Abstract
Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar ‘Gala’. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.
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References
Abu-Quaoud H, Skirvin RM, Chevreau E (1990) In vitro separation of chimeral pears into their component genotypes. Euphytica 48:189–196. https://doi.org/10.1007/BF00037199
Ampomah-Dwamena C, Dejnoprat S, Lewis D, Sutherland P, Volz RK, Allan AC (2012) Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis. J Exp Bot 63:4497–4511. https://doi.org/10.1093/jxb/ers134
Brown SC, Bergounioux C, Tallet S, Marie D (1991) Flow cytometry of nuclei for ploidy and cell cycle analysis. In: Negrutiu I, Gharti-Chetri GB (eds) Biomethods, a laboratory guide for cellular and molecular plant biology, vol 4. Birkhäuser, Basel, pp 326–345
Bubner B, Baldwin IT (2004) Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep 23:263–271. https://doi.org/10.1007/s00299-004-0859-y
Charrier A, Vergne E, Dousset N, Richer A, Petiteau A, Chevreau E (2019) Efficient targeted mutagenesis in apple and pear using the CRISPR/Cas9 system. Front Plant Sci. https://doi.org/10.3389/fpls.2019.00040
Chevreau E, Dupuis F, Taglioni JP, Sourice S, Cournol R, Deswartes C, Bersegeay A, Descombin J, Siegwart M, Loridon K (2011) Effect of ectopic expression of the eutypine detoxifying gene Vr-ERE in transgenic apple plants. Plant Cell Tiss Org Cult 106:161–168. https://doi.org/10.1007/s11240-010-9904-4
Czimmerer Z, Hulvely J, Simandi Z, Varallyay E, Havelda Z, Szabo E, Varga A, Dezso B, Balogh M, Horvath A, Domokos B, Torok Z, Nagy L, Balint BL (2013) A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules. PLoS ONE 8:e55168. https://doi.org/10.1371/journal.pone.0055168
Daccord N, Celton JM, Linsmith G, Becker C, Choisne N, Schijlen E, van de Geest H, Bianco L, Micheletti D, Velasco R et al (2017) High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet 49:1099–1106
Dai X, Zhuang Z, Zhao PX (2018) psRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Res 46:49–54. https://doi.org/10.1093/nar/gky316
Dominguez A, Cervera M, Perez R, Romero J, Fagoaga C, Cubero J, Lopez M, Juarez JA, Navarro L, Pena L (2004) Characterization of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of Citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol Breed 14:171–183. https://doi.org/10.1023/B:MOLB.0000038005.73265.61
Eamens AL, Agius C, Smith NA, Waterhouse PM, Wang MB (2011) Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana. Mol Plant 4:157–170. https://doi.org/10.1093/mp/ssq061
Faize M, Malnoy M, Dupuis F, Chevalier M, Parisi L, Chevreau E (2003) Chitinases of Trichoderma atroviridae induce scab resistance and some metabolic changes in two cultivars of apple. Phytopathology 93:1496–1504. https://doi.org/10.1094/PHYTO.2003.93.12.1496
FAOStat. http://www.fao.org/faostat/en/#data/QC. Accessed 20 Feb 2018
Flachowsky H, Riedel M, Reim S, Hanke MV (2008) Evaluation of the uniformity and stability of T-DNA integration and gene expression in transgenic apple plants. Electron J Biotechnol 11:1–14. https://doi.org/10.2225/vol11-issue1-fulltext-10
Flachowsky H, Tränkner C, Szankowski I, Waidmann S, Hanke MV, Treutter D, Fischer TC (2012) RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp. Int J Mol Sci 13:9992–10009. https://doi.org/10.3390/ijms13089992
Frizz A, Huang S (2010) Tapping RNA silencing pathways for plant biotechnology. Plant Biotechnol J 8:655–677. https://doi.org/10.1111/j.1467-7652.2010.00505.x
Fulton TM, Chunzoongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:207–209. https://doi.org/10.1007/BF02670897
Gambino G, Gribaudo I (2012) Genetic transformation of fruit trees: current status and remaining challenges. Transgenic Res 21:1163–1181. https://doi.org/10.1007/s11248-012-9602-6
Gilissen LJWJ, Bolhaar STH, Matos CL, Rouwendal GJA, Boone MJ, Krens FA, Zuidmeer L, van Leeuwen A, Akkerdaas J, Hoffmann-Sommergruber K, Knulst AC, Bosch D, van de Weg E, van Ree R (2005) Silencing the major apple allergen Mal d 1 by using the RNA interference approach. J Allergy Clin Immunol 115:364–369. https://doi.org/10.1016/j.jaci.2004.10.014
Gleave AP, Ampomah-Dwamena C, Berthold S, Dejnoprat S, Karunairetnam S, Nain B, Wang YY, Crowhurst RN, MacDiarmid RM (2008) Identification and characterisation of primary microRNAs from apple (Malus domestica cv. Royal Gala) expressed sequence tags. Tree Genet Genomes 4:343–358. https://doi.org/10.1007/s11295-007-0113-1
Hannon GJ (2002) RNA interference. Nature 418:244–251. https://doi.org/10.1038/418244a
Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795. https://doi.org/10.1101/gr.143000
Hood EE, Gelvin SB, Melchers L, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218. https://doi.org/10.1007/BF01977351
Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240. https://doi.org/10.1093/bioinformatics/btu031
Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195. https://doi.org/10.1016/S1360-1385(02)02251-3
Kumagai MH, Donson J, Della-Cioppa G, Harvey D, Hanley K, Grill LK (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci USA 92:1679–1683. https://doi.org/10.1073/pnas.92.5.1679
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Malnoy M, Korban S, Boresjza-Wysocka E, Aldwinckle HS (2008) Apple. In: Kole C, Hall TC (eds) Compendium of transgenic crops plants: transgenic temperate fruit and nuts. Wiley, Berlin, pp 11–52. https://doi.org/10.1002/9781405181099.k0401
