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Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection

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Abstract

Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of C. aurantifolia plants infected with a ‘Candidatus Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as GAPDH, CYCLOPHILIN and 18S rRNA, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in C. aurantifolia during phytoplasma infection.

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References

  • Alves MS, Silva FN, Guimarães DSPSF, Vital CE, Vidigal PMP, Al-Mahmooli I, Al-Sadi AM, Carvalho CM (2018) Differential expression and phytohormone unbalance in Citrus aurantifolia plants during "sudden decline of lime", a new phytoplasma disease of citrus. Tropical Plant Pathology In press

  • Al-Yahyai R, Khan I, Al-Said F, Al-Sadi A, Al-Wahaibi A, Deadman M (2012) Status of Citrus aurantifolia infected with witches’ broom disease of lime in Oman. Acta Horticulturae 928:375–381

    Article  Google Scholar 

  • Andersen CL, Jensen JL, Ørntoft 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 Research 64:5245–5250

    Article  CAS  PubMed  Google Scholar 

  • Askari N, Salehi Jouzani G, Mousivand M, Foroutan A, Hagh Nazari A, Abbasalizadeh S, Soheilivand S, Mardi M (2011) Evaluation of anti-phytoplasma properties of surfactin and tetracycline towards lime witches’ broom disease using real-time PCR. Journal of Microbiology and Biotechnology 21:81–88

    Article  CAS  PubMed  Google Scholar 

  • Bai X, Correa VR, Toruño TY, Ammar E-D, Kamoun S, Hogenhout SA (2009) AY-WB phytoplasma secretes a protein that targets plant cell nuclei. Molecular Plant-Microbe Interactions 22:18–30

    Article  CAS  PubMed  Google Scholar 

  • Biswas KK, Ooura C, Higuchi K, Miyazaki Y, Van Nguyen V, Rahman A, Uchimiya H, Kiyosue T, Koshiba T, Tanaka A, Narumi I, Oono Y (2007) Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots. Plant Physiology 145:773–785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 Reports 31:827–838

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55:611–622

    Article  CAS  PubMed  Google Scholar 

  • De Spiegelaere W, Dern-Wieloch J, Weigel R, Schumacher V, Schorle H, Nettersheim D, Bergmann M, Brehm R, Kliesch S, Vandekerckhove L, Fink C (2015) Reference gene validation for RT-qPCR, a note on different available software packages. PLoS One 10:e0122515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dekkers BJW, Willems L, Bassel GW, van Bolderen-Veldkamp RP, Ligterink W, Hilhorst HWM, Bentsink L (2012) Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds. Plant and Cell Physiology 53:28–37

    Article  CAS  PubMed  Google Scholar 

  • Ehya F, Monavarfeshani A, Mohseni Fard E, Karimi Farsad L, Khayam Nekouei M, Mardi M, Salekdeh GH (2013) Phytoplasma-responsive microRNAs modulate hormonal, nutritional, and stress signalling pathways in mexican lime trees. PLoS One 8:e66372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fan G, Cao X, Niu S, Deng M, Zhao Z, Dong Y (2015) Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa. BMC Genomics 16:896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoshi A, Oshima K, Kakizawa S, Ishii Y, Ozeki J, Hashimoto M, Komatsu K, Kagiwada S, Yamaji Y, Namba S (2009) A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium. Proceedings of the National Academy of Sciences, USA 106:6416–6421

    Article  CAS  Google Scholar 

  • Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16:319–331

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li J, Brader G, Kariola T, Palva ET (2006) WRKY70 modulates the selection of signaling pathways in plant defense. Plant Journal 46:477–491

    Article  CAS  Google Scholar 

  • Liu L-YD, Tseng H-I, Lin C-P, Lin Y-Y, Huang Y-H, Huang C-K, Chang T-H, Lin S-S (2014) High-throughput transcriptome analysis of the leafy flower transition of Catharanthus roseus induced by peanut witches'-broom phytoplasma infection. Plant & Cell Physiology 55:942–957

    Article  CAS  Google Scholar 

  • Luge T, Kube M, Freiwald A, Meierhofer D, Seemüller E, Sauer S (2014) Transcriptomics assisted proteomic analysis of Nicotiana occidentalis infected by 'Candidatus Phytoplasma mali' strain AT. Proteomics 14:1882–1889

    Article  CAS  PubMed  Google Scholar 

  • MacLean AM, Sugio A, Makarova OV, Findlay KC, Grieve VM, Tóth R, Nicolaisen M, Hogenhout SA (2011) Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants. Plant Physiology 157:831–841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • MacLean AM, Orlovskis Z, Kowitwanich K, Zdziarska AM, Angenent GC, Immink RGH, Hogenhout SA (2014) Phytoplasma effector SAP54 hijacks plant reproduction by degrading MADS-box proteins and promotes insect colonization in a RAD23-dependent manner. PLoS Biology 12:e1001835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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:e31263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manoli A, Sturaro A, Trevisan S, Quaggiotti S, Nonis A (2012) Evaluation of candidate reference genes for qPCR in maize. Journal of Plant Physiology 169:807–815

    Article  CAS  PubMed  Google Scholar 

  • Mardi M, Karimi Farsad L, Gharechahi J, Salekdeh GH (2015) In-depth transcriptome sequencing of mexican lime trees infected with 'Candidatus Phytoplasma aurantifolia. PLoS One 10:e0130425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Minato N, Himeno M, Hoshi A, Maejima K, Komatsu K, Takebayashi Y, Kasahara H, Yusa A, Yamaji Y, Oshima K, Kamiya Y, Namba S (2014) The phytoplasmal virulence factor TENGU causes plant sterility by downregulating of the jasmonic acid and auxin pathways. Scientific Reports 4:7399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Monavarfeshani A, Mirzaei M, Sarhadi E, Amirkhani A, Khayam Nekouei M, Haynes PA, Mardi M, Salekdeh GH (2013) Shotgun proteomic analysis of the Mexican lime tree infected with 'Candidatus Phytoplasma aurantifolia'. Journal of Proteome Research 12:785–795

