We collected seven melon leaves showing mosaic symptoms (Supplementary Fig. 1A) in the melon greenhouse of Jeonbuk State Agricultural Research & Extension Service in Iksan, Jeonbuk province, Korea in 2015. The samples collected from symptomatic plants were examined by leaf dip using transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR) using the WMV detection primers (Table 1). The isolate was purified biologically by three times of single local lesion passages using Chenopodium quinoa and were proliferated in zucchini (Cucurbita pepo). Fresh leaf samples were stored at −80°C until use.

Quick dip preparation was used for the observation of virus particle morphology in samples collected from the greenhouse and inoculated by infectious clone. Dip preparations were conducted by grinding a small piece (1 × 1 mm) of leaf tissue with 2-3 drops of 2% PTA (phosphotungstic acid), pH 7.0. The extract was mounted on a carbon-stabilized and formvar coated grid (200 mesh). After 1 min later, the excessive sap on grid was filtered off with a piece of filter paper. Electron microscopy was done with 80 KV with LEO 912AB (Carl Zeiss, Jena, Germany).

Total RNA was extracted from WMV-infected leaves using the EASY-spin (DNA-free) total RNA extraction kit (Intron, Seongnam, Korea) according to the manufacturer’s instructions. Specific primer pairs were designed to construct a full-length cDNA clone of WMV-IS-me2 and detect WMV infection (Table 1). For the amplification of three WMV genome fragments and the backbone pJL89 vector, RT-PCR reactions were carried out using the SuperScript IV One-step RT-PCR System (Invitrogen, Thermo Fisher Scientific, Seoul, Korea). The reaction was performed in a volume of 50 μl containing 1 μl total nucleic acids (0.5 μg/μl), 2.5 μl forward primer (10 μM), 2.5 μl reverse primer (10 μM), 25 μl 2× Platinum SuperFi RT-PCR master mix, 0.5 μl SuperScript IV RT mix, and nuclease-free water. The RT-PCR conditions were as follows: reverse transcription at 50°C for 10 min and RT inactivation/initial denaturation at 98°C for 2 min; amplification of 35 cycles of denaturation at 98°C for 10 s, annealing at 45°C (for WMV-1F/1R), 53°C (for WMV-2F/2R, -3F/3R), or 63°C (for pJL89-F1/R1) for 10 s, and extension at 72°C for 5 min; and a final extension at 72°C for 5 min. The amplified fragments were purified using a MEGAquick-spin agarose gel extraction kit (Intron), and the concentrations of the purified fragments were measured using the Nanodrop 2000c (Thermo Fisher Scientific). For WMV detection, one-step RT-PCR was conducted in a 20 μl final volume. The reaction mixtures included 10 μL of RT-PCR master mix 2× (Genetbio, Daejeon, Korea), 1 μl of total RNA (approx. 0.5 μg), 0.5 μl each of 10 μM forward and reverse primers, and 8 μl of ddH₂O. The RT-PCR conditions were as follows: 30 min at 50°C; 10 min at 94°C; and 35 cycles consisting of 30 s at 94°C, 30 s at 55°C, 60 s at 72°C, and 5 min at 72°C. PCR products were analyzed by electrophoresis on a 1% agarose gel at 100 V for 60 min, and DNA bands were visualized using a UV transilluminator.

To construct the infectious cDNA clone of WMV, three fragments covering the full-length genomic RNA of WMV-IS-me (GenBank accession no. MN854647, 10,031 nt) were amplified using the primer pairs WMV-1F/1R, −2F/2R, and −3F/3R, and linearized binary vector was prepared from pJL89 (4,707 nt, Addgene, Cambridge, MA, USA) by PCR using the primer pair pJL89-F1/R1 (Table 1). These four fragments share overlapping sequences of 40 bp at both the 5′ and 3′ ends (Table 1, Fig. 1). To generate the recombinant plasmid containing the whole WMV genome, the RT-PCR amplified genomic cDNA fragments of WMV-IS-me and PCR-linearized pJL89 vector were fused using Gibson Assembly Master Mix (New England BioLabs, Hitchin, UK). The reaction was performed in a total volume of 20 μl, containing 1.5 μl (~0.05 pmol) of the gel-purified pJL89 vector (113 ng/μl); 2 μl, 3.5 μl, and 3 μl (0.1 pmol each) of the gel-purified viral fragments for WMV-1F/1R (88 ng/μl), WMV-2F/2R (76 ng/μl), and WMV-3F/3R (77 ng/μl), respectively; and 10 μl of the 2× GA master mix. The reaction mix was incubated at 50°C for 1 h, and then GA products were dialyzed for 15 min using a cellulose membrane (VSWV, 0.025 μm) (Merck-Millipore, Darmstadt, Germany).

The dialyzed GA product (2 μl) was used for transformation of competent cells of the E. coli strain DH10B (Thermo Fisher Scientific) by heat shock according to a standard protocol. Positive colonies containing the expected cDNA clone of WMV were screened by kanamycin resistance and confirmed by colony PCR using detection primers (Table 1). The plasmids were extracted from PCR-positive colonies to sequence the full-length cDNA clone of WMV. Constructs of the WMV clone (pWMV-M, 2 μl, 1.7-2.8 μg/μl) were transformed into 100 μl of the A. tumefaciens strain GV3101 using the freeze-thaw method. The transformants were selected on yeast extract peptone (YEP) medium containing 50 mg/l kanamycin and 50 mg/l rifampicin for 2 days at 28°C. In agro-transformation of plasmid pWMV-M, three transformants were selected and confirmed via colony PCR.

