Virus source and inoculation
Diseased cucumbers showing severe symptoms of leaf mosaic and fruit malformation (Fig. 1a) were collected in Xizhou Township of Changhua County, Taiwan in June 2020. The virus isolate CX-2 was isolated from the diseased cucumber sample ‘2106-2’ through three successive single lesion transfers on C. quinoa leaves. Manual mechanical inoculation was performed for virus transfer, and crude sap of virus-infected leaf tissue ground in 10 mM potassium phosphate buffer (pH 7.0) containing 10 mM sodium sulfite was used as inoculum. The virus was propagated in C. quinoa and Nicotiana benthamiana plants under greenhouse conditions for future studies.
Transmission electron microscopy (TEM)
Small pieces (5 × 5 mm2) of C. quinoa leaves inoculated with viruses were ground and sap droplets were mixed with 2% glutaraldehyde for fixation. Copper grids were floated on the sample droplets for 1 min, the residual liquid on the copper grids was removed with filter paper, and then stained with 2% uranyl acetate. A JEM-2000EX transmission electron microscope (JEOL Ltd., Japan) was used for examination.
Viral dsRNA extraction
Double-stranded RNA was extracted from virus-inoculated C. quinoa leaf tissue as previously described [19]. Briefly, 200 mg of fresh leaf tissue ground with liquid nitrogen was immediately suspended in 600 µl of EBA-30% E buffer (50 mM Tris-HCl pH 8.5, 50 mM EDTA, 3% SDS, 1% β-mercaptoethanol, 1% PVPP-40, adjusted to 30% ethanol) by rolling for 20 min at room temperature and then centrifuged at 16,110 g for 15 min at 4 °C. The supernatant was collected and adjusted to a final concentration of 20% ethanol and then loaded to a micro-column, in which 600 µl of cellulose CF-11 (Whatman, Buckinghamshire, UK) had been equilibrated with 1× STE-20% E buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, pH 8.0, adjusted to pH 7.8 and 20% ethanol). The micro-column was centrifuged at 100 g for 2 min to remove liquid and then washed twice by adding 450 µl of 1× STE-20% E buffer and centrifuging at 100 g for 2 min. The dsRNA was eluted from the column by adding 400 µl of 1× STE buffer twice and centrifuging at 100 g for 2 min. The eluate was collected and mixed with an equal volume of isopropanol, rolled for 10 min at room temperature and centrifuged at 16,110 g for 30 min at 4 °C. The dsRNA pellet was washed with 70% ethanol, air-dried at room temperature, and dissolved in 50 µl of RNase-free water.
cDNA library construction and ONT nanopore sequencing
First strand cDNA was synthesized with SuperScript IV reverse transcriptase (ThermoFisher Scientific, Waltham, MA) and random hexamers starting from 200 ng of dsRNA. The second strand DNA was synthesized with Klenow fragment of DNA polymerase I (New England Biolabs, Ipswich, MA). The synthesized dsDNA was precipitated by 100% ethanol and then end-repaired and A-tailed by adding with the EA enzyme provided by the KAPA Hyper Prep kit (KAPA Biosystems, Wilmington, MA). The ligation of the treated dsDNA with adaptor motor mix was performed by the ONT ligation sequencing kit following the ONT protocol SQK-LSK109. The prepared dsDNA was loaded on flow cells and sequencing was performed with MinION for eight hours. Reads obtained from sequencing were real-time analyzed using the ONT EPI2ME WIMP workflow.
Metagenomic analysis for taxonomic classification
The taxonomic classification of sequence data was performed by Kraken 2 [20]. The non-redundant nt database was downloaded from the GenBank of National Center for Biotechnology Information (NCBI) and used for building a classification database for Kraken 2 (k = 35, ℓ = 31). Dustmasker and segmasker programs [21] provided as part of NCBI’s BLAST suite were used to mask low-complexity regions. Bracken was used to estimate abundance at standard taxonomy level [22]. The output results were confirmed by using BLASTn in NCBI with customized Python scripts.
Viral genome sequencing by Sanger sequencing
Total RNA was extracted from virus-infected C. quinoa leaf tissue using the Plant Total RNA Miniprep Purification kit (GeneMark, GMbiolab, Taichung, Taiwan) according to the manufacturer’s instructions. The nt sequences of primers used to amplify viral genome fragments in reverse transcription-polymerase chain reaction (RT-PCR) are shown in (see Additional file 2: Table S1). RT-PCR was performed according to the instructions of the One-Step RT-PCR kit (GeneMark), 2 µl of total RNA (1 µg), 12.5 µl of 2× RT buffer (dNTPs, Mg2+ and enzyme stabilizer), 0.5 µl of enzyme mix (reverse transcriptase and Taq DNA polymerase), 0.5 µl forward and reverse primers (200 nM for final concentration) and 0.1 µl of RNase block were mixed in a final volume of 25 µl. First strand cDNA was synthesized at 50 °C for 30 min, followed by inactivation of reverse transcriptase and activation of Taq DNA polymerase at 94 °C for 2 min. PCR was performed for 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 1 min at 72 °C and an additional final reaction at 72 °C for 7 min. PCR products were analyzed by 1% agarose gel electrophoresis and then eluted with the Micro-Elute DNA Clean/Extraction kit (GeneMark) following the manufacturer’s instructions. The amplicons were cloned by TOPO TA cloning (Invitrogen), ligated with the pCR2.1-TOPO vector and transformed into E. coli strain DH5α competent cells, according to standard protocols recommended by the manufacturer. Recombinant plasmids purified from the resulting clones were sequenced by an ABI3730XL DNA Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA) performed by Mission Biotech Company (Taipei, Taiwan). Three clones of each fragment were selected for sequencing.
