Identification of stable reference gene for transcript normalization in black pepper-Phytophthora capsici pathosystem

Umadevi, P.; Suraby, Erinjery Jose; Anandaraj, M.; Nepolean, T.

doi:10.1007/s12298-019-00653-9

Cited by 10 publications

(6 citation statements)

References 21 publications

(20 reference statements)

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“…However, an overestimated relative expression level in the HK genes transcription was obtained from qRT-PCR using PGK in normalization (Fig 4). The relative expression level of target gene is overestimated due to use unstable gene normalization is also found in other studies [45,46]. These results indicated that using unstable RG(s) for qRT-PCR normalization will lead to inconsistent results.…”

Section: Plos Onesupporting

confidence: 54%

Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris

Liu

et al. 2020

PLoS ONE

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The development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris. Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, respectively. Moreover, target genes, two-componentsystem histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as RGs leads to biased results. Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research.

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Section: Plos Onesupporting

confidence: 54%

Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris

Liu

et al. 2020

PLoS ONE

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“…4). The relative expression level of target gene is overestimated due to use unstable gene normalization is also found in other studies [39,40]. These results indicated that failure to statistically validate RG(s) will lead to inconsistent results.…”

Section: Discussionmentioning

confidence: 55%

“…RT-qPCR experiments were performed using a previously described method [42]. Briefly, all genes were amplified by initial heating at 95 °C for 10 min, followed by 40 [43] and ubiquitin-conjugating enzyme (UBC) [44]; and (3) novel stable reference genes in cold stress such as phosphoglycerate kinase (PGK) and serine/threonine protein phosphatase 2A (PP2A). Details of these genes are provided in Table 1.…”

Section: Real-time Quantitative Polymerase Chain Reaction Conditionsmentioning

confidence: 99%

Analysis of Reference Genes stability and Histidine Kinase Expression under Cold Stress in Cordyceps militaris

Liu

et al. 2020

Preprint

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Background: The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris.Results: Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris.Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long time, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable housekeeping genes for cold treatments in short and long time, respectively.Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as housekeeping genes leads to biased results. Conclusions:Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research. BackgroundCordyceps militaris, a famous traditional Chinese medicine, has been used as a healthy food for a long time in China. The C. militaris fruiting body has anti-inflammatory [1], anti-tumor [2], antiinfluenza virus [3] and radio-protection [4] functions. Methods for commercial production of fruiting bodies of this fungus have been established in artificial media [5,6] or with insects, such as silkworm Bombyx mori pupae [7]. The molecular mechanism of mushroom fruiting body formation is a basic biological problem. The essential role of light in fruit body development in C. militaris was demonstrated in previous study [8]. A blue-light receptor gene white collar-1 (wc-1) inactivation

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“…Piper nigrum , commonly known as black pepper is an important non-model spice crop that is susceptible to a highly destructive disease called quick-wilt or foot rot caused by the pathogen, Phytophthora capsici 39 , 40 . The suitability of different RGs in conducting expression studies in the host plant ( P. nigrum ) in response to P. capsici infection has been reported earlier 37 . However, to gain a comprehensive understanding of the P. nigrum – P. capsici phytopathosystem, it is crucial to uncover the molecular undulations of gene expressions associated with the infection steps employed by the pathogen to invade the host plant.…”

Section: Discussionmentioning

confidence: 76%

“…It is widely acknowledged that when determining the relative gene expression of a target gene, it is essential to employ multiple reference genes that exhibit stable expression under the specific biological conditions of interest in order to normalize the data 20 . However, there is a noticeable scarcity of studies focusing on this aspect, particularly in the field of crop plants 37 and phytopathogens 38 . Piper nigrum , commonly known as black pepper is an important non-model spice crop that is susceptible to a highly destructive disease called quick-wilt or foot rot caused by the pathogen, Phytophthora capsici 39 , 40 .…”

Section: Discussionmentioning

confidence: 99%

Validation of reference gene stability for normalization of RT-qPCR in Phytophthora capsici Leonian during its interaction with Piper nigrum L.

Vijayakumar,

Sakuntala

2024

Sci Rep

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The selection of stable reference genes for the normalization of reverse transcription quantitative real-time PCR (RT-qPCR) is generally overlooked despite being the crucial element in determining the accuracy of the relative expression of genes. In the present study, the stability of seven candidate reference genes: actin (act), α-tubulin (atub), β-tubulin (btub), translation elongation factor 1-α (ef1), elongation factor 2 (ef2), ubiquitin-conjugating enzyme (ubc) and 40S ribosomal protein S3A (ws21) in Phytophthora capsici has been validated. The validation was performed at six infection time points during its interaction with its susceptible host Piper nigrum, two developmental stages, and for the combined dataset. Four algorithms: geNorm, NormFinder, BestKeeper, and the ΔCt method were compared, and a comprehensive ranking order was produced using RefFinder. The overall analysis revealed that ef1, ws21, and ubc were identified as the three most stable genes in the combined dataset, ef1, ws21, and act were the most stable at the infection stages, and, ef1, btub, and ubc were most stable during the developmental stages. These findings were further corroborated by validating the P. capsici pathogenesis gene NPP1 expression. The findings are significant as this is the first study addressing the stability of reference genes for P. capsici–P. nigrum interaction studies.

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Identification of stable reference gene for transcript normalization in black pepper-Phytophthora capsici pathosystem

Cited by 10 publications

References 21 publications

Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris

Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris

Analysis of Reference Genes stability and Histidine Kinase Expression under Cold Stress in Cordyceps militaris

Validation of reference gene stability for normalization of RT-qPCR in Phytophthora capsici Leonian during its interaction with Piper nigrum L.

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