Plant Molecular Biology Reporter

, Volume 31, Issue 4, pp 936–945 | Cite as

Identification of Reference Genes for Normalizing RNA Expression in Potato Roots Infected with Cyst Nematodes

  • Patricio Castro-Quezada
  • Jawad Aarrouf
  • Michel Claverie
  • Bruno Favery
  • Didier Mugniéry
  • Véronique Lefebvre
  • Bernard Caromel
Original Paper
First Online:

Abstract

Potato cyst nematodes induce changes in plant host gene expression following root invasion. For an accurate comparison of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR), internal reference genes are necessary for transcript normalization. Very few experimental data on suitable reference genes are available for interactions between plant and root pathogens. In this study, we tested eight potential candidate reference genes for normalizing levels of potato gene transcripts by RT-qPCR after inoculation by nematodes. The ranking of candidate reference genes was performed using RefFinder WEB-based software. Four reference genes, RPN7 (26S proteasome regulatory subunit), UBP22 (ubiquitin-specific protease 22), OXA1 (OXA1 protein), and MST2 (mercaptopyruvate sulfurtransferase), were stably expressed in roots of susceptible or resistant potato plants, infected or uninfected by Globodera pallida. A normalization factor based on data from these four genes, highly homologous between potato and tomato, was used to normalize the expression of a chitinase gene, which was induced by nematodes in roots of potatoes carrying the resistance allele at a low-effect QTL, GpaXI spl .

Keywords

RT-qPCR Transcript normalization Compatible–incompatible interactions Globodera pallida Solanum Chitinase 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We warmly thank Dr MC Kerlan, who provided plant material, C. Garchery for advice on experimental procedure and Rebecca Stevens for critical reading of the manuscript. We also thank the anonymous reviewers for helpful suggestions to improve the manuscript. This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the European project APOPHYS (QLRT-2001-01849). PCQ was funded by a PhD grant of the Secretaria Nacional de Ciencia y Tecnología (SENESCYT) of Ecuador.

Supplementary material

11105_2013_566_MOESM1_ESM.doc (92 kb)
ESM 1 (DOC 92 kb)
11105_2013_566_MOESM2_ESM.doc (962 kb)
ESM 2 (DOC 962 kb)

