Generation of VIGS TRV2-AqPDS and TRV2-AqANS constructs
Total RNA was extracted from Aquilegia vulgaris leaf tissue using Plant RNA Isolation Reagent (Invitrogen, Carlsbad, CA) and mRNA was purified using Magnetite Oligo (dT) Particles (Novagen (EMD), San Diego, CA). cDNA was generated from 500 ng of mRNA using Superscript II Reverse Transcriptase™ (Invitrogen). The AqPDS sequence was isolated through PCR using degenerate primers from conserved regions of the PDS gene (PDS-F: 5'-TGGAARGARCAYTCIATGATWTTTGCWATG-3' and PDS-R: 5'-ACRACATGRTACTTIAVDATYTTWGCTTT-3', Figure 1). The primary AqPDS fragment was then sequenced to confirm its identity [DQ923721] using BigDye Terminator® (Applied Biosystems, Foster City, CA) and re-amplified using locus-specific primers with appended restriction sites (PDS-F2-XbaI: 5'-GGTCTAGACAGCCGATTTGATTTCCCAGAT-3' and PDS-R3-BamH1: 5'-AAGGATCCGAGAATTGAGTCGGACTTCACC-3', Figure 1). The AqANS sequence was obtained from the Aquilegia EST database [DR946275 and DR946276], and primers were designed to amplify a 440 bp fragment of the gene (ANS-F-Xba1: 5'-GGTCTAGATTGGGATTGGAAGAAGAAAGGC-3' and ANS-R-BamH1: 5'-AAGGATCCATGTTGAGCAAATGTGCGA-3', Figure 1).
Both gene products were double digested with XbaI and BamH1, as was the TRV2 vector, and ligated separately using T4 DNA ligase (New England Biolabs, Ipswich, MA). The resulting constructs were transformed into heat-shock competent E.coli (TOP10 cells, Invitrogen) and plated on selective LB media containing 50 μg/mL kanamycin. Colonies were PCR screened for the presence of the modified constructs using primers 156 F (5'-TTACTCAAGGAAGCACGATGAGC-3') and 156 R (5'-GAACCGTAGTTTAATGTCTTCGGG-3'), which span the multiple cloning site in TRV2. Both constructs were then purified (Fast-Plasmid Mini kit, Eppendorf, Hamburg, Germany) and the identity of the final constructs was verified by sequencing with the 156 F and 156 R primers and BigDye Terminator®
Germination of A. vulgaris seedlings and growth of adult plants
When germinating Aquilegia vulgaris seed, we found that seeds soaked in distilled water for 24 hours, planted in soil and then stratified at 4°C for at least 3 weeks show the best germination rates. Seeds germinate approximately 10 days after being removed from stratification. More mature seed, stored for 6–8 months at 4°C, had higher germination rates than fresh seed or seed stored for 1–2 months. Treatment of seed with gibberellic acid (GA) at varied concentrations did not improve germination of either fresh or aged seed, however it has been previously reported that seed stored at 20°C may have overall higher germination rates and benefit from GA treatment . Similar rates of germination were observed for seeds planted in all-purpose nursery soil (Fafard 3 B Mix) versus a 1:1 mixture of fine soil and vermiculite. For the TRV2-AqPDS-VIGS experiment, A. vulgaris seeds (collected from the Harvard University experimental garden) were soaked in distilled water for 24 hours at room temperature and then planted 1/8 inch deep in soil. They were lightly watered, covered, and stratified in the dark for 4 weeks at 4°C. After stratification, they were moved to a growth chamber at 20°C under long days until germination was observed. For the TRV2-AqANS-VIGS experiment, seedlings were transplanted and allowed to grow for 12 weeks or more, until they had at least 15 leaves, and then vernalized for 8 weeks either outdoors in winter or in a growth chamber on short days at 4°C. They were then removed to a 20°C chamber and treated 1–2 weeks afterward.