5´ and 3´ rapid amplification of cDNA ends (RACE)
The 5´- and 3´-ends of viral genome were confirmed by RACE [23]. Specific primers were designed from the determined nt sequences as shown in (see Additional file 2: Table S1). Total RNA used as template was denatured at 70 °C for 10 min and then put on ice for 1 min. First strand cDNA was synthesized by SuperScript IV reverse transcriptase (Invitrogen) mixing with 200 nM of each primer at 50 °C for 60 min, followed by stop reaction at 70 °C for 15 min. After the removal of template RNA by RNase H (Invitrogen), the cDNA products were precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volume of absolute ethanol at − 20 °C for overnight. After centrifugation at 17,000 g for 15 min, the pellet was resuspended in 20 µl DEPC-treated water. Subsequently, 200 nM of PolyG oligonucleotide [24] was tailed at the 3´ end of cDNA fragments by 20 U terminal deoxynucleotidy1 transferase (TdT) (New England Biolabs) at 37 °C for 30 min and the reaction was terminated at 70 °C for 10 min. The tailed cDNA fragments were mixed with 2.5 U Blend Taq-Plus (TOYOBO, Osaka, Japan), 200 nM PolyC [24] complementary to the PolyG tail and 200 nM another proper primer as shown in [see Additional file 2: Table S1] and Fig. 2. The PCR amplification was performed for 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 1 min at 72 °C and an additional final reaction of 7 min at 72 °C. The amplified DNA fragments were cloned by TOPO TA cloning (Invitrogen) for sequencing as mentioned above.
Viral genome sequence analysis
Genome sequences of different species of the genus Tombusvirus, including distinct CBLV isolates, were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/) as shown in (see Additional file 3: Table S2). Sequence identity analysis was performed by AlignX in Vector NTI Suite 10 (Invitrogen). Multiple sequence alignments were performed using the ClusalX 2.1 program in MEGA X [25]. Phylogenetic analyses were analyzed by the Neighbor-Joining method with 1000 bootstrap replicates using the Tree Explorer program in MEGA X.
Purification of viral coat protein (CP)
CX-2 CP was purified using the ultra-speed centrifugation method previously described [26] with modifications. Briefly, 100 g of CX-2-infected C. quinoa leaves harvested 3 days post-inoculation (dpi) were homogenized in 300 ml of TB buffer (10 mM Tris-HCl, pH 8.0, containing 10 mM sodium sulfite and 0.1% cysteine) in a blender and centrifugated at 10,000 rpm for 15 min (GRF-L-m2.0-30, Gyrozen, Korea). The supernatants were collected and treated with 1% Triton X-100 at 4 °C for 30 min, followed by centrifugation at 25,000 rpm in Beckman Type 45 Ti rotor for 2.5 h in 20% sucrose cushion. The pellets were then resuspended in TBG buffer (TB buffer containing 10 mM glycine) for isopycnic centrifugation through 32% cesium sulfate at 35,000 rpm in Beckman SW 41 rotor for 17 h. The opalescent zones were collected and precipitated by centrifugation at 45,000 rpm in Beckman Type 70 Ti rotor for 1 h. The pellets were resuspended in TBG buffer and treated with protein sample buffer (50 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 12% glycerol, 0.01% bromophenol blue and 2% β-mercaptoethanol) at 100 °C for 3 min. Proteins were separated in 12% SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by soaking the gels in cold 0.3 M KCl. The desired protein was cut and eluted from the gel using a Model 422 Elutro-Eluter (Bio-Rad, Hercules, CA). The yield of the purified CP was estimated by the software Spot Density of AlphaInnotech IS2000 (AlphaInnotech Corporation, San Leandro, CA) by comparison with the quantified bovine serum albumin (BSA) as previously described [27].
Production of rabbit antiserum
One hundred microgram of the purified CP dissolved in 1 ml of PBS buffer (136 mM NaCl, 1 mM KH2PO4, 8 mM Na2HPO4‧12H2O, 2 mM KCl and 3 mM NaN3) was emulsified with an equal volume of Freund’s complete adjuvant (BioSmart, South Korea) and injected subcutaneously into a New Zealand white rabbit. One week later, the rabbit was injected weekly with 100 µg of the same immunogen in 1 ml of PBS emulsified with an equal volume of Freund’s incomplete adjuvant (BioSmart) for two weeks. Blood was collected weekly from the ear marginal veins of the rabbit for one month, starting from 1 week after the third injection. The collected blood was incubated at 37 °C for 1 h and antiserum was collected from the supernatant after centrifugation at 8100 g for 10 min.
