Tropical Plant Pathology

, Volume 42, Issue 2, pp 76–85 | Cite as

Reference genes for RT-qPCR analysis in Citrus and Poncirus infected by zoospores of Phytophthora parasitica

  • Heros J. Máximo
  • Ronaldo J. D. Dalio
  • Carolina M. Rodrigues
  • Michèle C. Breton
  • Marcos A. Machado
Original Article


Phytophthora species are highly destructive phytopathogens, associated with massive damage in natural ecosystems and agriculture. Citrus production is also affected, mainly by the hemibiothrophic oomycete Phytophthora parasitica, which causes root rot and gummosis. Poncirus trifoliata and Citrus sunki (two rootstocks widely used in citrus orchards) pose a resistance and a susceptible interaction with P. parasitica, respectively, which makes them suitable models to study plant defense mechanisms. Gene expression analysis is a very important tool in this type of study, in particular PCR (RT-qPCR). Hence, it is crucial to use appropriate reference genes for expression normalization. Our aim was to evaluate the stability of several candidate reference genes to determine which set is best suited for normalization in citrus infected with P. parasitica. We evaluated five candidate reference genes selected from the database CitEST. GeNorm and NormFinder algorithms were used to assess the best reference genes. We found that the more stable genes to be used for RT-qPCR analysis in P. trifoliata plants were GAPC2 and F-BOX, while EGIDH and GAPC2 were more suitable to C. sunki. These four genes were found to be excellent normalizers, being stable throughout the infection regardless of pathogen attack or symptom development.


Oomycetes Gene expression Housekeeping genes Molecular biology 



RJDD thanks FAPESP for grant number 2015/14498-6. HJM thanks Anhanguera Educacional and Kroton Group. MAM thanks INCT citrus (Fapesp 2008/57909-2, CNPq 573848/08-4).


