|Cazzonelli, Christopher - Australian National University|
Submitted to: Keystone Symposia
Publication Type: Proceedings
Publication Acceptance Date: 2/21/2010
Publication Date: 2/26/2010
Citation: Velten, J.P., Cakir, C., Cazzonelli, C. 2010. Gene dosage induction of silencing directed against an Arabidopsis Myb transgene in tobacco[abstract]. Keystone Symposia: RNA Silencing Mechanisms in Plants, February 21-26, 2010, Santa Fe, New Mexico.
Technical Abstract: An unexpected reduction in petal pigmentation on petunia plants genetically engineered for enhanced flower color was one of the first experimental demonstrations of the natural process of RNA-associated gene silencing. The obvious visual nature of such alterations to pigment patterns of transgenic plant lines made a relatively infrequent event stand out, allowing researchers to begin the process of exploring the underlying molecular processes involved. We have been exploring the use of genetically engineered anthocyanin over-production as a visual indicator of gene activation and silencing in plants. Previous work has demonstrated that constitutive over-expression of genes encoding specific Arabidopsis transcription factors from the myb family (AtMYB75 and AtMYB90) can result in ectopic over-production of anthocyanin pigments in Arabidopsis and several other plant species (Xia, et. al , The Plant Cell,12:2383). The resulting pigment accumulation leads to a clearly visible red-to-purple color in the leaves, stems and flowers of select transgenic lines. As part of the process of examining the utility of MYB-induced ectopic pigmentation as a visible reporter of gene silencing we have generated several transgenic N. tabacum lines that constitutively over-express the Arabidopsis PAP2, or AtMyb90, gene (CaMV35S::AtMYB90). Several of the resulting transgenic lines display extensive anthocyanin pigmentation in nearly all tissues, but are otherwise identical to wild type plants. Transgene associated patterns and levels of anthocyanin production within these transgenic lines are were found to be subject to gene silencing initiated by the production of double stranded RNA (dsRNA) targeting the AtMYB90 transgene. Transgene silencing, as indicated by a clear reduction in pigment production and a concomitant drop in steady state transgene mRNA levels, can be induced by Agrobacterium infiltration with constructs that generate hairpin RNA covering most of the AtMyb90 coding region. Silencing is observed both locally, at the infiltration site, and systemically, in newly developing leaves above the site of infiltration. One line, Myb27, was found to show similar patterns of pigment loss (and mRNA reduction) when transgene dosage was doubled (homozygous at the transgene locus). Hemizygous Myb27 plants are dark purple, as opposed to homozygotes that show only limited anthocyanin pigmentation within segments of the vegetative tissues. The loss of pigmentation within homozygous Myb27 plants correlates with reduced transgene mRNA levels and the production of siRNAs targeting the AtMyb90 transgene.