A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts

Plant Methods - 2006
Rebecca Bart1, Mawsheng Chern1, Chang‐Jin Park1, Laura E. Bartley1, Pamela C. Ronald1
1Department of Plant Pathology, University of California at Davis, Davis, California, USA

Tóm tắt

Abstract Background Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. Results We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7–14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60–70% transformation efficiency. In contrast, using 50 μg of a 12 kb plasmid we obtained a maximum of 25–30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. Conclusion We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.

Từ khóa


Tài liệu tham khảo

De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inze D, Goossens A, Hilson P: Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J. 2005, 44: 1065-1076.

Gregory DW, Cocking EC: The Large-Scale Isolation Of Protoplasts From Immature Tomato Fruit. J Cell Biol. 1965, 24: 143-146.

Cocking EC: The isolation of plant protoplasts. Methods Enzymol. 1974, 31: 578-583.

Asai T, Stone JM, Heard JE, Kovtun Y, Yorgey P, Sheen J, Ausubel FM: Fumonisin Bl-induced cell death in arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways. Plant Cell. 2000, 12: 1823-1836.

Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J: MAP kinase signalling cascade in Arabidopsis innate immunity. Nature. 2002, 415: 977-983.

Hattori T, Terada T, Hamasuna S: Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1. Plant J. 1995, 7: 913-925.

Hobo T, Kowyama Y, Hattori T: A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription. Proc Natl Acad Sci USA. 1999, 96: 15348-15353.

Kagaya Y, Hobo T, Murata M, Ban A, Hattori T: Abscisic acid-induced transcription is mediated by phosphorylation of an abscisic acid response element binding factor, TRAB1. Plant Cell. 2002, 14: 3177-3189.

Sugimoto K, Takeda S, Hirochika H: Transcriptional activation mediated by binding of a plant GATA-type zinc finger protein AGP1 to the AG-motif (AGATCCAA) of the wound-inducible Myb gene NtMyb2. Plant J. 2003, 36: 550-564.

Meyer A, Eskandari S, Grallath S, Rentsch D: AtGATl, a high affinity transporter for gamma-aminobutyric acid in Arabidopsis thaliana. J Biol Chem. 2006.

Pih KT, Yi MJ, Liang YS, Shin BJ, Cho MJ, Hwang I, Son D: Molecular cloning and targeting of a fibrillarin homolog from Arabidopsis. Plant Physiol. 2000, 123: 51-58.

Yao N, Greenberg JT: Arabidopsis ACCELERATED CELL DEATH2 Modulates Programmed Cell Death. Plant Cell. 2005.

Sheen J: Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 2001, 127: 1466-1475.

Tabara H, Grishok A, Mello CC: RNAi in C. elegans: soaking in the genome sequence. Science. 1998, 282: 430-431.

Vanitharani R, Chellappan P, Fauquet CM: Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc Natl Acad Sci USA. 2003, 100: 9632-9636.

Chern M, Fitzgerald HA, Canlas PE, Navarre DA, Ronald PC: Overexpression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light. Mol Plant Microbe Interact. 2005, 18: 511-520.

Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH: A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science. 1995, 270: 1804-1806.

An CI, Sawada A, Fukusaki E, Kobayashi A: A transient RNA interference assay system using Arabidopsis protoplasts. Biosci Biotechnol Biochem. 2003, 67: 2674-2677.

An CI, Sawada A, Kawaguchi Y, Fukusaki E, Kobayashi A: Transient RNAi induction against endogenous genes in Arabidopsis protoplasts using in vitro-prepared double-stranded RNA. Biosci Biotechnol Biochem. 2005, 69: 415-418.

Zentella R, Yamauchi D, Ho TH: Molecular dissection of the gibberellin/abscisic acid signaling pathways by transiently expressed RNA interference in barley aleurone cells. Plant Cell. 2002, 14: 2289-2301.

Holzberg S, Brosio P, Gross C, Pogue GP: Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J. 2002, 30: 315-327.

Christensen AH, Quail PH: Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 1996, 5: 213-218.

Chern MS, Bobb AJ, Bustos MM: The regulator of MAT2 (ROM2) protein binds to early maturation promoters and represses PvALF-activated transcription. Plant Cell. 1996, 8: 305-321.

Park CJ, Kim KJ, Shin R, Park JM, Shin YC, Paek KH: Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J. 2004, 37: 186-198.

Thông tin
Thông tin xuất bản

Plant Methods

Thông tin tác giả