S1 nuclease

Plasmids were extracted (Promega, Fitchburg, WI, USA) and characterized by PCR as described previously [38 (link)]. Plasmids from clinical isolates were detected using PFGE. A single block was incubated at 55°C for 1 hour with 1 unit of S1 nuclease (New England Biolabs, Ipswich, MA, USA) in Zinc Buffer (200 μl of 50 mM NaCl, 30 mM sodium acetate and 5 mM ZnSO₄). Electrophoresis was performed at 6 V, 5-50s for 20 h [39 (link)].

S1-PFGE was conducted to determine the size of large plasmids. Briefly, agarose-embedded DNA was digested with S1 nuclease (New England BioLab) at 37°C for 1 h. The restriction fragments were separated by electrophoresis in 0.5 Tris-borate-EDTA buffer at 14°C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA, USA) with pulse times of 2.16 to 63.8 s. Phage Lambda PFGE ladder (New England BioLab) was used as DNA size marker. The gels were stained with GelRed, and DNA bands were visualized with UV transillumination (Bio-Rad). Chromosomal and plasmid DNA of S. typhimurium strains were transferred and cross-linked onto nylon membrane and hybridized with a DIG-labeled oqxAB probe using DIG High Prime DNA Labeling and Detection Starter Kit I (Roche) following manufacturer’s instructions to determine the localization of oqxAB and aac(6′)-Ib-cr genes in S. typhimurium genetic materials.

Cloning was done using standard molecular biology procedures (Sambrook & Russell, 2001 ). Escherichia coli XL1Blue (Stratagene, San Diego, CA, USA) cells were transformed by electroporation (Bio-Rad Laboratories, Hercules, CA, USA). Plasmid DNA was purified with the QIAprep or Maxiprep kits (Qiagen, Valencia, CA, USA). DNA fragments were purified with the QIAquick or QIAEX II kits (Qiagen, Valencia, CA, USA). Restriction enzymes, T4 DNA polymerase, T4 DNA ligase, S1 nuclease were purchased from New England Biolabs. PCR were performed with the proofreading PfuTurbo DNA polymerase (Stratagene, San Diego, CA, USA). Oligonucleotide synthesis and DNA sequencing were carried out by Eurofins MWG Operon. The construction and maps of the plasmids (Figs. S2–S12) generated during this study are provided in the supplemental material.

Total dsRNA (1–8) was extracted from mycelia of strain LT-3-1 and analyzed as described above. Aliquots of 2.0 μg of total dsRNA were digested with 20 U DNase I and 100 U S1 nuclease (New England Biolabs), as described above, dissolved in DEPC-treated water, and stored at −70 °C until use.
Protoplasts were prepared from actively growing mycelia of C. camelliae strain LT-3-1D2, a subisolate regenerated from a conidium of isolate LT-3-1, or strain DP-3-1 as previously described^{30 (link)}. The protoplasts were filtered through a Millipore filter, counted under a microscope (×100) using a hemocytometer, and used for dsRNA transfection using PEG 6000 as previously described^{31 (link)}. A total of 2.0×10⁶ protoplasts were transfected with ~5.0–7.0 μg of enzyme-treated dsRNAs (containing dsRNAs 1–8) or 70.0–80.0 μg virus-like particles. Following transfection, the protoplast suspensions were diluted with sterilized water and then spread onto PDA plates for colony formation. The new colonies were separately cultured on new PDA plates for dsRNA extraction.

Isolation of total RNA from S. lavendulae subsp. lavendulae CCM 3239 and high-resolution S1 nuclease mapping were performed according to [46 ]. RNA samples (40 μg) were hybridized to approximately 0.02 pmol DNA probe labelled at one 5′ end with [γ-³²P] ATP (producing approximately 10⁶ dpm/pmol of probe) and treated with 120 U of S1-nuclease (New England Biolabs, San Diego, CA, USA). The DNA probe for the aur1Ap promoter (274 bp DNA fragment) was prepared by PCR amplification using chromosomal DNA from S. lavendulae subsp. lavendulae CCM 3239 as a template and the 5′ end-labelled primer aur1AS1Rev from the aur1A coding region and the unlabeled primer aur1AS1Dir from the aur1A upstream region. The control hrdBp2 promoter DNA probe was described in [24 (link)]. Oligonucleotides were labelled at the 5′ end with [γ-³²P] ATP (ICN, Costa Mesa, CA, USA, 4500 Ci/mmol) and T4 polynucleotide kinase (New England Biolabs, San Diego, CA, USA) as described in [22 ]. The protected DNA fragments were analyzed on DNA sequencing gels (6% polyacrylamide containing 8 M urea) along with G+A and T+C sequencing ladders derived from the end-labeled fragments [25 (link)].

To determine whether bla_CMY-2 was located in the chromosome or on a plasmid, S1 nuclease (New England BioLabs) and BlnI (Takara Bio, Otsu, Japan) digestions were prepared from the total DNA of the four isolates selected for ISEcp1 analysis (harboring bla_CMY-2) (S. Infantis isolates 1993, 2127, and 2150, and S. Manhattan isolate 2179), as well as two susceptible isolates (S. Infantis isolate 1737 and S. Manhattan isolates 2129) for use as bla_CMY-2-negative controls. Total DNA was treated with 2 U/ml of S1 nuclease (incubated at 37°C for 45 min) or BlnI (incubated at 37°C for 16 h), followed by PFGE separation [30 (link)]. Separated fingerprints were transferred to positively-charged Nylon membranes (Roche Applied Science, Penzberg, Germany) and hybridized with PCR-generated bla_CMY-2 digoxigenin-labeled probes (Roche Diagnostics, Basel, Switzerland) using hybridization solution (Roche Diagnostics) according to the manufacturer’s instructions and a previous report [32 (link)].