Synthesis of CPNs
The fabrication of CPNs was previously described [23] (Figure 7). Briefly, an amine-containing poly(phenylene ethynylene) (PPE) was synthesized by polymerizing 13,13'-(2,5-diethynyl-1,4-phenylene)bis(oxy)bis(2,5,8,11-tetraoxatridecane) and 2,2'(2,2'-(2,5-dibromo-1,4-phenylene)bis(ethane-2,1-diyl))bis(oxy)diethanamine in a mixed solvent of N-methyl pyrrolidone and morpholine using palladium/copper catalysts. The PPE solution was treated with excess amounts of glacial acetic acid followed by dialysis (10,000 MWCO) against dH2O. Final PPE- dH2O solution was filtered using a syringe filter (0.45 μm) and stored at room temperature.
Compound (1) is 13,13'-(2,5-dibromo-1,4-phenylene)bis(oxy)bis(2,5,8,11-tetraoxatridecane) and was prepared by incubating at 80°C overnight a suspension of 2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}thyl 4-methylbenzenesulfonate1 (20.6 mM; 5.9 g), 2,5-dibromohydroquinone (10.3 mMl; 2.76 g) and K2CO3 (103 mM; 14 g) in 30 mL of dimethyl formamide (DMF). The mixture was concentrated in vacuo and diluted with 50 mL of dichloromethane. The solution was washed with three times with 20 mL dH2O, dried over Na2SO4, and evaporated in vacuo. The crude product was purified by column chromatography (silica gel, ethyl acetate/hexane (3:1, v/v). Yield : 3.7 g (55%). 1H NMR(600 MHz) : δ = 7.15 (s, 1H, Ar-H, J = 6), 4.12 (t, 4H, Ar-OCH2, J = 6), 3.87 (t, 4H, OCH2, J = 6), 3.76 (t, 4H, OCH2, J = 6), 3.69-3.63 (m, 16H, OCH2), 3.55 (t, 4H, OCH2, J = 6), 3.37 (s, 6H, CH3); 13C NMR(150 MHz) : δ = 150.5, 119.4, 111.6, 72.1, 71.3, 70.9, 70.8, 70.72, 70.4, 69.8, 59.2.
Compound (2) is (2,5-bis(2,5,8,11-tetraoxatridecan-13-yloxy)-1,4-phenylene)bis(ethyne-2,1-diyl)bis(trimethylsilane) and was prepared by adding Compound (1) (5 g, 7.7 mM) to a shrink flask fitted with a stir bar and Pd(PPh3)2Cl2 (0.54 g, 0.77 mM) and CuI (0.073 g, 0.39 mM). Thirty mL of a 2:1 mixture of tetrahydrofuran and diisopropylamine was added to the reaction. Following addition of trimethylsilylacetylene (4.4 mL, 31 mM), the reaction mixture was heated to 60°C for 12 h. After evaporating solvent, the crude mixture was dissolved in methylenechloride and washed twice with 30 mL saturated ammonium chloride, followed by drying over anhydrous MgSO4. The solvent was evaporated to produce dark brown oil, which was purified by column chromatography (silica gel, ethyl acetate/hexane (4:1, v/v)). Yield : 5 g (95%). 1H NMR(600 MHz) : δ = 6.91 (s, 1H, Ar-H), 4.12 (t, 4H, Ar-OCH2, J = 6), 3.87 (t, 4H, OCH2, J = 6), 3.78 (t, 4H, OCH2, J = 6), 3.68-3.66 (m, 12H, OCH2), 3.64 (t, 4H, OCH2, J = 6), 3.54 (t, 4H, OCH2, J = 6), 3.38 (s, 6H, CH3), 0.25 (s, 9H, SiMe3); 13C NMR(150 MHz) : δ = 154.0, 117.9, 114.3, 100.9, 100.4, 72.0, 71.2, 70.8, 70.7, 70.6, 69.7, 69.6, 59.0, 0.0.
