007 ◽

Vol 115 (1-2) ◽

pp. 43-55 ◽

Cited By ~ 10

Author(s):

Deborah L. Taylor ◽

Peter C. Thomson ◽

Kumudika de Silva ◽

Richard J. Whittington

Keyword(s):

Quantitative Pcr ◽

Mycobacterium Avium ◽

Expression Profiling ◽

Reference Genes ◽

Mycobacterium Avium Subsp ◽

Raw264.7 Cells ◽

Endogenous Reference ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Endogenous Reference Genes

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Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Applied and Environmental Microbiology ◽

10.1128/aem.00402-15 ◽

2015 ◽

Vol 81 (12) ◽

pp. 4120-4129 ◽

Cited By ~ 16

Author(s):

Raúl Castanera ◽

Leticia López-Varas ◽

Antonio G. Pisabarro ◽

Lucía Ramírez

Keyword(s):

Quantitative Pcr ◽

Reverse Transcription ◽

Pleurotus Ostreatus ◽

Reference Genes ◽

Time Course ◽

Extracellular Enzymes ◽

Expression Patterns ◽

Model Organism ◽

Culture Conditions ◽

Content Type

ABSTRACTRecently, the lignin-degrading basidiomycetePleurotus ostreatushas become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes inP. ostreatustime course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10andmnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.

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Selection of stable reference genes for real-time quantitative PCR in honey bee pesticide toxicity studies

Journal of Apicultural Research ◽

10.1080/00218839.2021.1950972 ◽

2021 ◽

pp. 1-11

Author(s):

Sanghyeon Kim ◽

Susie Cho ◽

Si Hyeock Lee

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Honey Bee ◽

Reference Genes ◽

Pesticide Toxicity ◽

Real Time Quantitative Pcr ◽

Toxicity Studies ◽

Selection Of

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Assessment of Xanthomonas arboricola pv. juglandis Bacterial Load in Infected Walnut Fruits by Quantitative PCR

Plant Disease ◽

10.1094/pdis-12-18-2253-re ◽

2019 ◽

Vol 103 (10) ◽

pp. 2577-2586

Author(s):

Leonor Martins ◽

Camila Fernandes ◽

Pedro Albuquerque ◽

Fernando Tavares

Keyword(s):

Quantitative Pcr ◽

Juglans Regia ◽

Bacterial Load ◽

Etiologic Agent ◽

Economic Losses ◽

Rapid Diagnostics ◽

In Planta ◽

Chromosomal Dna ◽

Fruit Samples ◽

Xanthomonas Arboricola

Xanthomonas arboricola pv. juglandis is the etiologic agent of important walnut (Juglans regia L.) diseases, causing severe fruit drop and high economic losses in walnut production regions. Rapid diagnostics and knowledge of bacterial virulence fitness are key to hinder disease progression and apply timely phytosanitary measures. This work describes an X. arboricola pv. juglandis-specific real-time quantitative PCR (qPCR) using X. arboricola pv. juglandis-specific DNA markers to quantify the bacterial load in infected walnut plant tissues. Method validation was achieved using calibration curves obtained with serial dilutions of X. arboricola pv. juglandis chromosomal DNA and standard curves obtained from walnut samples spiked with X. arboricola pv. juglandis cells. High correlations (R2 > 0.990 and > 0.995) and low limits of detection (35 chromosomes/qPCR reaction and 2.7 CFU/qPCR reaction) were obtained for both markers considering the calibration and standard curves, respectively. Assessment of qPCR repeatability, reproducibility, and specificity allowed us to demonstrate the reliability and consistency of the method. Furthermore, in planta quantification of X. arboricola pv. juglandis bacterial load using infected walnut fruit samples showed a higher detection resolution compared with standard PCR detection. By allowing quantification of virulence fitness of distinct X. arboricola pv. juglandis strains in planta, the proposed qPCR method may contribute to assertive risk assessment of walnut diseases caused by X. arboricola pv. juglandis and ultimately help to improve phytosanitary practices.

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