Plant Molecular Biology

, Volume 63, Issue 6, pp 803–813 | Cite as

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

  • Katsuyoshi Shimamura
  • Shin-ichiro Oka
  • Yumi Shimotori
  • Takashi Ohmori
  • Hiroaki Kodama
Article

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.

Keywords

Fatty acid desaturase RdRP RNA silencing Secondary siRNA Systemic gene silencing Tobacco 

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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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Katsuyoshi Shimamura
    • 1
  • Shin-ichiro Oka
    • 1
  • Yumi Shimotori
    • 1
  • Takashi Ohmori
    • 2
  • Hiroaki Kodama
    • 2
  1. 1.Graduate School of Science and TechnologyChiba UniversityChiba Japan
  2. 2.Department of Bioproduction Science, Faculty of HorticultureChiba UniversityChiba Japan

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