Constructs for plant transformation
The binary plasmid vectors pLX222GUS and pBI22SAR2E are based on the binary vector pLX222  and on the gus gene fusion plasmid pBI221. They both contain the kanamycin resistance gene nptII as selectable marker. The P35S-gus-Tnos reporter gene constructs present in these plasmids were described in previous work . Binary vectors were mobilized from Escherichia coli to the Agrobacterium tumefaciens strain LBA4404 for subsequent plant transformation.
Plant transformation and propagation
Transgenic plants were obtained after leaf disk transformation of N. tabacum var. W38, followed by shoot and root regeneration, as described previously . Homozygous F3 lines of transformants LX222GUS-A 6b, BI22SAR2E 13a and BI22SAR2E 16b were obtained by two back-crosses of primary transformants with W38 tobacco, followed by selfing. F1 progeny of a young (18 months) LX222GUS-A 9b primary transformant (short TC) were obtained from cuttings. After long-term in vitro cultivation (long TC) of transformant LX222GUS-A 9b on MSO agar for 10 years, leaf regenerants were transferred to the greenhouse for the production of `long TC´ T1 progeny by selfing. Seeds were surface sterilized with 0.25 % sodium hypochlorite, washed with distilled water and germinated on MSO agar supplemented with 100 mg/l kanamycin. Segregation ratios for antibiotic resistant and sensitive plants were recorded after 5 weeks. Kanamycin resistant (KanR) transgenic progeny plants were further propagated on MSO medium. Plants were kept in a climate chamber (16 h/8 h light period, 2400 lux) at 22°C, before they were transferred to the greenhouse for further crossings.
Greenhouse cultivation and cross-hybridization
To obtain hybrid progeny, a hemizygous F1 plant (F1-3) and a hemizygous T1 plant (T1-1) of the `short TC´ respectively the `long TC´ LX222GUS-A9b epigenetic variant was subject to cross-hybridizations with homozygous F3 plants of three different transgenic Nicotiana tabacum lines. In addition backcrosses with non-transgenic N. tabacum var. W38 were performed. Young kanamycin resistant seedlings were transferred from the climate chamber to the greenhouse, potted into TKS 1 soil and grown at 24°C (14 h light, 10.000 lux). At flowering time transgenic tobacco plants were emasculated and pollinated using pollen from the different transgenic lines or from non-transgenic N. tabacum var. W38. Mature seed pods were harvested and stored at room temperature.
Genomic DNA isolation and molecular analyses
Genomic DNA from plant leaves was extracted either according to  or using the NucleoSpin Plant II kit (Macherey-Nagel). Hybrid progeny plants were characterized with respect to the presence or absence of the LX222GUS-A9b transgene insert by Southern blot analysis or by differential polymerase chain reactions (PCR). For Southern blot analysis ten micrograms of plant DNA digested with the restriction enzyme HindIII were separated in a 0.8 % agarose gel and transferred to a Hybond-N (Amersham™/ GE Healthcare) nylon membrane . Membrane blots were hybridized according to the manufacturer´s protocol with a digoxigenin (DIG)-labeled DNA probe (Roche Applied Science) specific for nptII in order to detect left border fragments of individual LX222GUS transgenic lines. For differential PCR the primers 35S-2 (5´-GATAGTGGGATTGTGCG-3´) and Npt2 (5´-TAGCCAACGCTATGTCCT-3´) amplifying a 1920 base pair (bp) fragment were used to detect the LX222GUS-A9b insert, while GN151 (5´-TCAACTGACACCAAACTTAG-3´) and GN152 (5´-ACACAAACTGCTTATTATCT-3´) amplifying a 1096 bp fragment were used as primers for the detection of BI22SAR2E inserts. Amplification reactions containing 0.2 µM primers, 100 µM deoxynucleotides, 1.5 mM MgCl2, 1x GreenFlexi buffer and 1 unit GoTaq DNA polymerase (both Promega) were incubated after initial denaturation at 94°C in a thermocycler for 35 cycles with annealing temperatures between 52°C and 53°C and 5 min final elongation at 72°C. PCR products were analyzed in 1.8 % agarose gels.