Compound (3) is 13,13'-(2,5-diethynyl-1,4-phenylene)bis(oxy)bis(2,5,8,11-tetraoxatridecane). A 100 mL round-flask was charged with the compound (2) (2.5 g, 3.7 mM), 50 mL tetrahydrofuran, and 10 mL MeOH. Then 5 mL of 1 M KOH (aq) solution was added to the reaction mixture and stirred for 2 h. The solvent was evaporated and the reaction mixture was purified by column chromatography (silica gel, ethyl acetate). Yield : 1.1 g (90%). 1H NMR(400 MHz) : δ = 7.00 (s, 1H, Ar-H), 4.15 (t, 4H, Ar-OCH2, J = 6), 3.87 (t, 4H, OCH2, J = 6), 3.76 (dd, 4H, OCH2, J = 6), 3.73-3.63 (m, 16H, OCH2, J = 6), 3.55 (dd, 4H, OCH2, J = 6), 3.38 (s, 6H, CH3, J = 6), 3.35 (s, 2H, CH); 13C NMR(150 MHz) : δ = 154.1, 118.3, 113.6, 82.8, 79.6, 71.9, 71.1, 70.7, 70.6, 70.5, 69.6, 69.5,59.0.
Preparation of siRNAs for cellulose synthases, NtCesA-1a and NtCesA-1b
TRIzol® reagent was used to extract total RNA from tobacco (N. tabacum) leaves (Invitrogen Corp, Carlsbad, CA). cDNA of the putative cellulose synthase (AF233892), NtCesA-1a and NtCesA-1b was prepared using Superscript III reverse transcriptase (Life Technologies) and then a fragment of the gene was PCR amplified (640 base pairs (bp) using forward (5'- AGTGTATGTGGGTACCGGATG- 3') and reverse (5'- CCATATGGGACA ATGCCTAC - 3') primers. Following a 5-min denaturation at 94°C, PCR was performed for 34 cycles of 94°C for 2 min, 94°C for 15 s, 50°C for 30 s, and 68°C for 45 s, followed by final 5-min extension at 68°C [25]. The 640 bp PCR product was purified using PCR Preps DNA Purification system (Promega, Madison,WI) and cloned into the pGEM-T Easy vector (Promega) according to manufacturer's instructions. The nucleotide sequence of this cDNA fragment was confirmed as 100% and 98% identical for NtCesA-1a and NtCesA-1b, respectively. To generate sense and anti sense RNA, pGEM-T:NtCesA was linearized using NcoI or SalI and in vitro transcription was performed (RiboMAX, Promega) with SP6 and T7 RNA polymerases. Transcription products were purified using MEGAclear Kit (Ambion, Austin, TX). Sense and anti sense RNAs were annealed in annealing buffer (100 mM,KOAc, 4 mM MgCl2 and 60 mM HEPES- KOH, pH 7.4), boiled for 5 min, and incubated overnight at 37°C [37]. The resulting double strand RNAs were precipitated using ethanol and then dissolved in nuclease-free double distilled (dd) H2O. siRNAs were generated by treating double stranded NtCesA-1 with recombinant Dicer enzyme according to the manufacturer's instructions (Gene Therapy Systems, San Diego, CA). The reaction was stopped by adding the Dicer stop solution and 22 bp products were detected using 3% agarose gel electrophoresis [37]. The final siRNA products were purified using RNA purification column 1(Gene Therapy Systems) and dissolved in nuclease-free ddH2O.
BY-2 protoplast preparation
Typically, BY-2 cells are grown are subcultured from a 4 d-old liquid culture by transferring 10 mL BY-2 cells to 40 mL fresh BY-2 culture medium (Murashige and Skoog salts pH 5.6 (Sigma-Aldrich Co, St. Louis, MO), 30 g.L-1 sucrose, 256 mg.L-1 KH2PO4, 100 mg.L-1 myo-inositol, 1 mg.L-1 thiamine, and 0.2 mg.L-1 2,4-dichlorophenoxyacetic acid). Cells are grown on a rotary shaker that is maintained in the dark at 120 rpm at 28°C.
