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. 2007 Dec;145(4):1272-81.
doi: 10.1104/pp.107.106062. Epub 2007 Aug 31.

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

Mery Dafny-Yelin  1 , Sang-Min Chung, Ellen L Frankman, Tzvi Tzfira
Affiliations

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

Mery Dafny-Yelin et al. Plant Physiol. 2007 Dec.

Abstract

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.

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Figures

Figure 1.
Figure 1.
Structural features of the pSAT-RNAi vector system. A, The general structure of a pSAT-RNAi plasmid and its cloning compatibility with pRCS2-based binary plasmid. A typical pSAT-RNAi plasmid is composed of a promoter and terminator region, the ChsA intron, and two unique multicloning sites, MCS-I and MCS-II, which should be used for cloning the two target gene sequences in reverse orientations. The entire hpRNA cassette is flanked by AgeI and NotI, allowing its transfer between different pSAT plasmid backbones. The hpRNA cassette is also flanked with rare cutters (e.g. PI-PspI), which can be used for its cloning into the pRCS2-based binary plasmid. B, Outline of the different pSAT-RNAi plasmids produced in this study. Expression of hpRNA is controlled by various promoters and terminators and each of the various RNAi cassettes can be transferred to the pRCS2-based binary plasmid using different combinations of rare-cutting endonucleases, as indicated. Both MCS-I and MCS-II are present in all plasmids and restriction recognition sites that are not unique in all plasmids are indicated by asterisks. Plasmid names from top to bottom are: pSAT3.masP.RNAi, pSAT4.35SP.RNAi, pSAT5.nosP.RNAi, pSAT6.masP.RNAi, pSAT6.35SP.RNAi, pSAT6.supP.RNAi, and pSAT6.rbcP.RNAi. 35S, CaMV 35S promoter and terminator; sup, superpromoter; mas, manopine synthase promoter and terminator; rbc1, Rubisco small-subunit promoter and terminator; nos, nopaline synthase promoter and terminator; ags, agropine synthase terminator. One, two, and three asterisks represent BspEI, BamHI, and XbaI restriction sites, respectively.
Figure 2.
Figure 2.
Outline of the pRCS2-[EGFP][KAN][DsRed2] binary plasmid. The plasmids constructed to carry three cassettes for the stable overexpression of the EGFP, NPTII, and DsRed2 coding sequences. Expression cassettes were cloned as an AgeI-NotI fragment into the different pSAT plasmids and were successively transferred as I-PpoI, I-SceI, and PI-PspI fragments into the pRCS2 binary plasmid. 2x35S, Tandem CaMV 35S promoter; 35ST, CaMV 35S terminator; nos, nopaline synthase promoter and terminator; rbc1, Rubisco small-subunit promoter and terminator.
Figure 3.
Figure 3.
Confocal microscopy analysis of EGFP and DsRed2 expression in single- and double-transformed plants. A to D, Confocal imaging of pRCS2-[EGFP][KAN][DsRed2] (designated KGD plants). E to H, Confocal imaging of KGD transformed by GFPi construct. I to L, Confocal imaging of KGD transformed by DsRed2i construct. M to P, Confocal imaging of KGD transformed by GFPi and DsRed2i constructs. A, E, I, and M, EGFP signal (green, when present). B, F, J, and N, DsRed2 signal (red, when present). C, G, K, and O, Plastid autofluorescence in blue. D, H, L, and P, Merge signals of the corresponding EGFP, DsRed2, and plastid autofluorescence. Images are single confocal sections (bar = 50 μm).
Figure 4.
Figure 4.
KAN resistance regeneration assay of single- and double-transformed plants. Leaf discs of diverse transgenic tobacco plants were placed on shoot regeneration medium supplemented with KAN and documented following several weeks in culture. w.t., Wild type, nontransformed plants; KGD, single-transgenic plants produced by overexpressing the KAN, EGFP, and DsRed2 coding genes; KGD-Ki, KGD plants transformed with an nptII silencing cassette (KANi); KGD-Di, KGD plants transformed with a DsRed2-silencing cassette (DsRed2i); KGD-Gi, KGD plants transformed with a gfp silencing cassette (GFPi); KGD-GiDi, KGD plants transformed with GFPi and DsRed2i; KGD-GiDiKi, KGD plants transformed with GFPi, DsRed2i, and KANi; KGD-bar, KGD plants transformed with empty binary vector.
Figure 5.
Figure 5.
Northern-blot analysis of nptII expression in transgenic tobacco plants. Top, Detection of the nptII transcript by digoxigenin-labeled nptII probe; bottom, total RNA loading. w.t., Wild-type, nontransformed plants; KGD, single-transgenic plants produced by overexpressing the KAN, EGFP, and DsRed2 genes; KGD-Gi, KGD plants transformed with a gfp silencing cassette (GFPi); KGD-Ki, KGD plants transformed with an nptII silencing cassette (KANi); KGD-GiDi, KGD plants transformed with GFPi and dsRed2 silencing cassette (DsRed2i); KGD-GiDiKi, KGD plants transformed with GFPi, DsRed2i, and KANi.
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References

    1. Abbott JC, Barakate A, Pincon G, Legrand M, Lapierre C, Mila I, Schuch W, Halpin C (2002) Simultaneous suppression of multiple genes by single transgenes: down-regulation of three unrelated lignin biosynthetic genes in tobacco. Plant Physiol 128 844–853 - PMC - PubMed
    1. Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67 657–685 - PMC - PubMed
    1. Bruening G (1998) Plant gene silencing regularized. Proc Natl Acad Sci USA 95 13349–13351 - PMC - PubMed
    1. Bucher E, Lohuis D, van Poppel PM, Geerts-Dimitriadou C, Goldbach R, Prins M (2006) Multiple virus resistance at a high frequency using a single transgene construct. J Gen Virol 87 3697–3701 - PubMed
    1. Chabannes M, Barakate A, Lapierre C, Marita JM, Ralph J, Pean M, Danoun S, Halpin C, Grima-Pettenati J, Boudet AM (2001) Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. Plant J 28 257–270 - PubMed

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