Title: RNAi-directed post transcriptional gene silencing of an Arabidopsis Myb transgene in tobacco Authors
Submitted to: Keystone Symposia
Publication Type: Proceedings
Publication Acceptance Date: February 19, 2010
Publication Date: February 26, 2010
Citation: Cakir, C., Velten, J.P. 2010. RNAi-directed post transcriptional gene silencing of an Arabidopsis Myb transgene in tobacco[abstract]. Keystone Symposia: RNA Silencing Mechanisms in Plants, February 21-26, 2010, Santa Fe, New Mexico. Technical Abstract: The AtMyb90 gene encodes the 'production of anthocyanin pigment 2' (PAP2) transcription factor of Arabidopsis thaliana and is able to induce a visible hyper-pigmented phenotype when expressed in tobacco. Based upon this phenotype, we have used the AtMyb90 gene as a reporter gene to examine RNAi-directed post transcriptional gene silencing in transgenic tobacco. Transient expression (via Agrobacteria leaf infiltration) using binary vectors containing CaMV35S::AtMyb90, pKOMyb (produces double-stranded RNA targeting AtMyb90), and CaMV35S::HcPro (produces the HcPro suppressor of silencing protein from potato virus X) were used to examine local silencing in two AtMyb90 transgenic lines (Myb27 and Myb237), an intron-hairpin AtMyb90 line (pKOMyb) and wild type tobacco plants. Co-expression of HcPro with CaMV35S::AtMyb90 dramatically enhanced AtMyb90 mRNA levels by inhibiting silencing. Additionally, the quantity and size distribution of AtMyb90 smRNAs within infiltrated leaf tissue was impacted by co-expression of the HcPro suppressor. The size and accumulation of small RNAs were dramatically changed depending on the construct used in binary vector and the transgenic line infiltrated, most notably with the inverted hairpin AtMyb containing construct and transgenic plants. Also, HcPro suppressor inhibited accumulation of some sizes of small RNAs, but not 22 nucleotide RNAs.