title: Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.
abstract: The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1α, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1α and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1α, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1α, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola.
authors.affiliation: Plant Systems Biology Lab, Center of Biodiversity, Functional & Integrative Genomics (BioFIG), Science Faculty of Lisbon University, Lisboa, Portugal.
authors.forename: Filipa
authors.initials: F
authors.lastname: Monteiro
authors.forename: Mónica
authors.initials: M
authors.lastname: Sebastiana
authors.forename: Maria Salomé
authors.initials: MS
authors.lastname: Pais
authors.forename: Andreia
authors.initials: A
authors.lastname: Figueiredo
mesh_terms: D018506:Gene Expression Regulation, Plant; D005838:Genotype; D007113:Immunity, Innate / Q000235:genetics / Q000502:physiology; D044742:Peronospora / Q000472:pathogenicity; D010935:Plant Diseases / Q000235:genetics / Q000469:parasitology*; D016133:Polymerase Chain Reaction; D027843:Vitis / Q000235:genetics / Q000469:parasitology*
references.citation: J Mol Endocrinol. 2000 Oct;25(2):169-93
references.pmid: 11013345
references.citation: Methods. 2010 Apr;50(4):227-30
references.pmid: 19969088
references.citation: Planta. 2008 May;227(6):1343-9
references.pmid: 18273637
references.citation: BMC Res Notes. 2012 May 07;5:220
references.pmid: 22564373
references.citation: PLoS One. 2012;7(9):e46451
references.pmid: 23029521
references.citation: Mol Genet Genomics. 2011 Apr;285(4):273-85
references.pmid: 21340517
references.citation: Expert Rev Mol Diagn. 2010 Sep;10(6):713-5
references.pmid: 20843195
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6332-7
references.pmid: 8692815
references.citation: Cancer Res. 2004 Aug 1;64(15):5245-50
references.pmid: 15289330
references.citation: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references.pmid: 12184808
references.citation: BMC Plant Biol. 2006 Nov 14;6:27
references.pmid: 17105665
references.citation: Theor Appl Genet. 2011 Jun;123(1):43-53
references.pmid: 21404060
references.citation: J Plant Physiol. 2003 Nov;160(11):1393-400
references.pmid: 14658393
references.citation: BMC Plant Biol. 2010 Oct 28;10:234
references.pmid: 21029438
references.citation: Plant Physiol. 2000 Jan;122(1):67-74
references.pmid: 10631250
references.citation: Plant Biol (Stuttg). 2009 Nov;11(6):886-98
references.pmid: 19796366
references.citation: J Biol Chem. 1997 May 30;272(22):14412-9
references.pmid: 9162080
references.citation: Plant Cell Rep. 2012 Jan;31(1):205-16
references.pmid: 22006104
references.citation: Genome Biol. 2007;8(2):R19
references.pmid: 17291332
references.citation: Biotechnol Lett. 2006 Oct;28(19):1601-13
references.pmid: 16900335
references.citation: BMC Plant Biol. 2010 Jul 15;10:147
references.pmid: 20633270
references.citation: Genome. 2000 Apr;43(2):333-40
references.pmid: 10791822
references.citation: Biochem Biophys Res Commun. 2007 Feb 16;353(3):672-8
references.pmid: 17194440
references.citation: J Exp Bot. 2005 Nov;56(421):2907-14
references.pmid: 16188960
references.citation: BMC Genomics. 2010 Feb 18;11:117
references.pmid: 20167053
references.citation: BMC Genomics. 2008 Mar 26;9:142
references.pmid: 18366764
references.citation: Anal Biochem. 2002 Apr 1;303(1):95-8
references.pmid: 11906156
references.citation: Genes Immun. 2005 Jun;6(4):279-84
references.pmid: 15815687
references.citation: Biotechnol Lett. 2004 Mar;26(6):509-15
references.pmid: 15127793
references.citation: J Biochem Biophys Methods. 2000 Nov 20;46(1-2):69-81
references.pmid: 11086195
references.citation: J Mol Endocrinol. 2005 Jun;34(3):597-601
references.pmid: 15956331
references.citation: Mol Plant Microbe Interact. 2008 Feb;21(2):232-43
references.pmid: 18184067
references.citation: Nucleic Acids Res. 2001 May 1;29(9):e45
references.pmid: 11328886
references.citation: Theor Appl Genet. 2004 Feb;108(3):501-15
references.pmid: 14574452
references.citation: Curr Opin Plant Biol. 2004 Aug;7(4):400-7
references.pmid: 15231262
references.citation: BMC Plant Biol. 2011 Aug 12;11:114
references.pmid: 21838877
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Plant Cell. 2004 Apr;16(4):857-73
references.pmid: 15020745
references.citation: Theor Appl Genet. 2012 Jan;124(1):163-76
references.pmid: 21935694
references.citation: Physiol Plant. 2003 May;118(1):64-73
references.pmid: 12702015
references.citation: Int J Legal Med. 2012 Nov;126(6):943-52
references.pmid: 23010907
references.citation: Funct Integr Genomics. 2012 Jun;12(2):379-86
references.pmid: 22246600
references.citation: J Exp Bot. 2008;59(12):3371-81
references.pmid: 18648103
references.citation: Theor Appl Genet. 2009 Dec;120(1):163-76
references.pmid: 19821064
references.citation: J Plant Physiol. 2010 Jan 15;167(2):121-30
references.pmid: 19682768
references.citation: J Mol Endocrinol. 2002 Aug;29(1):23-39
references.pmid: 12200227
references.citation: J Biomol Tech. 2004 Sep;15(3):155-66
references.pmid: 15331581
references.citation: Plant Physiol. 2005 Sep;139(1):5-17
references.pmid: 16166256