Hybond n

For identification of T-DNA copy number in transgenic plants developed using both the binary vectors, 10 µg of genomic DNA from transgenic and wild-type plants was digested with HindIII (NEB high fidelity, New England Biolabs, Ipswich, Massachusetts, USA) overnight and separated on 0.8% agarose gel in 1X TAE buffer at constant voltage of 40 V. Restricted fragments were transferred onto a positively charged nylon membrane (Amersham™ Hybond™-N⁺) by capillary movement using 20× SSC and the membrane was later UV cross-linked. The membrane was hybridized with a DIG labeled 571 bp GFP and 750 bp nptII gene fragment for their corresponding transgenic plants. The blot was further processed with washing, blocking, and development as per manufacturer’s instructions (Roche Holding AG, Basel, Switzerland). The membranes were exposed to X-ray film for 1 h in dark and later observed for hybridization signal.

Total nucleic acids were extracted from infected protoplasts, separated by agarose gel electrophoresis, and transferred to nylon membrane (Hybond-N⁺, Amersham), after ensuring even loading via monitoring rRNA levels. Coding sense PEMV1 genome and sg mRNA were detected by blotting with nine oligonucleotide probes 5′-end-labeled with [γ -³²P] complementary to both the genome and sg mRNA (genome coordinates: 4004–4031, 4324–4359, 4401–4434, 4681–4708, 4749–4780, 4936–4968, 5122–5389, 5406–5439, 5679–5703). Northern blots, from three independent repeats, were captured using Typhoon FLA 9500 Variable Mode Imager and viral RNA bands were quantified using QuantityOne software. Sg mRNA levels of each mutant PEMV1 were calculated to generate average values with SE.

The 625-bp silver carp Thm3 probe was designed with primer pairs Thm3-f: 5′- AGATGCTGGCTGAAACTGGTAA- 3′ and Thm3-r: 5′-ATCAGGTTTGTAAGCCCTTCTC G-3′, which cover the major open reading frame (ORF) from 425 bp to 1049 bp of Thm3. The probes were digoxin-labeled in vitro using the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Southern blot analysis was performed as described previously by Koga et al. (2000) (link), with the following modifications. High-molecular-weight DNA (20–30 µg for each gel slot) were digested with BglII restriction enzyme, fractionated on 0.8% agarose gels, and transferred to nylon membranes (Hybond-N; Amersham), followed by fixation via baking at 80°. The membranes were then hybridized with a digoxin-labeled silver carp Thm3 probe. Finally, stripping and reprobing DNA blots were performed after immunological detection.

Total RNAs were extracted from cells incubated at 37 or 20°C to an OD₆₀₀ of 0.5–0.6 using the hot phenol method, as described previously (Sarmientos et al., 1983 (link)), and quantified by UV absorbance at 260 nm (NanoDrop^TM One^C Spectrophotometer, Thermo Fisher Scientific^TM). For 23S rRNA analyses, 4 μg of RNA was separated on 1.2% agarose gel in 0.5× TBE [45 mM Tris-borate (pH 8.3), 1 mM ethylenediaminetetraacetic acid (EDTA)] and transferred overnight to nylon membranes (Hybond N+, Amersham) by capillary action. After transfer, both sides of the membrane were cross-linked by exposure to UV light using CL-1000 UV crosslinker (UVP Inc.). The membrane was prehybridized for 4 h at 65°C in hybridization buffer [100 mM sodium phosphate (pH 7.2), 0.2 mM EDTA, 1% SDS, and 1 mg/ml bovine serum albumin] with 50 μg/ml salmon sperm DNA, which was boiled for 10 min, followed by hybridizing in the same buffer containing 500 ng/ml of biotin-labeled probes described in Supplementary Table S1. Hybridization and washing conditions are presented in Supplementary Table S1. Biotin signals were detected using a Biotin Chromogenic Detection Kit (Thermo Fisher Scientific^TM), following the manufacturer’s instructions. To visualize 16S and 17S rRNAs, 1 μg of total RNA was separated on 2% agarose gel electrophoresis in 1× TAE (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA).