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Silencing induced by inverted repeat constructs in protoplasts of Nicotiana benthamiana

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Double-stranded RNA interference can be used to silence gene expression in various organisms. Sustained RNAi-mediated gene silencing is typically triggered by hairpin RNAs (hpRNAs) generated by the transcription of inverted repeat (IR) DNA constructs. In this paper, we describe a transient expression assay that uses a green fluorescent protein marker gene (GFP) fused to the coat protein gene (CP) of papaya ring spot virus. This yields a nuclear and cytomembrane localized protein expression vector that can be used to monitor the silencing efficiency of inverted repeats. An ihpRNA was derived from a 215 nt IR segment of the 3′ end of the CP gene and recombined into the pHellsgate12 vector. We also recombined the 861 nt CP into the destination vectors pHellsgate12 and pWatergate using the GatewayTM technology. Nicotiana benthamiana protoplasts were co-transfected with (1) pGA, (2) pGA + pHellsgate12-CP IR, (3) pGA + pWatergate-CP IR, and (4) pGA + pHellsgate12-CP 215IR. Expression of the GFP fusion protein decreased by 77.7, 75.4, and 65.6% for the three constructs. Transfection with (2) and (3) yielded a silencing efficiency significantly higher than that of the (4) (P < 0.05), as measured by fluorescence intensity upon co-transfection, using the vector pGA-DsRed as an internal control. Depletions of 90.7, 91.5, and 87.5% of the CP transcript were observed by RT-PCR in protoplasts co-transfected with the (1) pGA, (2) pGA + pHellsgate12-CPIR, and (3) pGA + pWatergate-CPIR, (4) pGA + pHellsgate12-CP 215IR. Transient RNAi depletion of the target polypeptide was also detected by western blot analysis. Short RNA fragments complementary to the p6a and p7a antisense probes were detected by Ribonuclease Protection Assays.

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CP :

Coat protein


Double stranded RNA


Green fluorescence protein


Hairpin RNAs


Inverse repeat


Nuclear localization signal


Phosphate buffer solution


Post-transcriptional gene silencing


RNA interference


Ribonuclease protection assays


Small interfering RNA


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We thank CRIRO industry provided the destination vectors. We are grateful for the State Key Laboratory of Agricultural Microbiology of Huazhong Agriculture University provided the pGA482 vector and the equipments of phosphorimager and isotope label. Thanks for SRF (Student Researching Fund) project of Huazhong Agriculture University supported this work. This work was funded by the national natural science foundation of china projects (No. 30571277 and No. 30771477).

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Correspondence to Ling Jiang.

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Jiang, L., Wang, J., Liu, Z. et al. Silencing induced by inverted repeat constructs in protoplasts of Nicotiana benthamiana . Plant Cell Tiss Organ Cult 100, 139–148 (2010).

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