Effect of Rice stripe virus NS3 on Transient Gene Expression and Transgene Co-Silencing

Abstract

Nonstructural protein 3 (NS3) encoded by RNA3 of Rice stripe virus (RSV), known to be a suppressor of gene silencing, was cloned and sequenced. The cloned NS3 gene is composed of 636 nucleotides encoding 211 deduced amino acids, and showed a high degree of similarity with the equivalent genes isolated from Korea, Japan and China. The NS3 gene promoted the enhancement of transient gene expression and suppressed transgene co-silencing. In the transient GFP expression via agroinfiltration, GFP expression was dramatically enhanced in terms of both protein yield and expression period in the presence of NS3. The highest accumulation of GFP protein reached to 6.8% of total soluble proteins, which corresponded to a two-fold increase compared to that obtained in the absence of NS3. In addition, NS3 significantly suppressed the initiation of GFP co-silencing induced by the additive GFP infiltration in GFP-transgenic Nicotiana benthamiana. The NS3 gene was also found to be a stronger suppressor than Cucumber mosaic virus 2b. These observations are believed to be derived from the strong suppressive effect of NS3 on gene silencing, and indicate that NS3 could be used as an effective enhancer for the rapid production of foreign proteins in plants.

Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana

“…To determine the dominant pathway of silencing, Agrobacterium cells harbouring pGreen0229-35S:GUS (35S-GUS) or 35S-GFP were co-infiltrated with the presence of viral suppressors, pGreen0229-35S:2b (35S-2b; Choi et al, 2008) and pGreen0229-35S:NS3 (35S-NS3; Sohn et al, 2011a). The 2b protein has the ability to enhance the expression of a co-infiltrated foreign gene by suppressing the onset of PTGS in plant cells (Choi et al, 2008).…”

“…The 2b protein has the ability to enhance the expression of a co-infiltrated foreign gene by suppressing the onset of PTGS in plant cells (Choi et al, 2008). Non-structural protein 3 (NS3) of a rice stripe virus (RSV) also displays powerful enhancement of transient expression of foreign genes (Sohn et al, 2011a). Our detailed agroinfiltration procedures are as previously described (Choi et al, 2008).…”

“…The analysis by Sau96I digestion was a simple method to approximately examine the extent of DNA methylation in the 35S-GFP cassette (Jones et al, 1999;Vaistij et al, 2002). The steady-state level of GFP RNA transcripts and detection of siRNAs were performed as previously described (Sohn et al, 2011a). For hybridization and visualization of bands, GFP or bar genes labelled with 32 P-dCTPs were used as the probes, as previously reported (Sohn et al, 2011a).…”

“…The steady-state level of GFP RNA transcripts and detection of siRNAs were performed as previously described (Sohn et al, 2011a). For hybridization and visualization of bands, GFP or bar genes labelled with 32 P-dCTPs were used as the probes, as previously reported (Sohn et al, 2011a).…”

Phenotypic and Transcriptomic Analysis of Nicotiana benthamiana Expressing Cucumber mosaic virus 2b gene

“…2b RNA RNA(satellite RNA) (Ding , 1995). agroinfiltration , 2b (Choi , 2008;Sohn , 2011a (Choi , 2008) Agrobacterium tumefaciens AGL1 (Choi , 2008;Sohn , 2011b). 2b pGreen0229-…”