Phanta dna polymerase

All strains and plasmids used in this study are listed in Table S1. Plasmid pET28a (with T7 promoter) (Merck KGaA, Darmstadt, Germany) and pNV18.1 (National Institute of Infectious Diseases, Tokyo, Japan) were used as protein expression vectors. The nitrilase gene (EF467367.1) was from a mutant strain of R. rhodochrous tg1-A6 [6 (link)]. The RrGroES, RrGroEL and RrGroEL2 genes were cloned from R. ruber TH. E. coli Top10 for recombinant plasmid construction and cloning was purchased from Biomed (Beijing, China). E. coli BL21(DE3) was used to express target proteins. R. ruber TH3 is the amidase-deleted mutant of R. ruber TH. It carries the same chaperones RrGroES, RrGroEL and RrGroEL2 with strain TH. R. ruber TH3 also has a native nitrilase gene, but the enzyme activity of the natural nitrilase is too low to be detected.
Taq DNA polymerase, Phanta DNA polymerase and T4 DNA ligase were purchased from Vazyme (Nanjing, China). Restriction endonucleases were purchased from Takara (Shiga, Japan). All PCR primers are listed in Table S2.

P. pastoris GS115-Cas9 (his4::Cas9) constructed in previous studies was used as the parent strain [9 (link)]. Restriction enzymes and T4 DNA ligase were purchased from NEB (Ipswish, MA, UK). Phanta DNA polymerase and 2 × Taq PCR mix were purchased from Vazyme (Nanjing, China). DNA gel purification kit and plasmid extraction kit were purchased from Sangon Biotech (Shanghai, China). TAL standard was purchased from TCI (Shanghai, China). All chemicals were purchased from Sangon Biotech (Shanghai, China) unless stated otherwise.

To identify the presence of possible viruses and obtain the genomes of novel viruses, we extracted RNA and DNA from the leaves of the collected Physostegia virginiana plant utilizing an RNAiso Plus reagent (TaKaRa, Tokyo, Japan) and Fast Pure Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China). Using the reverse transcriptase M-MLV (TaKaRa, Kusatsu, Japan), total RNA was reverse-transcribed into cDNAs, followed by Super-Fidelity PCR using Phanta DNA polymerase (Vazyme, Nanjing, China) and specific detection primer pairs (Table S1). The DNA/RNA sequences of viral contigs obtained via RNA-seq and assembly were PCR/RT-PCR-amplified using Super-Fidelity PCR with specific primer pairs (Table S2). To amplify the 5′ and 3′ end sequences of the novel viruses, 5′/3′ RACE was performed using the HiScript-TS 5′/3′ RACE Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. For DNA viruses with circular structures, we utilized Super-Fidelity PCR with specific primer pairs (Table S3) to amplify the remaining unknown sequences outside the contigs. The PCR products were gel-purified and Sanger-sequenced, individually. The genomes of the four newly identified viruses were finally obtained from the diseased Physostegia virginiana plant through PCR, RT-PCR, and then DNA Sanger sequencing.

The dps gene was knocked out in D. wulumuqiensis R12 which was preserved in our laboratory, using the pK18mobSacB shuttle plasmid from Miaoling Biotechnology Co., Ltd (Wuhan, China). E. coli DH5α (Vazyme, Nanjing, China) was utilized for gene cloning. T4 DNA ligase, Phanta DNA polymerase, EcoRI and BamHI were purchased from Vazyme (Nanjing, China). Triton X-100, protease inhibitor, TEAB (tetraethylammonium bromide), trypsin, DTT (dithiothreitol), and IAA (iodoacetamide) were from Sangon (Shanghai, China). Yeast extract and tryptone were purchased from Oxoid (UK). Formic acid, acetonitrile, actone, and other chemicals were from Sigma-Aldrich (Shanghai, China). tryptone glucose yeast (TGY) medium (5 g/L tryptone, 3 g/L yeast extract, and 1 g/L glucose, pH 7.0) was used for D. wulumuqiensis R12 culture. Nutrient agar (NA) medium (10 g/L tryptone, 3 g/L beef extract, 5 g/L NaCl, and 15 g/L agar) was utilized for preparation and transformation of competent cells. Luria–Bertani (LB) medium (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl, pH 7.0) was used for E. coli DH5ɑ culture. When needed, 1.5% agar was added to obtain a solid medium.