    Article  CAS  PubMed  Google Scholar 

  • Nejat N, Cahill DM, Vadamalai G, Ziemann M, Rookes J, Naderali N (2015) Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection. Molecular Genetics and Genomics 290:1899–1910

    Article  CAS  PubMed  Google Scholar 

  • Oshima K, Maejima K, Namba S (2013) Genomic and evolutionary aspects of phytoplasmas. Frontiers in Microbiology 4:230

    Article  PubMed  PubMed Central  Google Scholar 

  • Pabinger S, Rödiger S, Kriegner A, Vierlinger K, Weinhäusel A (2014) A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomolecular Detection and Quantification 1:23–33

    Article  PubMed  PubMed Central  Google Scholar 

  • Pauwels L, Goossens A (2011) The JAZ proteins: a crucial interface in the jasmonate signaling cascade. Plant Cell 23:3089–3100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research 30:e36

    Article  PubMed  PubMed Central  Google Scholar 

  • 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. Biotechnology Letters 26:509–515

    Article  CAS  PubMed  Google Scholar 

  • Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 313:856–862

    Article  CAS  PubMed  Google Scholar 

  • Rose AB, Casselman AL, Last RL (1992) A phosphoribosylanthranilate transferase gene is defective in blue fluorescent Arabidopsis thaliana tryptophan mutants. Plant Physiology 100:582–592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative Ct method. Nature Protocols 3:1101–1108

    Article  CAS  PubMed  Google Scholar 

  • Shan X, Li C, Peng W, Gao B (2011a) New perspective of jasmonate function in leaf senescence. Plant Signaling & Behavior 6:575–577

    Article  CAS  Google Scholar 

  • Shan X, Wang J, Chua L, Jiang D, Peng W, Xie D (2011b) The role of Arabidopsis rubisco activase in jasmonate-induced leaf senescence. Plant Physiology 155:751–764

    Article  CAS  PubMed  Google Scholar 

  • Shim JS, Jung C, Lee S, Min K, Lee Y-W, Choi Y, Lee JS, Song JT, Kim J-K, Choi YD (2013) AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling. Plant Journal 73:483–495

    Article  CAS  Google Scholar 

  • Silva FN, Souza AN, Al-Mahmooli I, Al-Sa’di AM, Carvalho CM (2015) A new disease in Citrus aurantifolia in Oman, "sudden decline", is associated with a pathogen complex including a 16SrII group phytoplasma. Phytopathogenic Mollicutes 5:S101

    Article  Google Scholar 

  • Taheri F, Nematzadeh G, Zamharir MG, Nekouei MK, Naghavi M, Mardi M, Salekdeh GH (2011) Proteomic analysis of the Mexican lime tree response to 'Candidatus Phytoplasma aurantifolia' infection. Molecular BioSystems 7:3028–3035

    Article  CAS  PubMed  Google Scholar 

  • 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 Biology 3:RESEARCH0034

    Article  Google Scholar 

  • Yan J, Yuan F, Long G, Qin L, Deng Z (2012) Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Molecular Biology Reports 39:1831–1838

    Article  CAS  PubMed  Google Scholar 

  • Zamharir MG, Mardi M, Alavi SM, Hasanzadeh N, Nekouei MK, Zamanizadeh HR, Alizadeh A, Salekdeh GH (2011) Identification of genes differentially expressed during interaction of Mexican lime tree infected with 'Candidatus Phytoplasma aurantifolia'. BMC Microbiology 11:1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE (2013) The iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment. Methods in Molecular Biology 938:329–338

    Article  CAS  PubMed  Google Scholar 

  • Zreik L, Carle P, Bové JM, Garnier M (1995) Characterization of the mycoplasma-like organism associated with witches’-broom disease of lime and proposition of a Candidatus taxon for the organism, 'Candidatus Phytoplasma aurantifolia'. International Journal of Systematic Bacteriology 45:449–453

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was funded by Vale S.A. MSA was a Vale Technological Institute postdoctoral fellow. CMC is the recepient of a CNPq research productivity fellowship. We thank Sam Elliot (Dep. de Entomologia, UFV) for critically reviewing the English of the manuscript.

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Authors and Affiliations

  1. Dep. de Fitopatologia, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil

    Murilo S. Alves & Claudine M. Carvalho

  2. Dep. de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, 60440-900, Brazil

    Murilo S. Alves

  3. Dep. of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, Al-khod, 123, Sultanate of Oman

    Abdullah M. Al-Sadi

Authors
  1. Murilo S. Alves
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  2. Abdullah M. Al-Sadi
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  3. Claudine M. Carvalho
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Contributions

MSA and CMC conceived and designed the research. AMA assisted in the collection of infected samples. MSA conducted the experiments. MSA analyzed the data. MSA and CMC wrote the manuscript. All authors read and approved of the manuscript.

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Correspondence to Claudine M. Carvalho.

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The authors declare that they have no conflict of interest.

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Section Editor: Robert Miller

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Alves, M.S., Al-Sadi, A.M. & Carvalho, C.M. Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection. Trop. plant pathol. 43, 402–412 (2018). https://doi.org/10.1007/s40858-018-0224-2

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