References

  1. Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  2. Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250PubMedCrossRefGoogle Scholar
  3. Bustin SA, Nolan T (2004) Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomolecular Tech 15:155–166Google Scholar
  4. Caromel B, Mugniéry D, Kerlan MC, Andrzejewski S, Palloix A, Ellissèche D, Rousselle-Bourgeois F, Lefebvre V (2005) Resistance quantitative trait loci originating from Solanum sparsipilum act independently on the sex ratio of Globodera pallida and together for developing a necrotic reaction. Mol Plant-Microbe Interact 18:1186–1194PubMedCrossRefGoogle Scholar
  5. Chao WS, Dogramaci M, Foley ME, Horvath DP, Anderson JV (2012) Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula). PLoS One 7:e42839PubMedCrossRefGoogle Scholar
  6. Escobar C, Brown S, Mitchum M (2011) Transcriptomic and proteomic analysis of the plant response to nematode infection. In: Jones J, Gheysen G, Fenoll C (eds) Genomics and molecular genetics of plant–nematode interactions. Springer, Netherlands, pp 157–173CrossRefGoogle Scholar
  7. Goulao LF, Fortunato AS, Ramalho JC (2012) Selection of reference genes for normalizing quantitative real-time PCR gene expression data with multiple variables in Coffea spp. Plant Mol Biol Rep 30:741–759CrossRefGoogle Scholar
  8. Grover A (2012) Plant chitinases: genetic diversity and physiological roles. Crit RevPlant Sci 31:57–73CrossRefGoogle Scholar
  9. Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60:487–493PubMedCrossRefGoogle Scholar
  10. Guo JL, Xu LP, Fang JP, Su YC, Fu HY, Que YX, Xu JS (2012) A novel dirigent protein gene with highly stem-specific expression from sugarcane, response to drought, salt and oxidative stresses. Plant Cell Rep 31:1801–1812CrossRefGoogle Scholar
  11. Gupta N, Gupta AK, Singh NK, Kumar A (2011) Differential expression of PBF Dof transcription factor in different tissues of three finger millet genotypes differing in seed protein content and color. Plant Mol Biol Rep 29:69–76CrossRefGoogle Scholar
  12. Gutierrez L, Mauriat M, Pelloux J, Bellini C, van Wuytswinkel O (2008) Towards a systematic validation of references in real-time RT-PCR. Plant Cell 20:1734–1735PubMedCrossRefGoogle Scholar
  13. Hofmann J, Grundler FMW (2007) Identification of reference genes for qRT-PCR studies of gene expression in giant cells and syncytia induced in Arabidopsis thaliana by Meloidogyne incognita and Heterodera schachtii. Nematology 9:317–323CrossRefGoogle Scholar
  14. Hruz T, Wyss M, Docquier M, Pfaffl MW, Masanetz S, Borghi L, Verbrugghe P, Kalaydjieva L, Bleuler S, Laule O, Descombes P, Gruissem W, Zimmermann P (2011) RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics 12:156PubMedCrossRefGoogle Scholar
  15. Inigo S, Giraldez AN, Chory J, Cerdan PD (2012) Proteasome-mediated turnover of Arabidopsis MED25 Is coupled to the activation of FLOWERING LOCUS T transcription. Plant Physiol 160:1662–1673PubMedCrossRefGoogle Scholar
  16. Jammes F, Lecomte P, Almeida-Engler J, Bitton F, Martin-Magniette ML, Renou JP, Abad P, Favery B (2005) Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsis. Plant J 44:447–458PubMedCrossRefGoogle Scholar
  17. Jolivet K, Grenier E, Bouchet JP, Esquibet M, Kerlan MC, Caromel B, Mugniéry D, Lefebvre V (2007) Identification of plant genes regulated in resistant potato Solanum sparsipilum during the early stages of infection by Globodera pallida. Genome 50:422–427PubMedCrossRefGoogle Scholar
  18. Mugniéry D, Person F (1976) Methode d'élevage de quelques espèces de nématodes à kyste du genre Heterodera. Sciences agronomiques Rennes 217-220Google Scholar
  19. Mugniéry D, Fouville D, Dantec JP, Pellé R, Rousselle-Bourgeois F, Ellissèche D (2001) Résistance à Globodera pallida Pa2/3 chez Solanum sparsipilum. Nematology 3:619–626CrossRefGoogle Scholar
  20. Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914PubMedCrossRefGoogle Scholar
  21. Ovesna J, Kucera L, Vaculova K, Strymplova K, Svobodova I, Milella L (2012) Validation of the beta-amy1 transcription profiling assay and selection of reference genes suited for a RT-qPCR assay in developing barley caryopsis. PLoS One 7:e41886PubMedCrossRefGoogle Scholar