  1. Albrecht U, Bowman KD (2008) Gene expression in Citrus sinensis (L.) Osbeck following infection with the bacterial pathogen Candidatus Liberibacter asiaticus causing Huanglongbing in Florida. Plant Sci 175:291–306CrossRefGoogle Scholar
  2. Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative recerse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon câncer data sets. Cancer Res 64:5245–5250CrossRefPubMedGoogle Scholar
  3. Aritua V, Achor D, Gmitter FG, Albrigo G, Wang N (2013) Transcriptional and microscopic analyses of citrus stem and root responses to Candidatus Liberibacter asiaticus infection. PLoS ONE 8, e73742CrossRefPubMedPubMedCentralGoogle Scholar
  4. Boava LP, Laia ML, Jacob TR, Dabbas KM, Gonçalves JF, Ferro JA, Ferro MIT, Furtado EL (2010) Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR. BMC Res Notes 3:43CrossRefPubMedPubMedCentralGoogle Scholar
  5. Boava LP, Cristofani-Yaly M, Mafra VS, Kubo K, Kishi LT, Takita MA, Ribeiro-Alves M, Machado MA (2011) Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica. BMC Genomics 12:39CrossRefPubMedPubMedCentralGoogle Scholar
  6. Boiffin V, Hodges M, Galvez S, Balestrini R, Bonfante P, Gadal P, Martin F (1998) Eucalypt NADP-dependent isocitrate dehydrogenase - cDNA cloning and expression in ectomycorrhizae. Plant Physiol 117:939–948CrossRefPubMedPubMedCentralGoogle Scholar
  7. Borges A, Tsai SM, Caldas DGG (2012) Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses. Plant Cell Rep 31:827–838CrossRefPubMedGoogle Scholar
  8. Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cernadas RA, Camillo LR, Benedetti CE (2008) Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. aurantifolii. Mol Plant Pathol 9:609–631CrossRefPubMedGoogle Scholar
  10. Chai L, Ge X, Xu Q, Deng X (2011) CgSL2, an S-like RNase gene in ‘Zigui shatian’ pummelo (Citrus grandis Osbeck), is involved in ovary senescence. Mol Biol Rep 38:1–8CrossRefPubMedGoogle Scholar
  11. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17CrossRefPubMedPubMedCentralGoogle Scholar
  12. Endo T, Shimada T, Fujii H, Omura M (2006) Cloning and characterization of five MADS-box cDNAs isolated from citrus fruit tissue. Sci Hortic 109:315–321CrossRefGoogle Scholar
  13. Fan J, Chen C, Brlansky RH, Gmitter FG Jr, Li Z-G (2010) Changes in carbohydrate metabolism in Citrus sinensis infected with ‘Candidatus Liberibacter asiaticus’. Plant Pathol 59:1037–1043CrossRefGoogle Scholar
  14. Gachon C, Mingam A, Charrier B (2004) Real-time PCR: what relevance to plant studies. J Exp Bot 55:1445–1454CrossRefPubMedGoogle Scholar
  15. Gagne JM, Downes BP, Shiu SH, Durski AM, Vierstra RD (2002) The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc Natl Acad Sci U S A 99:11519–11524CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis for real-time quantitative PCR data. Genome Biol 8:R19Google Scholar
  17. Huerta L, Forment J, Gadea J, Fagoaga C, Peña L, Pérez-Amador MA, García-Martínez JL (2008) Gene expression analysis in citrus reveals the role of gibberellins on photosynthesis and stress. Plant Cell Environ 31:1620–1633CrossRefPubMedGoogle Scholar
  18. Ippolito A, Schena L, Nigro F (2002) Detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils by nested PCR. Eur J Plant Pathol 108:855–868CrossRefGoogle Scholar
  19. Iskandar H, Simpson RS, Casu RE, Bonnett GD, Maclean DJ, Manners JM (2004) Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol Biol Report 22:325–337CrossRefGoogle Scholar
  20. Jain M, Nijhawan A, Tyagi AK (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651CrossRefPubMedGoogle Scholar
  21. Jain M, Nijhawan A, Arora R, Agarwal P, Ray S, Sharma P, Kapoor S, Tyagi AK, Khurana JP (2007) F-box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiol 143:1467–1483CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kamoun S, Furzer O, Jones JDG, Judelson H S, Ali GS, Dalio RJD, Roy SG, Schena L, Zambounis A, Panabières F, Cahill D, Ruocco M, Figueiredo A, Chen X-R, Hulvey J, Stam R, Lamour K, Gijzen M, Tyler BM, Grünwald NJ, Mukhtar MS, Tomé DFA, Tör M, Van Den Ackerveken G, McDowell J, Daayf F, Fry WE, Lindqvist-Kreuze H, Meijer HJG, Petre B, Ristaino J, Yoshida K, Birch PRJ, Govers F (2015), The Top 10 oomycete pathogens in molecular plant pathology. Mol Plant Pathol 16:413–434Google Scholar
  23. Kruskal HW, Wallis AW (1952) Use of Ranks in One-Criterion Variance Analysis. Journal of the American Statistical Association 47(260):583–621Google Scholar
  24. Lechner E, Achard P, Vansiri A, Poutuschak T, Genschik P (2006) F-box proteins everywhere. Curr Opin Plant Biol 9:631–638CrossRefPubMedGoogle Scholar
  25. Legay S, Lacombe E, Goicoechea M, Brière C, Séguin A, Mackay J, Grimapettenati J (2007) Molecular characterization of EgMYB1, a putative transcriptional repressor of the lignin biosynthetic pathway. Plant Sci 173:542–549CrossRefGoogle Scholar
  26. Liu Q, Xu J, Liu Y, Zhao X, Deng X, Guo L, Gu J (2007) A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J Exp Bot 58:4161–4171CrossRefPubMedGoogle Scholar