Analysis of the molecular structure of T-DNA integration
For the determination of DNA sequences at left border (LB) and right border (RB) integration sites of LX222GUS-A 9b, inverse PCR assays were performed. T-DNA primers to be used in inverse PCR were pretested by PCRs to ensure the presence of binding sites in the transformed plant DNA. For RB analysis DNA of a `short TC´ plant was cut with the restriction enzyme AseI which has a recognition site about 500 bp from the RB, followed by re-ligation. The resulting fragment consisting of known transgene sequences and of unknown flanking sequences was cut with a second restriction enzyme yielding an RB-containing fragment which allowed amplification of flanking DNA sequences by using the T-DNA primers iPCR1 (5´-CATAAAGTGTAAAGCCTGG-3´) and iPCR2 (5´-GACTCCCTTAATTCTCCG-3´). For LB analysis DNA was restricted with MaeI, cutting about 490 bp from the putative left border, re-ligated and cut with StuI, before flanking DNA was amplified using primers LB1.1 (5´-GTCCGCAATGTGTTATTAAG-3´) and iPCRLB (5´-CGATTGCTCATCGTCATT-3´). Cycling conditions for inverse PCRs were: denaturation at 94°C for 2 min; 40 cycles of 94°C for 30 sec, 52°C for 30 sec, 72°C for 2 min + 2 sec/cycle; final elongation at 72°C for 10 min. The PCR products were cloned into a TA vector (pGEM-T; Promega), followed by sequence analysis of individual clones (GATC Biotech/Eurofins Genomics). For further T-DNA integration analysis the following primers were used: Tn3-1 (5´-GATCGGCGGAAAGGTCAACC-3´) from the newly identified DNA sequence flanking the right border, iPCR2, lacZ1 (5´-ATGCCTGCAGGTCGACTCTA-3´), GUSbi9 (5´-GTGGAGTGAAGAGTATTAGTGTG-3´) and the Tn5393-specific primers Tn3-a (5´- GAGCTTCATGGTGCTCCAGAA-3´), Tn3-c (5´-TGACCGCCTCATTTGGCTCAA-3´) and Tn3-d (5´-CATGATGCAGATCGCCATGTA-3´) reported by Kim and An .
Recombinant A. tumefaciens strain LBA4404 bacteria transformed with binary vectors pLX222GUS or pBI22SAR2E were grown overnight at 28°C and 200 rpm in 10 ml LB medium supplemented with 100 µg/ml rifampicin and 5 µg/ml tetracycline. Cells were harvested by centrifugation, washed with 20 ml 10 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 5.7; 10 mM MgCl2 and re-suspended in MMA medium (10 mM MES, pH 5.7; 10 mM MgCl2, 200 µM acetosyringone) to an OD600 of about 1.0. Subsequently, the cultures were incubated for 2-3 h at room temperature.
Leaves No. 4 to No. 6 from the top of different Nicotiana tabacum plants were infiltrated. The bacterial suspensions were applied to the lower side of the leaves using a 2 ml syringe without a needle. Agroinfiltrations were done in replicate for each plant and bacterial strain. Leaf samples were taken before infiltration and from infiltrated areas 6 days post infiltration for further testing.
GUS enzyme assays
b-glucuronidase (GUS) expression was analyzed by a spectrophotometric enzyme assay according to Jefferson . Samples from the sixth leaf from the top were collected from stably transformed plants. 100 mg leaf tissue was homogenized in 100 µl extraction buffer (50 mM sodium phosphate, pH 7.0, 1 mM EDTA, 0.1% Triton X-100). After centrifugation for 5 min at 15 800 g, 4°C the concentration of soluble protein in the supernatant was quantified using the BioRad Protein Assay . For the spectrophotometric GUS assay 35 µg of total protein was used with 1 mM p-nitrophenyl-ß-D-glucuronide as substrate. Reactions were stopped at 0, 30 and 60 min by adding 0.4 volumes of 1 M 2-amino-2-methyl-2,3-propanediol. Absorbance of the reaction product p-nitrophenol (p-NP) was measured at 415 nm and calibrated against a dilution series of p-nitrophenol (Sigma).