McGonigle B (2012) Down-regulation of gene expression using artificial microRNAs. United States Patent US 8,115,055 B2
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plantarum 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Murata M, Haruta M, Murai N, Tanikawa N, Nishimura M, Homma S, Itoh A (2000) Transgenic apple (Malus × domestica) shoot showing low browning potential. J Agric Food Chem 48:5243–5248. https://doi.org/10.1007/s00299-003-0668-8
Nishitani C, Hirai N, Komori S, Wada M, Okada K, Osakabe K, Yamamoto T, Osakabe Y (2016) Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep 6:31481. https://doi.org/10.1038/srep31481
Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua N (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428. https://doi.org/10.1038/nbt1255
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acid Res 29:e45. https://doi.org/10.1093/nar/29.9.e45
Qin G, Gu H, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid and gibberellin biosynthesis. Cell Res 17:471–482
Qu J, Ye F, Fang RX (2007) Artificial microRNA-mediated virus resistance in plants. J Virol 81:6690–6699. https://doi.org/10.1128/JVI.02457-06
Que Q, Wang HY, English JJ, Jorgensen RA (1997) The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. Plant Cell 9:1357–1368. https://doi.org/10.1105/tpc.9.8.1357
R Development Core Team (2016) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. ISBN: 3-900051-07-0. http://www.R-project.org/
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626
Rodermel SR, Abbott MS, Bogorad L (1988) Nuclear–organelle interactions: nuclear antisense gene inhibits ribulose biphosphate carboxylase enzyme levels in transformed tobacco plants. Cell 55:673–681. https://doi.org/10.1016/0092-8674(88)90226-7
Rogers K, Chen X (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25:2383–2399. https://doi.org/10.1105/tpc.113.113159
Sasaki S, Yamagishi N, Yoshikawa N (2011) Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple Latent Spherical Virus vectors. Plant Methods 7:15. https://doi.org/10.1186/1746-4811-7-15
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133. https://doi.org/10.1105/tpc.105.039834
Senthil-Kumar M, Mysore KS (2011) New dimensions for VIGS in plant functional genomics. Trends Plant Sci 16:656–665. https://doi.org/10.1016/j.tplants.2011.08.006
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407:319–320. https://doi.org/10.1038/35030305
Stoutjesdijk PA, Singh SP, Liu Q, Hurlstone CJ, Waterhouse PA, Green AG (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723–1731. https://doi.org/10.1104/pp.006353
Sun C, Zhao Q, Liu DD, You CX, Hao YJ (2013) Ectopic expression of the apple Md-miRNA156h gene regulates flower and fruit development in Arabidopsis. Plant Cell Tiss Org Cult 112:343–351. https://doi.org/10.1007/s11240-012-0241-7
Tiwari M, Sharma D, Trivedi PK (2014) Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Mol Biol 86:1–18. https://doi.org/10.1007/s11103-014-0224-7
Van der Fits L, Deakin EA, Hoge JH, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502. https://doi.org/10.1023/A:1006440221718
Vandesompele J, De Preter K, Pattyn F, Poppe B, Roy N, De Paepe A (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–11. https://doi.org/10.1186/gb-2002-3-7-research0034
Venisse JS, Malnoy M, Faize M, Paulin JP, Brisset MN (2002) Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora. Mol Plant Microbe Interact 15:1204–1212. https://doi.org/10.1094/MPMI.2002.15.12.1204
Vergne E, Dugé de Bernonville T, Dupuis F, Sourice S, Cournol R, Berthelot P, Barny MA, Brisset MN, Chevreau E (2014) Membrane targeted HrpNEa can modulate apple defense gene expression. Mol Plant Microbe Interact 27:125–135. https://doi.org/10.1094/MPMI-10-13-0305-R
Warthmann N, Chen H, Ossowski S, Weigel D, Hervé P (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS ONE 3:e1829. https://doi.org/10.1371/journal.pone.0001829
Zhang X, Li H, Zhang J, Zhang C, Gong P, Ziaf K, Xiao F, Ye Z (2011) Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner. Transgenic Res 20:569–581. https://doi.org/10.1007/s11248-010-9440-3
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415. https://doi.org/10.1093/nar/gkg595
Acknowledgements
This Project has received funding from the French Government managed by the Research National Agency (ANR) under the Investment for the Future program (GENIUS Project ANR11-BTBR-0001). The authors gratefully acknowledge B. Billy (SNES-GEVES) for technical assistance in flow cytometry. We also thank Anne Alderton (Ecolangues) for the English correction of the manuscript.
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AC and EV were the main investigators in this study. AC performed part of the experiments, analyzed the data and drafted the manuscript; EV analyzed the data and revised the manuscript; CJ, AR and ND performed part of the experiments and revised the manuscript; EC designed the study and revised the manuscript. All authors read and approved the final manuscript.
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Charrier, A., Vergne, E., Joffrion, C. et al. An artificial miRNA as a new tool to silence and explore gene functions in apple. Transgenic Res 28, 611–626 (2019). https://doi.org/10.1007/s11248-019-00170-1
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DOI: https://doi.org/10.1007/s11248-019-00170-1