  22. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST (c)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36PubMedCrossRefGoogle Scholar
  23. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotech Let 26:509–515CrossRefGoogle Scholar
  24. Qi JN, Yu SC, Zhang FL, Shen XQ, Zhao XY, Yu YJ, Zhang DS (2010) Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in chinese cabbage (Brassica rapa L. ssp pekinensis). Plant Mol Biol Rep 28:597–604CrossRefGoogle Scholar
  25. Rahimi S, Wright DJ, Perry RN (1998) Identification and localisation of chitinases induced in the roots of potato plants infected with the potato cyst nematode Globodera pallida. Fundam Applied Nematol 21:705–713Google Scholar
  26. Rivera-Vega L, Mamidala P, Koch JL, Mason ME, Mittapalli O (2012) Evaluation of reference genes for expression studies in ash (Fraxinus spp.). Plant Mol Biol Rep 30:242–245CrossRefGoogle Scholar
  27. Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:33PubMedCrossRefGoogle Scholar
  28. Stolf-Moreira R, Lemos EGM, Carareto-Alves L, Marcondes J, Pereira SS, Rolla AAP, Pereira RM, Neumaier N, Binneck E, Abdelnoor RV, de Oliveira MCN, Marcelino FC, Farias JRB, Nepomuceno AL (2011) Transcriptional profiles of roots of different soybean genotypes subjected to drought stress. Plant Mol Biol Rep 29:19–34CrossRefGoogle Scholar
  29. Vaghchhipawala Z, Bassuner R, Clayton K, Lewers K, Shoemaker R, Mackenzie S (2001) Modulations in gene expression and mapping of genes associated with cyst nematode infection of soybean. Mol Plant-Microbe Interact 14:42–54PubMedCrossRefGoogle Scholar
  30. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034Google Scholar
  31. Vuorinen AL, Gammelgard E, Auvinen P, Somervuo P, Dere S, Valkonen JPT (2010) Factors underpinning the responsiveness and higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes. Ann Appl Biol 157:229–241CrossRefGoogle Scholar
  32. Xie FL, Xiao P, Chen D, Xu L, Zhang BH (2012) miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80:75–84CrossRefGoogle Scholar
  33. Xu X, Pan SK, Cheng SF, Zhang B, Mu DS, Ni PX, Zhang GY, Yang S, Li RQ, Wang J, Orjeda G, Guzman F, Torres M, Lozano R, Ponce O, Martinez D, De la Cruz G, Chakrabarti SK, Patil VU, Skryabin KG, Kuznetsov BB, Ravin NV, Kolganova TV, Beletsky AV, Mardanov AV, Di Genova A, Bolser DM, Martin DMA, Li GC, Yang Y, Kuang HH, Hu Q, Xiong XY, Bishop GJ, Sagredo B, Mejia N, Zagorski W, Gromadka R, Gawor J, Szczesny P, Huang SW, Zhang ZH, Liang CB, He J, Li Y, He Y, Xu JF, Zhang YJ, Xie BY, Du YC, Qu DY, Bonierbale M, Ghislain M, Herrera MD, Giuliano G, Pietrella M, Perrotta G, Facella P, O'Brien K, Feingold SE, Barreiro LE, Massa GA, Diambra L, Whitty BR, Vaillancourt B, Lin HN, Massa A, Geoffroy M, Lundback S, DellaPenna D, Buell CR, Sharma SK, Marshall DF, Waugh R, Bryan GJ, Destefanis M, Nagy I, Milbourne D, Thomson SJ, Fiers M, Jacobs JME, Nielsen KL, Sonderkaer M, Iovene M, Torres GA, Jiang JM, Veilleux RE, Bachem CWB, de Boer J, Borm T, Kloosterman B, van Eck H, Datema E, Hekkert BTL, Goverse A, van Ham R, Visser RGF (2011) Genome sequence and analysis of the tuber crop potato. Nature 475:189–U194PubMedCrossRefGoogle Scholar
  34. Yang G, Zhou RC, Tang T, Chen XS, Ouyang JH, He L, Li WJ, Chen SF, Guo MM, Li XN, Zhong CR, Shi SH (2011) Gene expression profiles in response to salt stress in Hibiscus tiliaceus. Plant Mol Biol Rep 29:609–617CrossRefGoogle Scholar
  35. Zhu XY, Li XP, Chen WX, Chen JY, Lu WJ, Chen L, Fu DW (2012) Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions. PLoS One 7:e44405PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Patricio Castro-Quezada
    • 1
  • Jawad Aarrouf
    • 2
  • Michel Claverie
    • 1
  • Bruno Favery
    • 3
  • Didier Mugniéry
    • 4
  • Véronique Lefebvre
    • 1
  • Bernard Caromel
    • 1
  1. 1.INRA, UR1052 GAFL Génétique et Amélioration des Fruits et LégumesMontfavet CedexFrance
  2. 2.UAPV, Laboratoire de Physiologie des Fruits et des Légumes, Pôle AgrosciencesAvignon cedex 9France
  3. 3.INRA, UMR INRA 1355 – UNS - CNRS 7254 Institut Sophia-AgrobiotechSophia AntipolisFrance
  4. 4.INRA, UMR 1349 IGEPP INRA/Agrocampus Ouest/Université Rennes1Le RheuFrance

Personalised recommendations