Templiphi

DNA was extracted from gradient fractions 3–8 (encompassing roughly the middle third of the gradient, where polyomaviruses are expected to migrate). A 160 µl sample of each fraction was treated with 40 µl of digest buffer [250 mM Tris pH 8, 125 mM EDTA, 2.5 % SDS, 2.5 % proteinase K (Qiagen no. 191331) and 50 mM DTT]. The digestion was incubated at 50 °C for 15 min then at 72 °C for 10 min. DNA was precipitated by addition of 100 µl of 7.5 M ammonium acetate and 2.6 volumes of 95 % ethanol followed by overnight incubation at 4 °C (Crouse & Amorese, 1987 ). Samples were brought back to room temperature, centrifuged for 1 h at room temperature, washed with 70 % ethanol, briefly air-dried and the pelleted DNA was redissolved in 10 µl of the Sample Buffer supplied with the RCA kit (TempliPhi, GE Healthcare no. 25-6400-50). Samples were heated to 95 °C for 3 min, allowed to cool, then mixed with 10 µl of the instructed Reaction Buffer/Enzyme mix. The RCA reaction was allowed to proceed at 30 °C for 24 h, then the enzyme was heat-inactivated at 65 °C for 10 min. Amplified DNA was ethanol-precipitated and dissolved in 55 µl of 2 mM Tris pH 8, 0.2 mM EDTA.

Using tomato samples 331 and 332, a 1.1 kb fragment was cloned and subsequently sequenced. Since they shared 99.8% of identity, only sample 332 was considered for subsequent studies. DNA from this sample was used as a template to obtain the viral sequences by rolling circle amplification (RCA) using Φ29 DNA polymerase (TempliPhi, GE Healthcare, Munich, Germany). Restriction enzymes BamHI, EcoRI, HindIII, and PstI were used to follow a protocol of restriction fragment length polymorphism (RFLP) on RCA products (RFLP-RCA) according to Haible et al. [11 (link)]. The bands were excised from agarose gel and inserted into the appropriate restriction sites in pBluescript II SK (+) (La Jolla, Stratagene, CA, USA) for subsequent transformation of E. coli competent cells. The cloned products were sequenced at Macrogen Inc. (Amsterdam, Netherlands). A preliminary identification of viral sequences was based on a BLAST search and three different begomovirus DNA components were observed, suggesting a mixed infection. Subsequently, the three different full-length viral clones, referred to as isolates Be1.1B, Be6.6H, and B6.7H, were completely sequenced by primer walking and assembled using BioEdit v.7.1.9 [12 ]. The complete nucleotide sequence of clones Be6.6H, Be6.7H, and Be1.1B were deposited in GenBank under the accession numbers MK440292, MK440293, and MK440294, respectively.

Cell strains, bacteriophage reagents, and protocol have been previously described [36] (link). All reactions were performed in 40 mM Tris pH 8.0, 250 µg/mL BSA, 10 mM DTT, 10 mM MgCl₂, 60 mM KCl, and 1.25% glycerol. Reactions were supplemented with 100 µM final concentration of each dNTP. Restriction enzymes and nucleotides were purchased from New England Biolabs (Ipswich, MA). Reaction volumes for the lesion bypass assay were 50 µL. Reactions contained 10 pmoles substrate, 10 pmoles polymerase, and 50 pmoles RPA. RPA was added first and pre-incubated at 37°C for 30 minutes and synthesis was initiated by addition of polymerase. Reactions were incubated at 37°C for an additional 30 minutes. Reactions were stopped with the addition of 2 µL of 500 mM EDTA, and were processed according to previously described protocol [36] (link). After recovery, newly synthesized oligos were annealed to gapped M13 mp18 bacteriophage DNA (10–25 fold excess oligo over phage). Annealed gap DNA was transformed into MC1061 cells as previously described and plaque phenotype and numbers were counted. Dark blue plaques were amplified using Templiphi (GE Life Sciences) according to manufacturer protocol and resulting DNA was sequenced by Genewiz (Research Triangle Park, NC). Error rates were calculated as previously described [36] (link).