Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

McMillan, Mary; Pereg, Lily

doi:10.1371/journal.pone.0098162

Cited by 55 publications

(35 citation statements)

References 33 publications

(57 reference statements)

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“…Strategies employed for bacterial transcript quantification through qPCR are currently based on relative (23)(24)(25)(26) or absolute (27)(28)(29) quantification approaches. Phytoplasmas live their lives inside very different environments: the plant and the insect vector.…”

mentioning

confidence: 99%

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

Pacifico

Galetto

Rashidi

et al. 2015

Appl Environ Microbiol

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To highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of "Candidatus Phytoplasma asteris," chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatus and Euscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection of A. thaliana and M. quadripunctulatus but were more constant in E. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant. Phytoplasmas are wall-less plant-pathogenic bacteria, classified as "Candidatus Phytoplasma" spp. (1). They belong to the class Mollicutes and infect a wide variety of plants, causing heavy crop losses in many different countries (2). Phytoplasmas are phloem limited in the infected plant, and they cause severe symptoms (yellowing, dwarfism, and phyllody), often leading to plant death. They are transmitted by phloem-feeding Hemipteran vectors (leafhoppers, planthoppers, and psyllids) in a persistent propagative manner (3).Phytoplasmas are obligate parasites that depend on host cells for the uptake of essential compounds such as sugars, amino acids, ions, and nucleotide precursors (4). Consistent with this lifestyle, phytoplasmas have very small, A/T-rich genomes, ranging from 530 to 1,350 kb in size (5), that lack essential metabolic pathways, such as ATP synthesis. This genome condensation reflects the phytoplasma adaptation to nutrient-rich environments such as the plant phloem (6) and helps explain why these pathogens are not cultivable under axenic conditions (7).Although the pathogenicity mechanisms are still largely unclear, phytoplasmas influence plant metabolism both directly, through a set of membrane proteins acting as molecular carriers (6), and indirectly, through secretion of effector proteins (8,9). In vitro studies have also shown that phytoplasma immunodominant membrane proteins interact with vector proteins (10, 11) and plant proteins (12) and are subjected to strong positive selection (13-15). Moreover, phytoplasmas can modulate their genome express...

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mentioning

confidence: 99%

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

Pacifico

Galetto

Rashidi

et al. 2015

Appl Environ Microbiol

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“…A list of primers used in this study is given in Table 2 . The most stable reference genes ( GyrA and GlyA ) were selected, following a screen of 10 potential reference genes [26] . Total RNA was extracted from cell samples using a TRIzol Max Bacterial Isolation kit (Invitrogen, USA).…”

Section: Methodsmentioning

confidence: 99%

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

et al. 2014

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FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

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“…The wild-type strain EIB202 was cultivated in DMEM without shaking. Following centrifugation, the supernatant was used for the extracellular protein extraction by ultrafiltration after 6,8,10,12,14,16, and 22 h of culture. The samples were then analyzed by western blot assay using antibodies against FBA and GAPDH.…”

Section: Example Of Selecting Suitable Reference Genes For Different mentioning

confidence: 99%

“…Based on previous studies [13][14][15][16], seven extensively studied candidate reference genes (recA, rpoB, rho, topA, gyrA, rpoD, and 16S rRNA) were chosen, along with two additional genes of interest (uup, groEL), making a total of nine genes. The glycolytic enzyme genes gapA and fbaA were used as the target genes.…”

Section: Introductionmentioning

confidence: 99%

Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

Sun

Deng

et al. 2017

Journal of Microbiology and Biotechnology

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is a gram-negative pathogenic bacterium in aquaculture that can cause hemorrhagic septicemia in fish. Many secreted proteins have already been identified as virulent factors of . Moreover, since virulent phenotypes are based on the expression regulation of virulent genes, understanding the expression profile of virulent genes is important. A quantitative RT-PCR is one of the preferred methods for determining different gene expressions. However, this requires the selection of a stable reference gene in, which has not yet been systematically studied. Accordingly, this study evaluated nine candidate reference genes (, ,, ,, ,, , and) using the Excel-based programs BestKeeper, GeNorm, and NormFinder under different culture conditions. The results showed that was more stable than the other genes at different culture growth phases. However, at the same culture time, was identified as the reference gene under the conditions of different strains, different culture media, and infection, whereas was identified under the condition of different temperatures. Thus, in experiments, the expression of and in was analyzed by RT-qPCR using ,, and as the reference genes. The results showed that was the most suitable reference gene in this analysis, and that using unsuitable reference genes resulted in inaccurate results.

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Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

Cited by 55 publications

References 33 publications

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

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