Preparation of Agrobacterium
Electrocompetent cells of Agrobacterium strain GV3101 were prepared and transformed as described by Weigel and Glazebrook (2002). Agrobacterium and unmodified TRV vectors were kindly provided by S.P. Dinesh-Kumar, Yale University. Transformation was achieved using 1 μL of purified construct DNA per 50 μL competent cells shocked at 2.4 kV using an ECM399 electroporator (BTX Genetronics, San Diego, CA). After 1 hour incubation at 25°C, the cells were plated on selective media (50 μg/mL kanamycin, 50 μg/mL gentamycin, and 25 μg/mL rifampicin) and grown for 3–4 days at room temperature. Colonies were PCR screened using primers 156 F and 156 R for the presence of TRV2-AqPDS or TRV2-AqANS. Glycerol stocks were made from single positive transformants.
Vacuum infiltration of TRV1 and TRV2-AqPDS constructs into A. vulgaris
Agrobacterium transformants carrying TRV1 and either TRV2 or TRV2-AqPDS were streaked onto selective plates and single colonies were selected for growth in separate liquid cultures (50 μg/mL kanamycin, 50 μg/mL gentamycin, and 25 μg/mL rifampicin). Cultures were sequentially inoculated into a final volume of 550 mL and grown for 72 hours with shaking at room temperature. The cultures were collected by centrifugation at 4000 g for 10–15 minutes and each resuspended to an OD600 of approximately 2.0 in an infiltration buffer containing 10 mM MES (2-[N-morpholino]ethanesulfonic acid), 200 μM acetosyringone (3'5'Dimethoxy-4'-hydroxyacetophenone), and 10 mM MgCl2. The solutions were then incubated at room temperature for 3–4 hours.
Soil was removed from seedlings with 1–5 true leaves by gently floating them in distilled water. They were then immersed in a 1:1 mixture of the two Agrobacterium solutions with added 100 uL/L Silwet L-77 (Lehle Seeds, Round Rock, TX) and infiltrated under vacuum for 2 minutes. For each batch, approximately 15 seedlings were infiltrated per liter of bacterial mixture, with up to 126 seedlings treated per round. The seedlings were drained, transplanted to fresh soil, covered with clear plastic for at least 24 hours, and grown under long days (14 h light, 10 h dark). Over the course of four replicate experiments, a total of 229 A. vulgaris seedlings were mock-treated with TRV1 and the empty TRV2 construct, and 406 were treated with TRV1 and the TRV2-AqPDS construct.
Injection of TRV1 and TRV2-AqANS into adult A. vulgaris plants
Transformed TRV1 and TRV2-AqANS Agrobacterium cultures were grown, resuspended, incubated, and combined as described above for the vacuum infiltration procedure, except cultures were only grown for 36 hrs to a volume of 50 mL. 1–2 weeks after adult plants were removed from vernalization, a wound about 1 cm deep was made by applying a clean razor blade to a small area at the base of the basal rosette of each plant, where the leaf bases converge. Approximately 1 mL of Agrobacterium mix was injected at the site with a needle-less 1 mL syringe. Depending on the size of the plant and the number of crowns (lateral rosettes), this was done at multiple sites. The procedure was repeated at weekly intervals for 5–6 weeks with new injection sites created with each treatment.
RT-PCR analyses of viral transcripts and AqPDS expression
Leaves were scored for phenotype 5–6 weeks post infiltration. Leaves exhibiting a range of silencing phenotypes were collected and flash frozen at -80°C. Tissue was collected from a total of 96 plants (including untreated plants). Total RNA was extracted from the tissue and purified using the RNeasy Plant Mini kit (Qiagen, Valencia, CA). Each total RNA sample was DNAse treated to remove residual genomic DNA and the concentration was adjusted to 0.3 μg/μL. Three separate cDNA pools were prepared for each RNA sample using 3.3 μL of DNase-treated RNA, Superscript II Reverse Transcriptase (Invitrogen), and reverse primers specific for TRV1 (pYL156 R, ), TRV2 (OYL198, ), or a poly-T primer . A total of 61 samples were PCR screened for the presence of viral RNAs using primers pYL156F/R (for TRV2, ) and OYL195/198 (for TRV1, ). To determine relative levels of AqPDS expression, we first established the linear range of amplification for ACTIN and AqPDS in our polyT cDNA samples (25–29 cycles was optimal for ACTIN and 28–32 cycles for AqPDS) and then used semi-quantitative RT-PCR analysis on cDNAs from 7 to 9 tissue samples per seedling category for a total of 64 samples. ACTIN and AqPDS were amplified in separate reactions (ACTIN for 27 cycles, AqPDS for 29 cycles). Primers for the ACTIN control were ACT1/2  and for AqPDS were PDS-F2 (5'-CAGCCGATTTGATTTCCCAGATGTTCTTCCAGCAC-3') and PDS-R4 (5'-AATCTCTTTCCACTCCTCGGGCAG-3') (Figure 1). Note that due to the G/C rich sequence of the R4 primer region, a longer F2 primer was necessary to achieve comparable Tm values. PCR reactions were separated by electrophoresis in a 1% agarose gel containing 0.5 mg/L ethidium bromide. DNA band intensities were UV imaged on an Alpha Innotech ChemiImager at levels below saturation and calibrated against a low DNA mass ladder (Invitrogen) using AlphaEase FC imaging software (Alpha Innotech, San Leandro, CA). A subset of reactions was repeated twice for accuracy and the final AqPDS/ACTIN values were normalized to the lowest ratio.