  27. Liu Q, Zhu A, Chai L, Zhou W, Yu K, Ding J, Xu J, Deng X (2009) Transcriptome analysis of a spontaneous mutant in sweet orange [Citrus sinensis (L.) Osbeck] during fruit development. J Exp Bot 60:801–813CrossRefPubMedPubMedCentralGoogle Scholar
  28. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)). Methods 25:402–408CrossRefPubMedGoogle Scholar
  29. Mafra V, Kubo KS, Alves-Ferreira M, Ribeiro-Alves M, Stuart RM, Boava LP, Rodrigues CM, Machado MA (2012) Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS ONE 7, e31262CrossRefGoogle Scholar
  30. Martinelli F, Uratsu SL, Albrecht U, Reagan RL, Phu ML, Britton M, Buffalo V, Fass J, Leicht E, Zhao W, Lin D, D’Souza R, Davis CE, Bowman KD, Dandekar AM (2012) Transcriptome profiling of citrus fruit response to huanglongbing disease. PLoS ONE 7, e38039CrossRefPubMedPubMedCentralGoogle Scholar
  31. Maruyama T, Suzuki R, Furutani M (2004) Archaeal peptidyl prolyl cis-trans isomerases (PPIases) update 2004. Front Biosci 9:1680–1720CrossRefPubMedGoogle Scholar
  32. Matta BP, Bitner-Mathe BC, Alves-Ferreira M (2011) Getting real with real-time qPCR: a case study of reference gene selection for morphological variation in Drosophila melanogaster wings. Dev Genes Evol 221:49–57Google Scholar
  33. Miao HX, Qin YH, Silva JT, Ye ZX, Hu GB (2011) Cloning and expression analysis of S-RNase homologous gene in Citrus reticulata Blanco cv. Wuzisha- tangju. Plant Sci 180:58–367CrossRefGoogle Scholar
  34. 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 56:2907–2914Google Scholar
  35. Nishikawa F, Endo T, Shimada T, Fujii H, Shimizu T, Omura M (2009) Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarin. Tree Physiol 29:921–926CrossRefPubMedGoogle Scholar
  36. Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time PCR. Nat Protoc 1:1559–1582CrossRefPubMedGoogle Scholar
  37. Osswald W, Fleischmann F, Rigling D, Coelho AC, Cravador A, Diez J, Dalio RJ, Horta Jung M, Pfanz H, Robin C, Sipos G, Solla A, Cech T, Chambery A, Diamandis S, Hansen E, Jung T, Orlikowski LB, Parke J, Prospero S, Werres S (2014), Strategies of attack and defence in woody plant–Phytophthora interactions. For. Path., 44:169–190. Google Scholar
  38. Panabieres F, Ali GS, Allagui MB, Dalio RJD, Gudmestad NC, Kuhn M, Roy SG, Schena L, Zampounis A (2016) Phytophtora nicotianae diseases worldwide: new knowledge of a long-recognised pathogen. Phytopatologia Mediterr 55:20–40Google Scholar
  39. Paux E, Tamasloukht M, Ladouce N, Sivadon P, Grima-Pettenati J (2004) Identification of genes preferentially expressed during wood formation in Eucalyptus. Plant Mol Biol 55:263–280CrossRefPubMedGoogle Scholar
  40. Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:4856–4862CrossRefGoogle Scholar
  41. Remans T, Smeets K, Opdenakker K, Mathijsen D, Vangronsveld J, Cuypers A (2008) Normalization of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227:1343–1349CrossRefPubMedGoogle Scholar
  42. Rodrigues CM, De Souza AA, Takita MA, Kishi LT, Machado MA (2013) RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response. BMC Genomics 14:1CrossRefGoogle Scholar
  43. Sharifi-Sirchi GR, Beheshti B, Hosseinipour A, Mansouri M (2011) Priming against Asiatic citrus canker and monitoring of PR genes expression during resistance induction. Afr J Biotechnol 10:3818–3823Google Scholar
  44. Tan FC, Swain SM (2007) Functional characterizationof AP3, SOC1 and WUS homologues from citrus (Citrus sinensis). Plant Physiol 131:481–495CrossRefGoogle Scholar
  45. Timmer LR, Garnsey SM, Graham JM (2000) Compendium of Citrus Diseases. APS Press, Sant PaulGoogle Scholar
  46. Vandesompele J, De Preter K, Patyn F (2002) Accurate normalisation of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:0034.1-0034.11CrossRefGoogle Scholar
  47. Volkov RA (2003). Heat-stress-dependency and developmental modulation of gene expression, the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR. J Exp Bot 54:2343–2349Google Scholar
  48. Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39:1–9CrossRefGoogle Scholar
  49. Zheng TG, Qiu WM, Fan GE, Zheng BB, Guo WW (2011) Construction and characterization of a cDNA library from floral organs and fruitlets of Citrus reticulata. Biol Plant 55:431–436CrossRefGoogle Scholar
  50. Zhong Y, Cheng C-z, Jiang N-h, Jiang B, Wu B, Zhang Y-y, Hu M-l, Zeng J-w, H-x Y, G-j Y, Zhong G-y (2015) Comparative transcriptome and iTR AQ proteome analyses of citrus root responses to Candidatus Liberibacter asiaticus infection. PLoS ONE 10, e0126973Google Scholar

Copyright information

© Sociedade Brasileira de Fitopatologia 2017

Authors and Affiliations

  • Heros J. Máximo
    • 1
  • Ronaldo J. D. Dalio
    • 1
  • Carolina M. Rodrigues
    • 1
  • Michèle C. Breton
    • 1
  • Marcos A. Machado
    • 1
  1. 1.Lab. de Biotecnologia, Centro de Citricultura Sylvio MoreiraInstituto Agronômico de CampinasCordeirópolisBrazil

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