Exnase 2

The full-length coding sequences (CDS) of potato AREB/ABF/ABI5 genes with termination codon were amplified using potato cDNA as a template (refer to Table S1 for the primers used) and cloned into plasmid pH7LIC-N-eGFP with Exnase II (Vazyme, Nanjing, China) to produce the GFP-StAREB/ABF/ABI5s vectors for generating the GFP fused target protein in living cells. Subcellular localization of the GFP-StAREB/ABF/ABI5s was investigated at 60 h after infiltration by Agrobacterium tumefaciens GV3101 harboring the plasmid DNA with laser confocal fluorescence microscopy (Leica TCS-SPE, Wetzlar, Germany) in Nicotiana benthamiana epidermal cells.

PA2146 mutant strain construction was performed according to a previously described method with minor modifications (Luo et al., 2015 (link)). The primers used are listed in Table 2. Briefly, the upstream and downstream fragments of PA2146 were amplified with PA2146-up-F/R and PA2146-down-F/R, respectively. The amplified products were then fused by overlapping PCR, and the fused fragment was cloned into pLP12 (KnoGen Biotech, Guangzhou, China) using recombinant enzyme Exnase II (ClonExpress II, Vazyme) to generate recombinant plasmid pLPPA2146. The resulting plasmid was then transformed into E. coli DH5α, and the recombinant plasmids were confirmed by PCR using the primer pair pLP-U-F/R. Next, pLPPA2146 was transferred into E. coli β2163 and selected on LB agar containing 0.3 mM daptomycin (DAP) with 0.3% D-glucose. E. coli β2163 was then co-cultured with PAO1, and the insertion mutation was selected on LB agar containing 36 μg/mL tetracycline (TC) with 0.3% D-glucose and confirmed using the primer pair PA2146-up-F and PA2146-down-R. The PAO1ΔPA2146 strain was selected on LB agar with 0.4% L-arabinose. The PA2146 mutant was confirmed by sequencing using the primers PA2146-TF and PA2146-down-R.

Plasmid pBRAd4 carrying the HAdV-4 GZ01 genome (GenBank accession no. KF006344.1) was constructed with a previously described strategy (45 (link)). Plasmids that carry the HVR mutants rAd4-A3R7, rAd4-A3R7-1, and rAd4-A3R7-2 were then constructed as shown in Fig. 9. Briefly, the mutated fragment H4A3R7 was produced by overlapping PCR extension mutagenesis with primer pairs A4-3R7-1u (5′-GTTAAAACAGATGACGCTAATGGATGGGACAAAGATGACACCACAG-3′)/Ad4-20204r (5′-CTCTTTGGTCTTGAGACGCGTGAAG-3′) and A4-3R7-1r (5′-TCATCTTTGTCCCATCCATTAGCGTCATCTGTTTTAACTTTAACACCCT-3′)/Ad4-17833u (5′-ACTGGGCCTGCCCACCACGCGTCCCA-3′), using pBRAd4 as the DNA template, and then with primer pair Ad4-17833u/Ad4-20204r. The H4A3R7 fragment was then cloned into the MluI-digested pBRAd4 vector to generate the mutagenesis vector pBRAd4-A3R7 with homology recombination in vitro with Exnase II (Vazyme, China). The vector was then transferred into E. coli for confirmation. Plasmids pBRAd4-A3R7-1 and pBRAd4-A3R7-2 were generated with the same method. The successful creation of these constructs was confirmed with restriction digestion and DNA sequencing analyses. Mutant viruses rAd4-A3R7, rAd4-A3R7-1, and rAd4-A3R7-2 were then rescued and purified in AD293 cells, as described above.