Enzyme-linked immunosorbent assay (ELISA)
Indirect ELISA was conducted as previously described with modifications [28] for antiserum titration and virus detection. Aliquots of 200 µl of purified protein or crude sap of plant tissue diluted with coating buffer (15 mM Na2CO3, 35 mM NaHCO3 and 3 mM NaN3) were loaded in each well of polystyrene microtitration plates. Sample-coated plates were incubated at 37 °C for 30 min and then washed with PBST (PBS buffer containing 0.05% Tween 20) for three times, each time for 3 min. Antiserum diluted in enzyme-conjugate buffer (PBST containing 2% PVP-40 and 0.2% ovalbumin) was loaded to the plates, 200 µl for each well. The plates were incubated at 37 °C for 30 min and then washed three times with PBST. The secondary antibody, alkaline phosphatase (AP)-conjugated affinipure goat anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA) for rabbit antiserum, was diluted at a 1/5000 dilution in enzyme-conjugate buffer, and aliquots of 200 µl were loaded to each well. After incubation at 37 °C for 30 min and washing with PBST, 180 µl of color-developing solution (9.7% diethanolamine and 3 mM NaN3) containing 1 mg/ml of ρ-nitrophenyl phosphate disodium hexahydrate (ρ-NPP-Na) (GMbiolab) was loaded to each well for colorization. The absorbances at 405 nm (A405) were recorded by a Model 680 microplate reader (Bio-Rad) for 1 h after the addition of enzyme substrate.
Immunoblotting
Plant leaf tissues were ground in protein sample buffer at a 1/50 dilution, denatured by boiling for 3 min, put on ice for 1 min and centrifuged at 16,110 g for 3 min. The supernatants were collected and separated in 12% SDS-PAGE, and then transferred onto nitrocellulose (NC) membranes in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) at 120 V for 30 min. The NC membranes were washed with TSW buffer (10 mM Tris-HCl, pH 7.4, 154 mM NaCl, 0.25% gelatin, 0.1% Triton X-100 and 0.02% SDS) for 3 times, each time for 3 min, and then shaken with TSW buffer-diluted antibodies at room temperature for 30 min. After washing for 3 times with TSW buffer, each for 3 min, the NC membranes were incubated at room temperature with AP-conjugated affinipure goat anti-rabbit IgG (Jackson Immuno Research Laboratories) at a 1/5000 dilution in TSW buffer for 30 min. The NC membranes were then washed twice with TSW buffer for 3 min and rinsed with substrate buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl2) for 3 min. Color development was conducted by adding 50 µl of 50 mg/ml NBT (nitro blue tetrazolium chloride) and 25 µl of 50 mg/ml BCIP (5-bromo-4-chloro-3-indoyl phosphate) in 7.5 ml substrate buffer. The reaction was stopped by submerging the NC membranes in water.
Virus detection in fields
Symptomatic cucurbit samples were collected in fields of Yunlin, Changhua and Taichung in central Taiwan. Total RNA extracted from plant tissue by the Plant Total RNA Miniprep Purification kit (GeneMark) was used for RT-PCR analysis. The primer pairs CBLV3900F/CBLV4576R specific to CBLV (see Additional file 2), Crini-hsp70-f (5′-GCCATAACCATTACGGGAGA-3′)/Crini-hsp70-r (5′-CGCAGTGAAAAACCCAAACT-3′) to CCYV [4], MYSV-N-f (5′-GCCATGGCATGCATGTCTACCGTTACTAAGCTGACA-3′)/MYSV-N-r (5′-GTCTAGAGGTACCAACTTCAATGGACTTAGCTCTGGA-3′) to MYSV [29], WN2963 (5′-AATAATCGGTGCCAGTCCCCTT-3′)/WN3469c (5′-ATGTCTAACGTTAAGCAGCTCACA-3′) to WSMoV [29] and SLCuPV-AV1-74 F (5′-GCCCCTATGTTTCCCGTGCAGT-3′)/SLCuPV-AV1-760R (5′-CCGAATCATAAAATAGATCCGG-3′) to SLCuPV, designed in this study, were used for nucleic acid amplification. The primer pair nad5-s (5′-GATGCTTCTTGGGGCTTCTTGTT-3′)/nad5-as (5′-CTCCAGTCACCAACATTGGCATAA-3′) for amplifying NADH dehydrogenase subunits 5 (nad5) gene was used as plant internal control [30]. The One-Step RT-PCR kit (GeneMark) was used in RT-PCR analysis as described by the manufacturer. The amplification conditions were set as 50 °C for 30 min, followed by 94 °C for 2 min, and then 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 1 min at 72 °C and a final reaction at 72 °C for 7 min. Indirect ELISA was performed as mentioned above to detect MYSV, WSMoV, PRSV-W and ZYMV using individual antisera described previously [9].