DNA methylation analysis through bisulfite sequencing
DNA methylation was analyzed through bisulfite conversion of unmethylated cytosine residues and subsequent DNA sequencing. DNA was digested with an appropriate restriction enzyme to allow full denaturation during the conversion step. Bisulfite treatments were carried out using the EZ DNA Methylation-GoldTM Kit (Zymo Research) following the manufacturer’s instructions. Bisulfite-treated DNA was subjected to PCR amplifications as described above. Amplification with primers 35Sbi-3 (5’-TAAGGTAAGTAATAGAGATTGGAG -3’) and 35Sbi-6 (5’-TCTCTCCAAATAAAATAAACTTC-3’) resulted in a 584 bp fragment covering the entire P35S promoter sequence as well as vector sequences. With the primers GUSbi-1 (5’-AGGAAGTTTATTTTATTTGGAG-3’) and GUSbi-4 (5’-CACCACTTACAAAATCCC-3’) a 777 bp fragment was amplified comprising the 5’ region of the gus coding sequence. For bisulfite methylation analysis of the 3´ region of the gus coding sequence and the adjacent Tnos terminator, the primers GUSbi9 (5´-GTGGAGTGAAGAGTATTAGTGTG-3´) and LXbi1 (5´-ACCTCTTCACTATTACRCCRC-3´) were used to amplify a 692 bp fragment specific for LX222GUS-A inserts, while GUSbi9 (5´-GTGGAGTGAAGAGTATTAGTGTG-3´) and SARbi1 (5´-AATACTCCCACTAATCATAATTTC-3´) were used to amplify a 719 bp fragment specific for the BI22SAR2E inserts. The PCR products were cloned into pGEM-T (Promega) and 2 to 10 clones from each amplification reaction were sequenced (GATC Biotech/Eurofins Genomics). Sequence data were analyzed and methylation densities were calculated using CyMATE software . As is shown in Fig. 2a, CyMATE analysis of the 35S promoter started at position -343 of the promoter sequence and ended at the TATA box (-24). For the 5´gus gene, analysis started 43 bp upstream of the start codon and continued up to position 693 of the coding sequence. For the 3´gus-Tnos region it started at position 1583 of the coding sequence, encompassed the complete nos-terminator and ended about 95 bp respectively 120 bp downstream.
RNA extraction and siRNA detection by Northern blot analysis
RNA from plant leaves was extracted using NucleoZOL (Macherey-Nagel), thereby separating small RNAs (10 – 200 nt) from the large RNA fraction (> 200 nt). For Northern blot analysis 2 µg of denatured small RNAs were separated overnight on a 15% denaturing polyacrylamide/7 M urea gel and transferred onto a positively charged nylon membrane through electroblotting (Semi-dry Blotter/Hoefer). Following chemical crosslinking with 1-Ethyl-3(3-dimethylaminopropyl)-Carbodiimid (EDC)  the membrane was hybridized with digoxigenin (DIG)-labeled RNA probes (Roche Applied Science) specific for the 3´-end (660 bp fragment) of the gus gene. Hybridizations were conducted overnight at 40°C in DIG Easy Hyb (Roche). The membrane was washed twice with 2x saline-sodium citrate (SSC); 0.1% SDS for 10 min at 40°C and then twice with 0.1× SSC; 0.1 % SDS for 15 min at 40°C. Immunological detection was performed with 1:10,000 anti-DIG-alkaline phosphatase (AP) (Roche). For AP detection the chemiluminescent substrate CDP-Star (Roche/Sigma-Aldrich) was used, followed by signal exposure on X-ray film (Agfa).