Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Jeff Velten,Eunseog Youn,Cahid Cakir,Christopher I Cazzonelli,Junping Chen

doi:10.1371/journal.pone.0030141

Abstract

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.

Highlights

Dramatic variability of transgene expression, including complete silencing of the introduced gene or genes, has been a factor impacting the success of plant genetic engineering since its inception
Following out-crossing of the R1 progeny to wild type tobacco, the partially pigmented Myb27 plants were all found to be homozygous for the introduced AtMYB90 transgene (27Hmo)
Silencing of single copy transgenes occurs in a subset of plant transformation events and has occasionally been found to be triggered by the doubling of transgene copy number associated with plants made homozygous for the introduced DNA [24,25,26,27,28,29,30,31, 32]

Summary

Introduction

Dramatic variability of transgene expression, including complete silencing of the introduced gene or genes, has been a factor impacting the success of plant genetic engineering since its inception. The observed variability in expression levels of what appear to be identical transgene constructs has been linked to multiple molecular factors such as high transcription levels, alterations to the copy number and orientation of introduced DNA, and the characteristics of closely linked plant genetic material [1,2,3,4,5,6]. Co-suppression of unlinked homologous plant genes is often associated with transgene silencing and represents one of the first published observations of RNA-based gene regulation [7,8,9]. Silencing of introduced transgenes is frequently attributed to post-transcriptional gene silencing (PTGS), one of many small RNA (smRNA) based molecular processes occurring in plants. Most of the regulatory activities associated with these smRNAs appear to involve direct alterations togene activity, impacting mRNA production, message stability, and/or translation

Methods

Results

Conclusion