Protoplasts were prepared from 3 d old tobacco BY-2 suspension cells using standard methods [52, 53]. Ten ml of packed BY-2 cells (sedimented by centrifugation at 100-g for 5 min) were resuspended in 100 mL of enzyme solution (1.5% cellulase "Onozuka RS" (Yakult Pharmaceutical Ind. Co. Ltd., Tokyo, Japan), 0.2% macerase (Calbiochem-Novabiochem Corp., La Jolla, CA), 0.5 M mannitol, and 3.6 mM 2-(N-morpholino) ethanesulfonic acid (pH 5.5) ) in a 1 L flask and incubated for 3-4 h at 28°C on a rotary shaker at 100 rpm. Protoplasts were recovered by filtration through 41 μm nylon mesh (Spectrum Laboratories, Inc., Rancho Dominguez, CA), and washed twice with Protoplast Wash Solution (0.5 M mannitol, 3.6 mM 2-(N-morpholino) ethanesulfonic acid (pH 5.5) at 59 g for 5 min. Protoplasts were resuspended in Protoplast Resuspension Solution (BY-2 culture media plus 0.45 M mannitol) to a density about 1 × 106 protoplasts mL-1. Protoplast viability was measured using 0.1% fluorescein diacetate prepared in 1 ml of 50 mM phosphate buffer (pH 7.4). For siRNA delivery experiments, protoplasts were cultured in cell wall regeneration medium.
siRNA delivery to protoplasts and intact BY-2 cells
Protoplasts (1 × 106mL-1) or intact BY-2 cells (1 × 106mL-1) were mixed with CPNs in Protoplast Resuspension Solution to a final concentration of 0, 5, 10, 15 and 20 μM and added to 6-well culture plates (Corning Inc., Corning, NY). Each well of the culture plate was lined with Protoplast Resuspension Solution plus 1.0% agarose (pH 5.6). Protoplasts and BY-2 cells were cultured at 28°C. Samples were harvested at 2, 5, 10 and 24 h and the proportion of CPN containing protoplasts and intact BY-2 cells were determined using a haemocytometer. In addition, the proportion of protoplasts or cells for which fluorescence was seen to be associating with the cell wall, plasma membrane, nucleus, and/or cytoplasm was recorded.
Protoplasts were resuspended in Protoplast Resuspension Solution (BY-2 culture media plus 0.45 M mannitol) to a density about 1 × 105 protoplasts.mL-1. CPNs (10 or 25 μM) were incubated with 200 nM siGLO Red for 3 h (Thermo Fisher Scientific, Pittsburgh, PA) and the complex was delivered to protoplasts. CPNs were incubated with 200 nM NtCesA-1 siRNAs overnight and then delivered to protoplasts. We found that mixing CPNs and siGLO Red for 3 h was sufficient to demonstrate transfection of CPN-siRNA complexes, but overnight mixing of CPNs and NtCesA-1 siRNAs improved complexation and improved siRNA delivery to plant protoplasts resulting in a measurable phenotype. Protoplast cultures were maintained in the dark at 28°C for 2 h and 24 h. Controls included treating protoplasts with Protoplast Resuspension Solution, 200 nM siGLO Red only, NtCesA-1 siRNAs or CPNs only. The protoplast containing both CPNs and siGLO Red were counted using a haemocytometer.
Propidium iodide and FM4-64 dye treatment
Propidium iodide, contained in the Plant Cell Viability Assay Kit (Sigma-Aldrich Co), was solubilized according to manufacturer's instructions. FM4-64 (Invitrogen Corp) staining was carried out to monitor CPN uptake, as previously described [54]. Protoplasts (1 × 105.mL-1) were incubated with 10 μM CPNs for 24 h at 28°C. 20,000 protoplasts (which were previously treated with medium or 10 μM CPNs and incubated for 24 h at 28°C) were incubated with 10 μM FM4-64 at room temperature for 10 min and then monitored using confocal microscopy.
