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. 2008 Mar;36(4):1220-6.
doi: 10.1093/nar/gkm1133. Epub 2007 Dec 26.

Phytohormone abscisic acid control RNA-dependent RNA polymerase 6 gene expression and post-transcriptional gene silencing in rice cells

Ji Hyun Yang  1 , Hyun Hee Seo, So Jung Han, Eun Kyung Yoon, Moon Sik Yang, Woo Sung Lee
Affiliations

Phytohormone abscisic acid control RNA-dependent RNA polymerase 6 gene expression and post-transcriptional gene silencing in rice cells

Ji Hyun Yang et al. Nucleic Acids Res. 2008 Mar.

Abstract

RNA-dependent RNA polymerase 6 (RDR6) catalyses dsRNA synthesis for post-transcriptional gene silencing (PTGS)-associated amplification and the generation of endogeneous siRNAs involved in developmental determinations or stress responses. The functional importance of RDR6 in PTGS led us to examine its connection to the cellular regulatory network by analyzing the hormonal responses of RDR6 gene expression in a cultured cell system. Delivery of dsRNA, prepared in vitro, into cultured rice (Oryza sativa cv. Japonica Dongjin) cells successfully silenced the target isocitrate lyase (ICL) transcripts. Silencing was transient in the absence of abscisic acid (ABA), while it became persistent in the presence of ABA in growth medium. A transcription assay of the OsRDR6 promoter showed that it was positively regulated by ABA. OsRDR6-dependent siRNA(ICL) generation was also significantly up-regulated by ABA. The results showed that, among the five rice OsRDR isogenes, only OsRDR6 was responsible for the observed ABA-mediated amplification and silencing of ICL transcripts. We propose that ABA modulates PTGS through the transcriptional control of the OsRDR6 gene.

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Figures

Figure 1.
Figure 1.
Formation of polyarginine(POA)/dsICL complex for silencing of ICL. (A) 10 mM acetate in medium gradually up-regulated the level of ICL transcripts during the 72 h period examined. (B) Formation of the dsRNA(ICL)/POA complex. To evaluate optimum complex formation, various concentrations of POA were mixed with a fixed concentration of dsRNA (lane 1, 100 ng of dsRNA only; lanes 2–11, 12.5, 25, 37.5, 50, 62.5, 75, 87.5, 100, 112.5 and 125 ng of POA mixed with 100 ng of dsRNA, respectively). As the POA concentration increased, dsRNA mobility was gradually retarded. The POA concentration in lane 8 was chosen as the most effective delivery-complex.
Figure 2.
Figure 2.
PTGS of ICL transcripts and generation of siRNA. (A) Silencing of ICL transcripts in the absence (upper) and presence (bottom) of ABA. Following delivery of 10 μg of dsICL into cells pre-cultured for 48 h in acetate, the levels of ICL transcripts were monitored by RT–PCR. (B) Detection of siRNA in cells grown in the absence (upper) and presence (bottom) of ABA. Following the delivery of dsICL, appearance of siRNA was monitored. In dsICL-free control cells, siRNA did not appear at 48 h following the transfer into ABA-containing medium (C48).
Figure 3.
Figure 3.
ABA regulation of OsRDR6 gene expression. RT–PCR analyses of changes of OsRDR6 transcript level following the transfer into acetate medium without ABA (A) and with ABA (B). (C) Northern analyses of ABA up-regulation of OsRDR6 gene expression in cells grown in acetate (10 mM) or sucrose (3%). (D) Transcription assays of OsRDR6 promoter (1.2 kb). A promoter:luciferase construct was transfected into Arabidopsis protoplasts, and they were incubated with different plant hormones (upper), and with different concentrations of ABA (bottom). Variations of the three independent assays are shown.
Figure 4.
Figure 4.
ABA responses of rice RDR homologues. (A) RT–PCR analysis of ABA responsiveness of rice RDR homologues. (B) Silencing of OsRDR3a transcripts by delivering dsOsRDR3a, together with dsICL, into cells growing in acetate in the presence of ABA. Following the delivery of dsOsRDR3a, levels of ICL, OsRDR3a and OsRDR6 transcripts were evaluated by RT–PCR.
Figure 5.
Figure 5.
Characterization of RNA polymerization, performed with cell extracts in the presence (A) and absence (B) of siRNA primers. Sense-stranded RNA corresponding to the internal ICL region (0.5 kb) was used as a template for polymerization in the polymerization mixture containing α-32P-UTP. In the presence of ABA (right columns), both primed- and unprimed-polymerization appeared to be induced (see 150 and 500 bp expected polymerization products, respectively), while only unprimed polymerization operated in dsICL-delivered cells in the absence of ABA (B, left).
Figure 6.
Figure 6.
ABA regulation of tasiRNA-ARF generation. The tasiRNA-ARF level was measured by monitoring cleavage of ARF3 mRNA by primer extension (PE) assays. Two primers were designed to extend to the predicted cleavage sites and generate products of 260 bp (PE1) and 150 bp (PE2) (arrows).
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