Sample collection and plant material
A P. frutescens plant sample with typical TuMV-like symptoms including mosaic and chlorosis was observed in Chuncheon city, South Korea (sample collected by Professor Jin-Sung Hong, Kangwon National University). The N. benthamiana, and Chinese cabbage and radish plants used in this study were incubated in 25oC ± 2oC with 16 hours of light and 8 hours of dark. All the soil used was sterilized before use.
RNA extraction, cDNA synthesis and PCR detection
For virus detection, total RNAs of plant tissues were extracted using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA), and then the extracted samples were preserved at -70oC. The cDNAs were produced using LeGene Express 1st Strand cDNA Synthesis System with an oligo dT primer. Then detection was conducted by PCR using TuMV-CP-forward primer (5’-TCT CAA TGG TTT AAT GGT CTG G-3’) and reverse primer (5’-AAC CCC TTA ACG CCA AGT AAG-3’) .
Construction of full-length clones of TuMV
To obtain infectious clones, we performed full-length PCR. The PCR mixture of 50 μl was composed of 2 μl of the template cDNA, 25 μl of 2 x PCR buffer for KOD FX Neo, 10 pmol of forward primer containing ApaI site and T7 RNA polymerase promoter sequence (5’-GAG GGG CCC TAA TAC GAC TCA CTA TAG GAA AAA TAT AAA AAC TCA ACA CAA CAT ACA CAA AAC G), 0.4mM dNTPs, 10 pmol of reverse primer containing XmaI site (5’-GAG CCC GGG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTC CCT TGC ATC CTA TCA AAT G), 1 μl of Taq polymerase (KOD FX Neo, Toyobo, Osaka, Japan). A cDNA template of TuMV that has been successfully amplified before was used as a positive control. Conditions of full-length PCR were 94 °C for 2 min followed by 5 cycles of 10 s at 98 °C, 30 s for annealing at 59 °C, 6 min for extension at 68°C, and then by 30 cycles of 10s at 98 °C, 30s at 65°C, 6 min at 68°C, and finally incubation at 4 °C. Analysis of full-length PCR products was conducted by 0.8% agarose gel electrophoresis with dye incorporated in the gel. The PCR product was digested by ApaI and XmaI and subsequently cloned into the binary vector pJY which was digested by the same enzymes [23, 24]. The recombinants were screened by colony PCR and double enzyme digestion. Then the recombinant plasmids were transformed into E.coli competent cell (DH5a).
Agrobacterium infiltration and sap inoculation
The recombinant plasmids were transformed into Agrobacterium tumefaciens GV2260. The colonies of each clone were incubated on LB plates supplemented with Kanamycin and Rifamycin and then the Agrobacterium cells collected from fresh plates were diluted to approximately OD600 = 0.6 in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone). N. benthamiana plants inoculated with constructed clones were incubated in a growth chamber at 24-26oC (16/8h, light/dark cycle) . Leaves of the infiltrated N. benthamiana plants with symptoms were used to inoculate Chinese cabbage cv. CR Victory and radish cv. Iljin as described previously .
Sequencing of TuMV infectious cDNA clones
After the infectivity of each full-length cDNA clone was assessed by agroinfiltration, the full-length clones that have infectivity were sequenced by BIONEER CORPORATION (Daejeon, South Korea). Sequencing was initiated from each terminus using vector specific primers, and continued using primers designed from the obtained sequences (Table 1). The complete genome sequences were compared and assembled in DNAMAN software (Version 5.2, Lynnon BioSoft).
Construction of phylogenetic tree.
The Maximum-likelihood method was utilized to construct phylogenetic tree with 1000 bootstrap replicates in MEGA software (version 7.0). The complete genome sequences of TuMV strains utilized in this study were collected from NCBI (Table 2), including isolates previously reported in South Korea [16, 17, 25] and 6 newly collected isolates (two each from Perilla, radish, and Chinese cabbage)
Firstly, complete genome sequences of all Korean TuMV isolates were aligned by clustal X in MEGA 7.0. Recombination analysis was performed using RDP4 software package and employed seven detection methods: RDP , GENECONV , Bootscan , Maxchi , Chimaera , SiScan , and 3SEQ . Recombination events were noted if supported by at least four different methods (p-values < 1.0x10 -6) .