Constructs, bacterial strain, antibodies and recombinant proteins
A random piece of 50-kb yeast genomic DNA was isolated using CHEF Yeast Genomic DNA Plug Kit (Cat#170–3593, Bio-Rad, Hercules, CA) and a 5 kb Arabidopsis genomic DNA fragment was individually cloned into BIBAC2 between the two NotI sites as single copy binary vectors pB50 and pB5, respectively. The same 5 kb Arabidopsis genomic DNA fragment was cloned into pCAMBIA1301 at the HindIII site to serve as a multi-copy binary vector pC5. Each binary vector was introduced into Agrobacterium strain AGL1 individually. AGL1 is a recA-deficient derivative of A. tumefaciens strain C58  and was acquired from Dr. Lynn Dahleen, USDA, ARS, Fargo, ND. Binary vectors BIBAC2 and pCAMBIA1301 were acquired from Cornell University, Center for Technology Enterprise and Commercialization (CCTEC), and CAMBIA (Canberra, Australia), respectively. The antibody against VirD2 was kindly provided by Dr. Zambryski at UC-Berkeley  and Dr. C. Baron at the University of Montreal (Montreal, Canada).
For expression and purification of the recombinant protein, a full-length gene of virD2 (amplified with primers virD2Fwd: 5’-GGGTCCATGGATATCGGGATGCCCG ATCGCGCTCA AG-3′, and virD2Rev: 5’-TGCTCGAGTGCGGCCGCACTAGGTC CCCCCGCGCC CA-3′) was cloned into the bacterial expression vector pET42b (Novogen, Hornsby Westfield, Australia) between BamHI and HindIII using Gibson assembly (New England Biolabs, Ipswich, MA). 6xHis-tagged GST fusion protein was expressed in Escherichia coli strain BL21 (DE3) (Agilent Technologies, Santa Clara, CA). The cell pellets were lysed by French press in the lysis buffer containing 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The cell extract was prepared by centrifugation at 20,000 g for 20 min at 4 °C. The cleared cell extract was incubated with His60 Ni Superflow Resin (Clontech, Mountain View, CA) equilibrated in the lysis buffer at 4 °C for 2 h. The recombinant proteins were subsequently purified in the gravity-flow column following the manufacturer’s instructions (Clontech).
For Agrobacterium samples, Agrobacterium strain AGL1 harboring various binary vectors was grown overnight in 150 ml YEP medium containing 25 mg/L rifampicin and 50 mg/L kanamycin at 28 °C. Cells were then centrifuged and re-suspended in 150 ml AB induction medium . The OD600 reading of the culture was adjusted to 0.6, and six ml from each culture was collected at this point as non-induced samples. Then acetosyringone (AS) was added (final concentration 200 μM) to each of the remaining cultures. Cultures were grown at room temperature (RT). Agrobacterium samples were collected at 6, 9, 12 and 24 h after AS addition, immediately washed by 20 mM sodium phosphate buffer (pH 6.8) and pelleted for lysate extraction. When sampling, the OD600 value of each sample was measured and adjusted to 0.6.
Lysate extraction from Agrobacterium cells was prepared after sampling . Briefly, cells were re-suspended in 200 μl TES buffer (50 mM Tris-HCl pH 6.8; 2 mM EDTA; 1% β-mercaptoethanol; 1% SDS) and shaken for 30 min at 37 °C. Then, 900 μl of NP1 buffer (150 mM Tris-HCl pH 8.0, 0.5 M sucrose, 10 mM EDTA) containing 1 mg/ml lysozyme was added. The mixture was incubated on ice for at least 1 h, followed by 30 min shaking at 37 °C. triton X-100 was then added to a 4% final concentration and the mixture was incubated for 15 min at RT. Three hundred and forty-three μl of 5× EDTA-free protease inhibitors cocktail (in 25 mM MgCl2, Roche Applied Science, Penzberg, Germany) was added and the mixture was rotated for 15 min at 37 °C followed by 2–3 h at 4 °C. The insoluble material was removed by centrifugation at 18,400 g for 15 min and the supernatant was collected. The protein concentration of the total soluble lysate was determined by the Bradford reagent (Bio-Rad) and diluted to a final concentration of 0.5 mg/ml. One mL of the diluted lysate was used for T-strand immune-precipitation (T-strand-IP). Another 100 μL of the same diluted lysate was treated with DNase-free RNase (Thermo Fisher Scientific, Waltham, MA), Proteinase K (Thermo Fisher Scientific) and then precipitated by ethanol to obtain the genomic DNA, which would be used as the “input” in the T-strand-IP and qPCR assays described later.
