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Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco

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Abstract

Small interfering RNA (siRNA) species with 21–25 nucleotides in length guide mRNA cleavage, translational arrest, and heterochromatin formation in RNA interference (RNAi). To delineate the target region of RNAi, a construct harboring a transcriptional fusion between parts of the target mRNA and the β-glucuronidase gene was biolistically delivered into tobacco leaves showing an RNAi phenotype and the assay sequence was transiently expressed. The RNAi effect was monitored by amplification of this chimeric transcript. By using this assay method, we addressed the transitive RNA silencing of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3). In the NtFAD3 RNAi plants, the target region of RNAi was restricted in the inducer region corresponding to a stem sequence of the hairpin double-stranded RNA, indicating that endogenous NtFAD3 mRNA was not a template for an RNA-dependent RNA polymerase. The secondary NtFAD3 siRNAs were produced in the crossbred plants between the NtFAD3 overexpressed plant and the NtFAD3 RNAi plant. Similarly, the secondary siRNAs were generated in the systemically silenced scion. Although these secondary siRNAs originated preferentially from the 3′ region downstream of the inducer region, the secondary siRNAs produced in the silenced scion (non-cell autonomous secondary siRNAs) resulted in the strong degradation of the target mRNA, but the secondary siRNAs in the crossbred plants (cell-autonomous secondary siRNAs) showed limited RNA degradation activity. These results showed that this in vivo assay for determination of RNAi efficiency is a useful tool to delineate RNAi mechanisms.

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Abbreviations

CaMV:

Cauliflower mosaic virus

cRNA:

Complimentary RNA

dsRNA:

Double-stranded RNA

IR-PTGS:

Inverted repeat-post-transcriptional gene silencing

PTGS:

Post-transcriptional gene silencing

RdRP:

RNA-dependent RNA polymerase

RISC:

RNA-induced silencing complex

RNAi:

RNA interference

siRNA:

Small interfering RNA

S-PTGS:

Sense-post-transcriptional gene silencing

VIGS:

Virus-induced gene silencing

WT:

Wild type

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Acknowledgement

This study was supported partly by a Grant-in Aid for Scientific Research (17570030) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba , 263-8522, Japan

    Katsuyoshi Shimamura, Shin-ichiro Oka & Yumi Shimotori

  2. Department of Bioproduction Science, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba , 271-8510, Japan

    Takashi Ohmori & Hiroaki Kodama

Authors
  1. Katsuyoshi Shimamura
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  2. Shin-ichiro Oka
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  3. Yumi Shimotori
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  4. Takashi Ohmori
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  5. Hiroaki Kodama
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Correspondence to Hiroaki Kodama.

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Shimamura, K., Oka, Si., Shimotori, Y. et al. Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco. Plant Mol Biol 63, 803–813 (2007). https://doi.org/10.1007/s11103-006-9124-9

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  • DOI: https://doi.org/10.1007/s11103-006-9124-9

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