Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct Jeff Velten 1 *, Cahid Cakir 1 , Eunseog Youn 2 , Junping Chen 1 , Christopher I. Cazzonelli 3 1 United States Department of Agriculture - Agricultural Research Service, Lubbock, Texas, United States of America, 2 Department of Computer Science, Texas Tech University, Lubbock, Texas, United States of America, 3 Australian Research Council - Centre of Excellence in Plant Energy Biology, Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia 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 39 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. Citation: Velten J, Cakir C, Youn E, Chen J, Cazzonelli CI (2012) Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct. PLoS ONE 7(2): e30141. doi:10.1371/journal.pone.0030141 Editor: Georg Stoecklin, German Cancer Research Center, Germany Received October 19, 2011; Accepted December 10, 2011; Published February 17, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: Funding for this research is provided by the United States Department of Agriculture. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jeff.Velten@ars.usda.gov 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 ori- entation 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. Short, 21–24 nucleotide (nt), RNA molecules are increasingly im- plicated as important regulators of critical biological processes, includ- ing; tissue development, pathogen defense, stress response, and epi- genetic gene silencing in plants (for recent reviews see [10,11,12,13, 14,15,16,17,18]). Most of the regulatory activities associated with these smRNAs appear to involve direct alterations togene activity, impacting mRNA production, message stability, and/or translation. The ‘gold standard’ for inducing gene silencing in plants involves the production of double-stranded RNA (dsRNA), usually from genetic constructs engineered to produce self-complementary hairpin RNA transcripts [19,20,21]. The initiation of naturally occurring transgene silencing is also generally believed to involve the production of some form of double-stranded RNA. However, despite dramatic advances in our understanding of the molecular and biochemical components of plant gene silencing, exactly how and why it is initiated often remains unclear. Transgenes that have been rearranged or duplicated during integration into the host genome appear to be prone to silencing, possibly due to the direct production of complementary RNA. In systems involving virus-induced gene silencing (VIGS), it is thought that replicative intermediates, and viral RNA secondary structures, induce the production of small interfering RNA (siRNA), a process that may be amplified by RNA-dependent RNA polymerase (RdRP) activity to enhance disruption of virus replication and spread [6,22]. It becomes less clear what factor(s) trigger the initiation of silencing with transgenes that lack evidence of PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30141