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Silencing induced by inverted repeat constructs in protoplasts of Nicotiana benthamiana

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Abstract

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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Abbreviations

CP :

Coat protein

dsRNA:

Double stranded RNA

GFP:

Green fluorescence protein

hpRNA:

Hairpin RNAs

IR:

Inverse repeat

NLS:

Nuclear localization signal

PBS:

Phosphate buffer solution

PTGS:

Post-transcriptional gene silencing

RNAi:

RNA interference

RPA:

Ribonuclease protection assays

siRNA:

Small interfering RNA

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Acknowledgments

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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Authors and Affiliations

  1. College of the Department of Horticulture and Foresty, National Indoor Conservation Center of Virus-free Gemplasms of Fruit Crops, Huazhong Agricultural University, Wuhan, China

    Ling Jiang, Zhuo Liu, Li Wang, Fan Zhang & Guo-cheng Liu

  2. College of the Life Science, Huazhong Agricultural University, Wuhan, China

    Jia Wang

  3. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 43007, Wuhan, Hubei, China

    Qi Zhong

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  1. Ling Jiang
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  2. Jia Wang
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  3. Zhuo Liu
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  4. Li Wang
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  5. Fan Zhang
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  6. Guo-cheng Liu
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  7. Qi Zhong
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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). https://doi.org/10.1007/s11240-009-9629-4

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