To test the pathogenicity of pWMV-M clone, each of three Agrobacterium cultures was infiltrated into host plants. A positive single colony of Agrobacterium harboring a full-length WMV cDNA clone was grown at 28°C overnight in YEP medium containing kanamycin (50 mg/l), rifampicin (50 mg/l), and acetosyringone (20 μM). The cultures were harvested by centrifugation at 5,000 rpm for 10 min, resuspended in MMA infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, 200 μM acetosyringone) to a final OD₆₀₀ of 0.6, and incubated at 28°C for 2 h. The resuspended Agrobacterium cultures containing each plasmid were infiltrated into the abaxial surface of cotyledons of melon (C. melo), zucchini (C. pepo), watermelon (C. lanatus), and true leaves of N. benthamiana using a 1-ml syringe without a needle. Transformed Agrobacterium containing only the binary vector pJL-89 were used as a negative control. All of the inoculated plants were kept in an aphid-proof greenhouse at 25-28°C, and after 30 days of inoculation the symptoms of inoculated plant leaves were photographed and subjected to RT-PCR using WMV detection primers (Table 1).

The sequence of the clone was determined by a commercial company (Bionics, Daejeon, Korea). The sequences were assembled using SeqMan of the DNAStar v. 5.02 program (Lasergene, Madison, WI, USA). The nucleotide sequences and the deduced amino acid sequence of the clone were aligned using the Geneious method in Geneious Pro 10 software and compared with those of WMV-IS-me (wild type) and previously reported WMV isolates. The phylogenetic relationships of the clone were analyzed using the maximum likelihood method in MEGA-X (Kumar et al., 2018) with best-fit nucleotide substitution models (TN93+G+I for the CP gene). Bootstrap values were calculated using 1,000 random replications. All positions containing gaps and missing data were eliminated. A soybean mosaic virus isolate (accession no. NC_002634) was used as an outgroup.

The melon samples collected from symptomatic plants were examined by leaf dip using TEM and revealed filamentous virus particles about 700 nm long (Supplementary Fig. 1B). The RT-PCR result using the WMV detection primers (Table 1) showed all tested samples were positive for WMV (Supplementary Fig. 1C). One (WMV-IS-me2) of the seven WMV-positive samples was purified biologically and proliferated in zucchini.

Four overlapping PCR fragments of the full-length genome of this isolate WMV-IS-me2 and the pJL89 binary vector were assembled in an isothermal GA reaction, and the resulting construct was named pWMV-M (Fig. 1). After E. coli (DH10B strain) transformation of GA reaction product, three colonies were randomly picked and confirmed to be positive for WMV via colony PCR. The sequences of plasmid DNA purified from positive colonies were identical. The full sequence of the cloned pWMV-M was determined using the dideoxy chain termination method and deposited in GenBank (GenBank accession no. OR134098, 10,081 nt [containing polyA]).

To examine the infectivity of the cloned pWMV-M, agroinfiltration was performed as described previously (Kwak et al., 2016). At 10 days post-inoculation (dpi), the upper leaves of plants agroinfiltrated with pWMV-M began to develop mosaic symptoms. After 3 weeks post-inoculation, N. benthamiana and watermelon showed mild mosaic symptoms, and melon and zucchini showed mosaic symptoms compared to mock-inoculated plants (Fig. 2). No symptoms were observed in the mock-control plants (Fig. 2). To confirm WMV infection of the inoculated plants, total RNAs extracted from the upper non-inoculated leaves were subjected to RT-PCR using the WMV detection primers (Table 1). A band of the predicted size (approximately 392 bp) was observed only from symptomatic plants (Fig. 3A), indicating that these plants were systemically infected with WMV. In addition, the typical WMV virion, a filamentous rod of about 700 nm in length, was visualized by TEM in symptomatic plants (Fig. 3B). Only two of the three agro-transformants were confirmed to infect all four plants with WMV (Table 2, Supplementary Fig. 2A), when the first agro-transformant was re-inoculated into six N. benthamiana plants, five out of the six plants were confirmed to be infected with WMV (Supplementary Fig. 2B). We further evaluated the infectivity of the pWMV-M clone by mechanical inoculation to three identical plants with inoculum from each plant previously agroinoculated by pWMV-M clone. All the plants were infected and confirmed to be positive for WMV detection (Supplementary Fig. 2C). Therefore, the results demonstrated that pWMV-M is fully infectious. The pWMV clone was deposited into the Korean Agricultural Culture Collection (KACC, Wanju, Korea) under accession no. KACC95145P.

The clone sequence was aligned with those of 46 other WMV isolates, including the WMV-IS-Me2 wild-type isolate, from NCBI GenBank to examine the phylogenetic relationships among WMV isolates. A maximum likelihood tree was constructed based on the CP sequences of 47 WMV isolates; it indicated that the pWMV-M clone and WMV-IS-me isolate clustered with the EM1 subgroup of EM isolates (Fig. 4). The pWMV-M clone had the highest nucleotide sequence identity, 99.8%, with the WMV-IS-Me2 wild-type isolate (MN854647) and showed 95-99% identities with other EM groups and 93-94% identities with the CL and G2 groups.

To confirm the pathogenicity and infectivity of the pWMV-M clone, the Agrobacterium culture was repeatedly infiltrated into cucurbit crops and tobacco and then compared to the original wild-type WMV-IS-me2 isolate. The cucurbits melon, zucchini, and watermelon agroinfiltrated with the pWMV-M clone showed milder symptoms of mosaic without vein banding compared to the original wild-type isolate (Table 2, Fig. 2). Full-length sequencing of the clone confirmed that out of 13 nucleotide changes, eight amino acid substitutions were present in seven proteins as compared to the original wild-type WMV-IS-me2 isolate (Table 3).