For rice calli samples, rice (Oryza sativa L.) cultivar ‘Taipei 309″ seeds were used for callus induction as previously described . The calli were sub-cultured every two weeks. Two- to three-month-old healthy friable rice calli were selected for Agrobacterium transformation. Agrobacterium culture was used to infect 100–120 pieces of rice calli according to Patel et al. . Infected calli were blotted onto three layers of sterile filter paper to remove the excessive Agrobacterium suspension and then put on clean sterile filter papers for sample collection. Healthy rice calli without Agrobacterium infection were used as controls.
Concentration of the total soluble proteins was determined by the Bradford reagent (Bio-Rad). Equal amounts of total proteins (20 μg) from each sample were subjected to SDS-PAGE and subsequently transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking with 2% non-fat milk in TBST buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% Tween-20), the membrane was incubated in the same buffer containing 1/1000 dilution of rabbit anti-VirD2 antibodies. After three times of washing with TBST buffer, the membrane was incubated with 2% non-fat milk in TBST buffer containing 1/5000 dilution of the goat anti-rabbit HRP (Thermo Fisher Scientific). The signal was detected with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
T-strand-immunoprecipitation and qPCR assay of Agrobacterium cells
One mL of diluted lysates (0.5 mg/mL) extracted from Agrobacterium cells were incubated with the antibodies against VirD2 at 4 °C overnight. Meanwhile, the Dynabeads™ Protein G magnetic beads (Thermo Fisher Scientific) were blocked with 20 μg BSA (New England BioLabs) and 20 μg glycogen (Thermo Fisher Scientific) at 4 °C overnight. The next day, the magnetic beads were washed twice using the extraction buffer NP1, and then re-suspended in appropriate volume of NP1 buffer. Forty μL of equilibrated magnetic beads were then added to the mixture of lysate and antibody against VirD2, and the total mixture was incubated for four hours at 4 °C. After washing with NP1 buffer for four times, freshly prepared elution buffer (1% SDS, 0.168 g NaHCO3/20 ml buffer) was used to elute the products of immuno-precipitation at 65 °C. The eluate was digested by Proteinase K at 55 °C overnight and then purified using phenol-chloroform extraction. The final products (T-strand DNA) were precipitated by ethanol.
qPCRs were performed with iTaq Universal SYBR Green Supermix (Bio-Rad) to analyze T-strand formation in Agrobacterium cells on a Real-Time PCR Instrument (Agilent). Agrobacterium chromosome marker dnaK (with primers of dnaKFwd: 5’-TACCTTCCTCGGTGGTGAAG-3′, and dnaKRev: 5’-CGAGGACGAAAGTTC GATC-3′) was amplified from the 100 μL diluted lysate prepared previously and used as an internal control. T-strand marker gene HYG (HYGFwd: 5’-GGTCGCCAACATCTT CTTCT-3′, HYGRev: 5’-CGAAATTGCCGTCAACCAAG-3′) or GUS (GusFwd: 5’-ACGTCTGGTATCAGCGCGAAGTC-3′, GusRev: 5’-TATAGCCGCCCTGATGCTCC ATC-3′) was amplified from the purified T-strand-IP products. Relative quantification using comparative CT calculation method was applied in qPCR data analysis.
Transient GUS transcripts analysis in the infected calli
Agrobacterium cells harboring the binary vector of pB50 were grown overnight in 15 ml of YEP medium containing 25 mg/L rifampicin and 50 mg/L kanamycin at 28 °C. The next day, the overnight culture was divided into 3 equal aliquots (5 ml each). Forty-five ml of fresh infection medium containing 200 μM AS was added to each aliquot for induction at RT. The cultures were sampled at three time points (3, 9, and 24 h). The OD600 of each 50 ml Agrobacterium suspension was examined and adjusted to 0.6 before rice calli infection. The rice transformation was performed as previously described . After a three-day co-cultivation, samples were collected, washed thoroughly with 200 mg/L timentin (GlaxoSmithKline, Research Triangle Park, NC) and stored at − 80 °C.
Total RNA was isolated from the infected rice calli using the Quick-RNA MicroPrep (Zymo Research, Irvine, CA), and then treated with DNase I (RNase-free DNase set; Zymo Research) to clean up potential contamination of the genomic DNA. Concentration of the total RNA was quantified using a NanoDrop 2000C spectrophotometer. The first-strand cDNAs were synthesized using iScript cDNA Synthesis Kit (Bio-Rad). Subsequent real-time RT-PCRs were performed with iTaq Universal SYBR Green Supermix (Bio-Rad) to analyze GUS transcripts on a Real-Time PCR Instrument (Agilent). The housekeeping gene UBQ5 was used as an internal control . The relative fold change of GUS transcripts is calculated against the GUS expression level with 3-h AS induction before transformation.
Two-tailed student t-test was carried out to evaluate significant differences in values of qPCR and qRT-PCR. “*” indicates significant differences: ***p < 0.001, **p < 0.01, *p < 0.05. Data presented are means ± standard errors (n = 3).