silencing in single copy transgenic tobacco plants having the same T-DNA construct was observed; therefore, the silencing phenom- enon might occur in a species-specific manner. Furthermore, the Cauliflower mosaic virus (CaMV) 35S promoter (35Spro) [12,13] seemed to be the target for silencing in gentian, because methylated cytosines were frequently detected in the 35Spro region, and that transgenic gentian introduced with the rolC promoter did not exhibit silencing [11,14]. However, it is difficult to discuss causal factor(s) for the silencing phenomenon, because the previous study used T-DNA constructs containing two 35Spro sequences and an endogenous MADS-box gene, GtMADS4. Hence, the possibility that the tandem structure of the 35Spro and/or the use of an endogenous gene might be a potent inducer for gentian silencing still remains. To clarify this, the present study used a vector containing a sole 35Spro connected to a sGFP reporter gene within the T-DNA region, and obtained transgenic gentian plants having single copies of the 35S construct within their genome. We also studied de novo methylation using asymmetric sequence contexts, in which cytosine methylation is frequently observed in higher plants [15,16]. Maintenance DNA methyltransferase (e.g. MET1 in Arabidopsis) recognizes hemimethylated CpG sites and methylates the cytosine in the unmethylated DNA strand [17]; therefore, the methylation status is likely to be retained at the CpG dinucleotide context. In plants, cytosine methylation on other sequence contexts, such as CpWpG (W is A or T) and asymmetric cytosine sequences (designated as CpHpH; H is A, C or T), is also found. Domains rearranged methyltransferase (DRM) class and chromomethylase (CMT) class of DNA methyltransferases in 35S De Novo Methylation Arabidopsis are involved in methylation of non-CpG sequences [16]. DRM class methyltransferases have homology to mamma- lian Dnmt3 de novo methyltransferase, whereas CMT class methyltransferases are unique to plants and maintain non-CpG (primarily CpWpG) methylation [2,18]. Asymmetric (CpHpH) methylation can only be maintained by de novo methylation [19], supporting the idea that monitoring of asymmetric methylation might reveal a ‘‘hot spot’’ region of de novo methylation in the plant genome. Conversely, the state of symmetric methylation is difficult to determine when de novo methylation has occurred. Herein, we describe the strict sequence-specific transgene silencing phenomenon that occurs in gentian. A defined region of the 35S enhancer was revealed as a possible target sequence for the strict silencing apparatus by analysis of asymmetric methyla- tion in transgenic plants having a single copy of unmodified- and modified 35S promoters. Silencing did not occur in transgenic tobacco, therefore the silencing machinery is thought to be specifically diversified in gentian and might contribute to genomic homeostasis against parasitic sequences. Results Production of single copy transgenic gentian and tobacco plants We produced transgenic gentian plants into which a single copy of the 35Spro was introduced by Agrobacterium-mediated transfor- mation. The T-DNA region of the binary vector, pSMABR35SsGFP (Figure 1A), did not use any endogenous sequence from the gentian genome or any (inverted) repeat Figure 1. Structures of the T-DNA regions and molecular characteristics of transgenic gentians. (A) A T-DNA construct of the binary vector pSMABR35SsGFP (above) and schematic diagrams of the modified 35S promoters. Modified nucleotides are indicated as underlined letters [35S(Das-1)]. Altered cytosine residues are marked with asterisks [35S(nos-1)]. The gray regions indicate CHS promoter core regions of petunia [35S(PhCHS)] and gentian [35S(GtCHS)], respectively. 59 UTRs of the CHS promoters are indicated as thick black arrows. Positions of primers used for bisulfite-PCR are indicated as small arrows. (B) Representation of single copy transgene in the unmodified 35S transgenic gentian plants by Southern blot analysis using HindIII-digested genomic DNAs with the bar gene probe. (C) Northern blots of sGFP and bar genes in leaf tissues of the single copy unmodified 35S transgenic gentian and tobacco plants. Wild-type (WT) gentian plant was also used as a control. doi:10.1371/journal.pone.0009670.g001 PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9670