RT-PCR analyses of viral transcripts and AqANS expression
Floral tissue exhibiting a range of silencing was collected and flash frozen at -80°C 1–2 weeks after the last injection treatment. Tissue was collected from a total of 27 plants, and 52 flowers (including untreated plants). Total RNA was extracted from Aquilegia combined sepal and petal tissue using Plant RNA Isolation Reagent (Invitrogen, Carlsbad, CA).). Each total RNA sample was DNAse treated to remove residual genomic DNA. Three separate cDNA pools were prepared for each RNA sample using 0.9 μg of DNase-treated RNA, Superscript II Reverse Transcriptase (Invitrogen), and reverse primers specific for TRV1 (pYL156R), TRV2 (OYL198) or the poly-T primer. A total of 29 samples from 22 separate plants were PCR screened for the presence of viral RNAs as described above.
To determine relative levels of AqANS expression, we first established the linear range of amplification for ACTIN and AqANS in our polyT cDNA samples, using cDNA diluted 1:200 (30–38 cycles was optimal for ACTIN and 27–33 cycles was optimal for AqANS). We then performed semi-quantitative RT-PCR analysis on cDNAs from 30 tissue samples (Figure 7). ACTIN and AqANS were amplified in separate reactions (ACTIN for 34 cycles, AqANS for 31 cycles). Primers for the ACTIN control were ACT1/2, and for AqANS were ANS-F2 (5'-AGTTCATTCCCAAGGAGTATGTGC-3') and ANS-R2 (5'-TACTTTTTACCCACTGACGGT-3') (Figure 1). ANS-F2 is outside of the TRV2-AqANS insert region (to avoid detecting background plasmid ANS sequences), and the screen region also spans a genomic intron to help avoid detection of any residual genomic AqANS sequences (Figure 1). Similar to the AqPDS analysis, PCR reactions were separated by electrophoresis in a 1% agarose gel containing 0.5 mg/L ethidium bromide. DNA band intensities were UV imaged on an Alpha Innotech ChemiImager at levels below saturation and calibrated against a low DNA mass ladder. A subset of reactions was repeated twice to test for precision and improve accuracy, and the final AqANS/ACTIN values were normalized to the lowest ratio.
Morphological characterization of TRV2-AqANS silenced flowers
Floral organs showing strong silencing of AqANS were identified based on their bright white color. Light photographs of dissected organs were prepared using a Kontron Elektronik ProgRes 3012 digital camera mounted on a Leica WILD M10 dissecting microscope (Harvard Imaging Center). For scanning electron microscopy (SEM) studies, dissected floral organs were fixed under vacuum in FAA (50% ethanol, 4% formalin, and 5% glacial acetic acid) and then transferred to 70% EtOH for storage. Before imaging, organs were dehydrated through an ethyl alcohol series and critical point-dried with CO2 in a Tousimis/Autosamdri-815 dryer. Material was mounted on aluminum stubs with carbon conductive adhesive tabs (Electron Microscopy Sciences, Ft. Washington, PA), sputter-coated with gold palladium in a Denton/Desk II, and studied in a model Quanta 200 SEM (FEI company, Hillsboro, OR, USA) at 5–20 kv.
The generation of the TRV2-AqPI-AqANS construct is described in Kramer et al. . Culture preparation and injection was performed as described for TRV2-AqANS. RT-PCR was performed as described for TRV2-AqANS and in Kramer et al. .