Abstract
Eschscholzia californica (California poppy), a member of the basal eudicot family of the Papaveraceae, is an important species to study alkaloid biosynthesis and the effect of alkaloids on plant metabolism. More recently, it has also been developed as a model system to study the evolution of plant morphogenesis. While progress has been made towards establishing methods for generating genetically modified cell culture lines, transcriptome data and gene expression analysis, the stable transformation and subsequent regeneration of transgenic plants has proven extremely time consuming and difficult. Here, we describe in detail a method to transiently down regulate expression of a target gene by virus-induced gene silencing (VIGS) and the subsequent analysis of the VIGS treated plants. VIGS in E. californica allows for the study of gene function within 2 to 3 weeks after inoculation, and the method proves very efficient, enabling the rapid analysis of gene functions.
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References
Wang W, Lu A-M, Ren Y et al (2009) Phylogeny and classification of Ranunculales: evidence from four molecular loci and morphological data. Perspect Plant Ecol Evol Syst 11(2):81–110
Kadereit JW, Blattner FR, Jork KB et al (1995) The phylogeny of the Papaveraceae s.l.: morphological, geographical and ecological implications. Plant Syst Evol 9:133–145
Becker A, Gleissberg S, Smyth DR (2005) Floral and vegetative morphogenesis in California poppy (Eschscholzia californica Cham). Int J Plant Sci 166(4):537–555
Bennett MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Philos Trans R Soc Lond B Biol Sci 274(933):227–274
Barakat A, Wall K, Leebens-Mack J et al (2007) Large-scale identification of microRNAs from a basal eudicot (Eschscholzia californica) and conservation in flowering plants. Plant J 51(6):991–1003
Carlson J, Leebens-Mack JH, Wall PK et al (2006) EST database for early flower development in California poppy (Eschscholzia californica Cham., Papaveraceae) tags over 6000 genes from a basal eudicot. Plant Mol Biol 62(3):351–369
Wall PK, Leebens-Mack J, Chanderbali AS et al (2009) Comparison of next generation sequencing technologies for transcriptome characterization. BMC Genomics 10:347
Zahn LM, Ma X, Altman NS et al (2010) Comparative transcriptomics among floral organs of the basal eudicot Eschscholzia californica as reference for floral evolutionary developmental studies. Genome Biol 11(10):R101
Park SU, Facchini PJ (2000) Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis. Plant Cell Rep 19(10):1006–1012
Orashakova S, Lange M, Lange S et al (2009) The CRABS CLAW ortholog from California poppy (Eschscholzia californica, Papaveraceae), EcCRC, is involved in floral meristem termination, gynoecium differentiation and ovule initiation. Plant J 58(4):682–693
Angelova S, Buchheim M, Frowitter D et al (2010) Overproduction of alkaloid phytoalexins in California poppy cells is associated with the co-expression of biosynthetic and stress-protective enzymes. Mol Plant 3(5):927–939
Liscombe DK, Ziegler J, Schmidt J et al (2009) Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species. Plant J 4:729–743
Vogel M, Lawson M, Sippl W et al (2010) Structure and mechanism of sanguinarine reductase, an enzyme of alkaloid detoxification. J Biol Chem 285(24):18397–18406
Weiss D, Baumert A, Vogel M et al (2006) Sanguinarine reductase, a key enzyme of benzophenanthridine detoxification. Plant Cell Environ 29(2):291–302
Donaire L, Barajas D, Martínez-García B et al (2008) Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs. J Virol 82(11):5167–5177
Waterhouse PM, Fusaro AF (2006) Plant science. Viruses face a double defense by plant small RNAs. Science 313(5783):54–55
Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130(3):413–426
Kalantidis K, Schumacher HT, Alexiadis T et al (2008) RNA silencing movement in plants. Biol Cell 100(1):13–26
Liu Y, Schiff M, Marathe R et al (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30(4):415–429
Yellina A, Orashakova S, Lange S et al (2010) Floral homeotic C function genes repress specific B function genes in the carpel whorl of the basal eudicot California poppy (Eschscholzia californica). EvoDevo 1(1):1–13
Koncz C, Schell J (1986) The promoter of TLDNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204(3):383–396
Mersereau M, Pazour GJ, Das A (1990) Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90(1):149–151
Shen WJ, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17(20):8385
Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622
Dorak MT (2006) Real-time PCR. In: Owen E (ed) BIOS Advanced Methods. Taylor & Francis Group, New York, p 127
Acknowledgments
The E. californica work in A.B.’s lab is largely funded by the German Research Foundation (DFG grants BE 2547/6-1, 6-2, 7-2, 8-1).
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Tekleyohans, D.G., Lange, S., Becker, A. (2013). Virus-Induced Gene Silencing of the Alkaloid-Producing Basal Eudicot Model Plant Eschscholzia californica (California Poppy). In: Becker, A. (eds) Virus-Induced Gene Silencing. Methods in Molecular Biology, vol 975. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-278-0_7
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DOI: https://doi.org/10.1007/978-1-62703-278-0_7
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