Epifluorescence and confocal microscopy
A Nikon E600 (Nikon Corp., Tokyo, Japan) epifluorescence microscope with a B2A filter cube (470- to 490-nm excitation filter), a DM505 dichroic mirror, and a BA520 barrier filter was used to monitor FDA staining following enzymatic digestion of BY-2 cells and to study uptake of CPNs protoplasts and intact BY-2 cells. Propidium iodide was detected in protoplasts using a UV filter cube. siGLO Red fluorescence (absorption/emission maximum at 557 nm/570 nm) was viewed using a Y-2E/C TX red filter cube containing a 540- to 580-nm excitation filter, a DM595 dichroic mirror, and a BA600-660 barrier filter. Images were captured using the Optronics Magnafire camera (Optronics Inc., Goleta, CA) and were edited using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA). Haemocytometer observations were recorded using Microsoft Excel software.
A Leica TCS SP2 (Leica Microsystems, Bannockburn, IL) confocal imaging system attached to a Leica DME 14 upright microscope equipped with Ar/Kr lasers were used to study BY-2 cells treated with CPNs and FM4-64 staining protoplasts. Serial images were collected using 0.3 μm steps and 3-D images of 100 μm thick sections were compiled.
Fluorescence activated cell sorting (FACS) flow-cytometry of treated BY-2 protoplasts
A Becton Dickinson FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) equipped with an Ar laser (excitation of 488 nm) was used to assess CPN-uptake by protoplasts. Protoplasts (1 × 106.mL-1) were mixed with 10 μM and 20 μM CPNs in Protoplast Resuspension Solution and added to 6-well culture plates (Corning Inc., Corning, NY) containing Protoplast Resuspension Solution plus 1.0% agarose (pH 5.7). Protoplasts treated with buffer, or a 1:1 mixture of untreated plus CPN-treated protoplasts were used as controls. Protoplasts were cultured at 28°C and FACS was performed at 2 h and 24 h of incubation.
The sorting capability of 10000 cells.s-1 and fluorescence emission (FL1-H, FL2-H) was detected using a 530 nm and 585 nm band pass filters. The percentages of fluorescence-emitting protoplasts were assessed as evidence of CPNs and siGLO Red uptake by protoplasts. Data were analyzed on a Macintosh computer equipped with BD CellQuest Pro program (Becton Dickinson) and were presented as two dimensional dot plots which represent CPN fluorescence emissions on the X-axis and siGLO Red fluorescence emissions on Y- axis. Data was compiled using Adobe Photoshop software.
Semi-quantitative RT-PCR of silenced protoplasts
Semi-quantitative RT-PCR was utilized to monitor NtCesA transcriptional levels following siRNA delivery. Extraction of total RNA from BY-2 protoplasts was carried out using SV Total RNA Isolation System (Promega, Madison, WI). The first strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen Corp), 1 μg total RNA and oligo(dT) primers. PCR was performed using NtCesA-1 specific forward primer (5'-AGTGTA TGTGGGTACCGGATG-3') and NtCesA reverse (5'-CCATATGGGACAATGCCTAC-3') primer that also shares homology with NtCesA-2. Forward (5'-GCCTCCGTGGTGGTG CTAAG- 3'), and reverse (5'-TCAATCGGCACC GGCCTT G-3') primers were used to amplify ubiquitin (AY912494) cDNA (261 bp) as the internal control. Following 10-min denaturation at 95°C, PCR was performed for 30, 35, 40, 45 cycles of 95°C for 15 s and 60°C for 60 s. PCR products were analyzed using ethidium bromide stained 1% agarose gel. Gels were scanned using Alpha Image system (Alpha Innotech, San Leandro, CA) and the images were recorded. Densitometry was performed by Alpha Ease FC software (Alpha Innotech).