Umut TOPRAK, Cathy COUTU, Doug BALDWIN, Martin ERLANDSON, Dwayne HEGEDUS

Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation

Plant-mediated RNA interference (RNAi) has shown a great potential in pest control and requires i) subcloning of sense/antisense regions in compatible vectors, ii) transfer of the silencing cassette into a binary vector, iii) transformation of Agrobacterium tumefaciens with desired binary plasmids, and iv) transformation of plants with Agrobacterium. The procedure is long and should ensure plasmid backbone stability; however, plasmid recombination due to antibiotic selection is a common problem. pGSA1252 is an RNAi silencing binary vector allowing direct cloning of hairpin structure; however, it possesses a chloramphenicol selection marker leading to plasmid recombination in various Agrobacterium strains. To solve this selection marker/Agrobacterium compatibility problem and to shorten the cloning process, we developed a new RNAi vector system containing the RNAi cassette of pGSA1252 in a plant expression vector, pMDC32, which has kanamycin as the selection marker. A T7 RNA polymerase promoter was also incorporated adjacent to the multiple cloning site, allowing for in vitro dsRNA synthesis. This vector was tested by transforming Arabidopsis thaliana with 4 different dsRNA constructs specific to insect midgut genes: insect intestinal mucin 1/4, peritrophic matrix protein 1, chitin deacetylase 1, and chitin synthase-B. This improved system shows no recombination and shortens the entire cloning procedure.

Anahtar Kelimeler:

Key words: RNA interference, plant transformation, Agrobacterium tumefaciens, chloramphenicol, kanamycin, Arabidopsis thaliana

Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation

Keywords:

Key words: RNA interference, plant transformation, Agrobacterium tumefaciens, chloramphenicol, kanamycin, Arabidopsis thaliana,

PDF

___

Arakane Y, Muthukrishnan S, Kramer KJ, Specht CA, Tomoyasu Y, Lorenzen MD, Kanost MR, Beeman RW (2005). The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix, respectively. Insect Mol Biol 14: 453–463.
Ballester S, Lopez P, Alonso JC, Espinosa M, Lacks SA (1986). Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids. Gene 41: 153–163.
Ballester S, Lopez P, Espinosa M, Alonso JC, Lacks SA (1989). Plasmid structural instability associated with pC194 replication functions. J Bacteriol 171: 2271–2277.
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M et al. (2007). Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25: 1322–1326.
Bechtold N, Ellis J, Pelletier G (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci Paris, Life Sciences 316: 1194– 1199.
Bent AF (2006). Arabidopsis thaliana floral dip transformation method. In: Wang K, editor. Agrobacterium Protocols. Totowa, NJ, USA: Humana Press, pp. 87–103.
Fairbairn DJ, Cavallaro AS, Bernard M, Mahalinga-Iyer J, Graham MW, Botella JR (2007). Host-delivered RNAi: an effective strategy to silence genes in plant parasitic nematodes. Planta 226: 1525–1533.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998). Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 391: 806–811.
Gordon KHJ, Waterhouse PM (2007). RNAi for insect-proof plants. Nat Biotechnol 25: 1231–1232.
Huang G, Allen R, Davis EL, Baum TJ, Hussey RS (2006). Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc Natl Acad Sci USA 103: 14302–14306.
Huvenne H, Smagghe G (2010). Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J Insect Physiol 56: 227–235.
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY (2007). Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25: 1307–1313.
Pitino M, Coleman AD, Maffei ME, Ridout CJ, Hogenhout SA (2011). Silencing of aphid genes by dsRNA feeding from plants. PLoS ONE 6:e25709.
Rajagopal R, Sivakumar S, Agrawal N, Malhotra P, Bhatnagar RK (2002). Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. J Biol Chem 277: 46849–46851.
Rogers EJ, Rahman MS, Hill RT, Lovett PS (2002). The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation. J Bacteriol 184: 4296–4300.
Shi X, Chamankhah M, Visal-Shah S, Hemmingsen SM, Erlandson M, Braun L, Alting-Mees M, Khachatourians GG, O’grady M, Hegedus DD (2004). Modeling the structure of the type I peritrophic matrix: characterization of a Mamestra configurata intestinal mucin and novel peritrophin containing 19 chitin binding domains. Insect Biochem Mol Biol 34: 1101–1115.
Szakasits D, Siddique S, Bohlmann H (2007). An improved pPZP vector for Agrobacterium-mediated plant transformation. Plant Mol Biol Rep 25: 115–120.
Tennigkeit J, Matzura H (1991). Nucleotide sequence analysis of a chloramphenicol-resistance determinant from Agrobacterium tumefaciens and identification of its gene product. Gene 98: 113–116.
Toprak U, Baldwin D, Erlandson M, Gillott C, Hou X, Coutu C, Hegedus DD (2008). A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix. Insect Mol Biol 17: 573–585.
Toprak U, Baldwin D, Erlandson M, Gillott C, Hegedus DD (2010). Insect intestinal mucins and serine proteases associated with the peritrophic matrix from feeding, starved and molting Mamestra configurata larvae. Insect Mol Biol 19: 163–175.
Toprak U, Baldwin D, Erlandson M, Gillott C, Harris S, Hegedus DD (2013). In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system. Insect Science 20: 92–100.
Turner CT, Davy MW, MacDiarmid RM, Plummer KM, Birch NP, Newcomb RD (2006). RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by doublestranded RNA feeding. Insect Mol Biol 15: 383–391.
Zha W, Peng X, Chen R, Du B, Zhu L, He G (2011). Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. PLoS ONE 6: e20504.
Zhang M, Wang Q, Xu K, Meng Y, Quan J, Shan W (2011). Production of dsRNA sequences in the host plant is not sufficient to initiate gene silencing in the colonizing oomycete pathogen Phytophthora parasitica. PLoS ONE 6: e28114.
Zhu Q, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S (2008). Functional specialization among insect chitinase family genes revealed by RNA interference. Proc Natl Acad Sci USA 105: 6650–6655