Abstract
Black rot is a cruciferous disease caused by Xanthomonas campestris pv. campestris (Xcc) and results in significant economic losses worldwide; therefore, elucidation of the mechanism of Xcc pathogenesis is urgently required. In this study, we aimed to select optimized reference genes to verify the relative quantification of virulent genes in Xcc. Xcc strains were cultured in three different media [basic medium (MMX), hrp-inducing medium (MMXC) and rich medium (NYG)] and the expression stability of five candidate genes [thymidylate synthase (thyA), DNA gyrase subunit B (gyrB), DNA-directed RNA polymerase subunit beta, glyceraldehyde-3-phosphate dehydrogenase and 16S ribosomal RNA (16S rRNA)] was evaluated using BestKeeper, GeNorm, and NormFinder software programs. Quantitative real-time PCR (qRT-PCR) analysis confirmed that two Xcc effector genes were hrpX/hrpG-regulated in MMXC using selected genes as controls. Finally, gyrB and thyA were validated as the optimized reference genes of Xcc cultured in MMXC, and qRT-PCR analysis was demonstrated to be an efficient alternative to Gus-activity detection for the analysis of Xcc expression. This information will be useful in the future studies of Xcc, especially those seeking new functional genes.
Similar content being viewed by others
References
Vicente JG, Holub EB (2013) Xanthomonas campestris pv. campestris (cause of black rot of crucifers) in the genomic era is still a worldwide threat to brassica crops. Mol Plant Pathol 14:2–18
Lee PC, Rietsch A (2015) Fueling type III secretion. Trends Microbiol 23(5):296–300
Sun W, Liu L, Bent AF (2011) Type III secretion-dependent host defence elicitation and type III secretion-independent growth within leaves by Xanthomonas campestris pv. campestris. Mol Plant Pathol 12:731–745
Dean P (2011) Functional domains and motifs of bacterial type III effector proteins and their roles in infection. FEMS Microbiol Rev 35:1100–1125
Feng F, Yang F, Rong W, Wu X, Zhang J, Chen S, He C, Zhou JM (2012) A Xanthomonas uridine 5′-monophosphate transferase inhibits plant immune kinases. Nature 485:114–118
Liu L, Wang Y, Cui F, Fang A, Wang S, Wang J, Wei C, Li S, Sun W (2016) The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis. Mol Plant Pathol 18:768–782
Koebnik R, Kruger A, Thieme F, Urban A, Bonas U (2006) Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes. J Bacteriol 188:7652–7660
Jiang BL, Jiang GF, Liu W, Yang LY, Wang L, Hang XH, Tang JL (2018) RpfC regulates the expression of the key regulator hrpX of the hrp/T3SS system in Xanthomonas campestris pv. campestris. BMC Microbiol 18:103
Mansfield JW (2009) From bacterial avirulence genes to effector functions via the hrp delivery system: an overview of 25 years of progress in our understanding of plant innate immunity. Mol Plant Pathol 10:721–734
Wengelnik K, Rossier O, Bonas U (1999) Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes. J Bacteriol 181(21):6828–6831
Jiang W, Jiang BL, Xu RQ, Huang JD, Wei HY, Jiang GF, Cen WJ, Liu J, Ge YY, Li GH, Su LL, Hang XH, Tang DJ, Lu GT, Feng JX, He YQ, Tang JL (2009) Identification of six type III Effector genes with the PIP Box in Xanthomonas campestris pv. campestris and five of them contribute individually to full pathogenicity. Mol Plant Microbe Interact 22:1401–1411
Meng QL, Tang DJ, Fan YY, Li ZJ, Zhang H, He YQ, Jiang BL, Lu GT, Tang JL (2011) Effect of interactions between Mip and PrtA on the full extracellular protease activity of Xanthomonas campestri pathovar campestris. FEMS Microbiol Lett 323:180–187
Rong W, Feng F, Zhou J, He C (2010) Effector-triggered innate immunity contributes Arabidopsis resistance to Xanthomonas campestris. Mol Plant Pathol 11:783–793
Kim SM (2018) Identification of novel recessive gene xa44 (t) conferring resistance to bacterial blight races in rice by QTL linkage analysis using an SNP chip. Theor Appl Genet 131(12):2733–2743
Marin MJ, Ambrosio N, Herrera D, Sanz M, Figuero E (2018) Validation of a multiplex qPCR assay for the identification and quantification of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: In vitro and subgingival plaque samples. Arch Oral Biol 88:47–53
Chen L, Shahid MQ, Wu J, Chen Z, Wang L, Liu X (2018) Cytological and transcriptome analyses reveal abrupt gene expression for meiosis and saccharide metabolisms that associated with pollen abortion in autotetraploid rice. Mol Genet Genomics 293(6):1402–1407
Lin J, Li JP, Yuan F, Yang Z, Wang BS, Chen M (2018) Transcriptome profiling of genes involved in photosynthesis in Elaeagnus angustifolia L. under salt stress. Photosynthetica 56(4):998–1009
Mohanpuria P, Duhan N, Sarao NK, Kaur M, Kaur M (2018) In silico identification and validation of potential microRNAs in Kinnow Mandarin (Citrus reticulata Blanco). Interdiscip Sci 10(4):762–770
Nammo T, Udagawa H, Funahashi N, Kawaguchi M, Uebanso T, Hiramoto M, Nishimura W, Yasuda K (2018) Genome-wide profiling of histone H3K27 acetylation featured fatty acid signalling in pancreatic beta cells in diet-induced obesity in mice. Diabetologia 61(12):2608–2620
Zhang X, Hashem MA, Chen X, Tan H (2018) On passing a non-newtonian circulating tumor cell (CTC) through a deformation-based microfluidic chip. Theor Comp Fluid Dyn 32(60):753–764
Link-Lenczowska D, Pallisgaard N, Cordua S, Zawada M, Czekalska S, Krochmalczyk D, Kandula Z, Sacha T (2018) A comparison of qPCR and ddPCR used for quantification of the JAK2 V617F allele burden in Ph negative MPNs. Ann Hematol 97(12):2299–2308
Mehle N, Dreo T (2019) Quantitative analysis with droplet digital PCR. Methods Mol Biol 1875:171–186
Uthicke S, Lamare M, Doyle JR (2018) eDNA detection of corallivorous seastar (Acanthaster cf. solaris) outbreaks on the Great Barrier Reef using digital droplet PCR. Coral Reefs 37(4):1229–1239
Appukuttan B, Ashander LM, Ma Y, Smith JR (2018) Selection of reference genes for studies of human retinal endothelial cell gene expression by reverse transcription quantitative real-time polymerase chain reaction. Gene Rep 10:123–134
Mackay IM (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10(3):190–212
Manimekalai R, Narayanan J, Ranjini R, Gokul M, Selvi A, Kumar P, Gomathi R (2018) Hydrogen peroxide-induced oxidative stress in sugarcane and response expression pattern of stress-responsive genes through quantitative RT-PCR. Sugar Tech 20(6):681–691
Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalization: strategies and consideration. Genes Immun 6(4):279–284
Valasek MA, Repa JJ (2005) The power of real-time PCR. Advan Physiol Edu 29(3):151–159
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(15):5245–5250
Zampieri D, Nora LC, Basso V, Camassola M, Dillon AJP (2014) Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR. Curr Genet 60(3):231–236
Bevitori R, Oliveira MB, Grossi-de-Sa MF, Lanna AC, Dasilveira RD, Petrofeza S (2014) Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought. Genet Mol Res 13(4):9795–9805
Niaz Z, Sui Z, Riaz S, Liu Y, Shang E, Xing Q, Khan S, Du Q, Zhou W, Wang J (2018) Identification of valid reference genes for the normalization of RT-qPCR gene expression data Alexandrium catenella under different nutritional conditions. J Appl Phycol. https://doi.org/10.1007/s10811-1664-2
Wu BY, Ye JR, Huang L, He LM, Li DW (2017) Validation of reference genes for RT-qPCR analysis in Burkholderia pyrrocinia JK-SH007. J Microbiol Methods 132:95–98
Pessoa DDV, Vidal MS, Baldani JI, Simoes-Araujo JL (2016) Validation of reference genes for RT-qPCR analysis in Herbaspirillum seropedicae. J Microbiol Methods 127:193–196
Ren H, Wu X, Lyu Y, Zhou H, Xie X, Zhang X, Yang H (2017) Selection of reliable reference genes for gene expression studies in Botrytis cinerea. J Microbiol Methods 142:71–75
Jin L, Wu X, Ko SR, Jin FJ, Li T, Ahn CY, Oh HM, Lee HG (2018) Description of Hymenobacter daejeonensis sp. nov., isolated from grass soil, based on multilocus sequence analysis of the 16S rRNA gene, gyrB and tuf genes. J Microbiol 111(12):2283–2292
Wei S, Chelliah R, Park BJ, Park JH, Forghani F, Park YS, Ch MS, Park DS, Oh DH (2018) Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB. Microb Pathogenesis 115:312–320
Jacob TR, Laia ML, Ferro JA, Ferro MI (2011) Selection and validation of reference genes for gene expression studies by reverse transcription quantitative PCR in Xanthomonas citri subsp. citri during infection of Citrus sinensis. Biotechnol Lett 33:1177–1184
Pfaffl MW, Tichopad A, Prgomet C, Neuviansm TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper–excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515
Abou-Jawdah Y, Aknadibossian V, Jawhari M, Tawidian P, Abrahamian P (2019) Real-Time PCR protocol for phytoplasma detection and quantification. Methods Mol Biol 1875:117–130
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(7): research0034.1
Long X, He B, Gao X, Qin Y, Yang J, Fang Y, Qi J, Tang C (2015) Validation of reference genes for quantitative real-time PCR latex regeneration in rubber tree. Gene 563:190–195
Ye X, Zhang L, Dong H, Tian Y, Lao H, Bai J, Yu L (2010) Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR. Biotechnol Lett 32(8):1031–1038
Tang R, Dodd A, Lai D, McNabb WC, Love DR (2007) Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin (Shanghai) 39(5):384–390
Acknowledgements
This work was supported by the Innovation Subject of Hainan Province (Grant No. B201304) and Priming Scientific Research Foundation of Hainan University (KYQD1546). We thank Dr Bin He [42] for the aid of software programs.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The author declare that they have no competing interest.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Yan, X., Zhang, Q., Zou, J. et al. Selection of Optimized Reference Genes for qRT-PCR Normalization in Xanthomonas campestris pv. campestris Cultured in Different Media. Curr Microbiol 76, 613–619 (2019). https://doi.org/10.1007/s00284-019-01667-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-019-01667-y