Infectious in Vitro Transcripts From cDNA Clone of a Japanese Gentian Isolate of Sikte Waterborne Virus, Which Shows Host-specic Low-temperature-preferred Multiplication

Tombusviruses have been identied in several crops, which include gentian virus A (GeVA), in Japanese gentians. In this study, we isolated another tombusvirus, Sikte waterborne virus strain C1 (SWBV-C1) from Japanese gentian. Although SWBV-C1 and GeVA are not closely related among tombusviruses, SWBV-C1, like GeVA, showed host-specic low-temperature-preferred multiplication in gentians and Arabidopsis. The use of in vitro transcripts from full-length cDNA clones of SWBV-C1 genomic RNA as inocula conrmed these properties, which indicates that the identied genomic RNA sequences encode viral factors underlying characteristic SWBV-C1 features.


Introduction
Plant viruses form a group of pathogens that cause serious damage to economically important crops.
Although many plant viral diseases have been reported in natural elds, new unknown viral diseases have arisen, owing, in part, to current agricultural practices, such as the cultivation of diverse new crops and environmental amendments.
Japanese gentians (Gentiana tri ora, Gentiana scabra and their hybrids) are important ornamental owering plants in Japan [1], and multiple viruses, including cucumber mosaic virus, broad bean wilt virus 2, clover yellow vein virus, impatiens necrotic spot virus, and gentian mosaic virus [2][3][4], infect Japanese gentians in farms. By investigating cultivated gentians in northeastern Japan that had symptoms of an unknown disease, novel plant viruses, such as gentian ovary ring-spot virus, gentian kobu-sho-associated virus, and gentian virus A (GeVA), have been identi ed [5][6][7]. Tombusviruses, including GeVA, have monopartite positive-sense RNA genomes that encode ve proteins, p33 and its read-through product p92, which are replication proteins, as well as p41, p21, and p19, which represent a coat protein (CP), movement protein, and silencing suppressor, respectively [8]. A phylogenetic analysis of the amino acid sequences of these proteins suggests that GeVA is a novel tombusvirus [7]. In addition, GeVA e ciently multiplies at a low temperature (18°C) and induces symptoms in gentians and Arabidopsis, but GeVA multiplication and virulence have not been detected at 23°C [9]. To our knowledge, GeVA was the rst tombusvirus reported to show virulence against Japanese gentians.
In this study, to understand more about tombusvirus-related diseases of gentians, we determined the presence of viruses in gentian plants using the double-stranded RNA (dsRNA) isolation, exhaustive ampli cation, cloning, and sequencing (DECS) method [10,11]. In the DECS analysis, dsRNA was puri ed from the total RNA of gentians using glutathione S-transferase-tagged dsRNA-binding protein 4, and the cDNAs were cloned and sequenced as described previously [7]. Of the 96 clones produced by the DECS method from a Japanese gentian plant showing necrotic symptoms, 34 cDNA fragments were homologous to the nucleotide sequence of the Sikte waterborne virus (SWBV) isolate Eckbach CP gene (92.8% coverage). On the basis of the nucleotide sequences, cDNA fragments containing full-length CPcoding sequences were ampli ed by reverse transcription polymerase chain reaction (RT-PCR) using the primer pair GVCPf1 (5′-ATGTCGATGGTAAGAAGAAATCAG-3′) and GVCPr1 (5′-TTAAGGGAATGTGACCGAGTTTAT-3′), and the resulting PCR products were directly sequenced using the Sanger method. The deduced CP amino acid sequence shared a 98.8% identity, which is greater than the 87% taxonomic criterion for tombusvirus species, with SWBV-Eckbach CP ( Figure 1a) [12,13], suggesting that the identi ed virus belongs to the same species as SWBV. Single lesion isolations were repeated three times from inoculated leaves of Chenopodium quinoa, and viruses were nally propagated in Nicotiana benthamiana and puri ed as described previously [6]. We named this virus SWBV strain C1 (SWBV-C1).
SWBV is a pathogen of Limonium sinuatum [13], but SWBV infections in gentians have not been reported. During SDS-PAGE, the CP band of puri ed SWBV-C1 migrated more rapidly than that of GeVA ( Figure 1b), and a phylogenetic analysis using the deduced amino acid sequences of tombusvirus CPs ( Figure 1c) revealed a distance between SWBV-C1 and GeVA (50.6% shared identity between CP amino acid sequences), indicating that GeVA and SWBV-C1 belong to different tombusvirus species. To characterize SWBV-C1, its genomic structure was determined. On the basis of the partial tombusvirus sequences identi ed using the DECS method, we designed the rapid ampli cation of cDNA ends (RACE) primers G-SWBV-RACErev (5′-CCTGCCGCCAGTCGCAATTG-3′) and G-SWBV-RACEfw (5′-AGCGTCTCATTGAGATGGCA-3′). SWBV-C1 virion RNA was polyadenylated by poly A polymerase (NEB), and the 5′ and 3′ terminal cDNA ends were ampli ed by the RACE method using a GeneRacer kit with Superscript III (ThermoFisher Scienti c). A Zero Blunt TOPO PCR cloning kit (ThermoFisher Scienti c) was used for the cloning and sequencing of the RACE fragments. Then, cDNA was synthesized using a reverse-transcription reaction with primer SWBV-3′Rv (5′-GGGCTGCATTTCTGCAATGT-3′) and ReverTra Ace (TOYOBO). The full-length cDNA of SWBV genomic RNA was ampli ed by PCR using the primer pair GtSWBV-FW (5′-AAGCTTGCATGCCTGCAGGAAATTCTCCAGGATTTCTC-3′) and GtSWBV-RV (5′-ACCCGGGGATCCTCTAGAACGCGTGGGCTGCATTTCTGCAATGT-3′). pUC19 was digested with PstI and XbaI, and a 4-5-kb cDNA fragment was cloned using an In-Fusion HD cloning Kit (TaKaRa). Cloned plasmids were ampli ed in Escherichia coli strain JM109, and plasmid vectors carrying SWBV cDNA were sequenced using the Sanger method.
The low-temperature (18°C) multiplication preference of GeVA in gentians and A. thaliana ecotype Columbia-0 (Col-0) has been reported [9]. Therefore, we examined the effects of temperature on SWBV-C1 multiplication and virulence in gentians and Arabidopsis. For the infection assay, gentian plants grown in vitro, which are available all year, were inoculated with SWBV-C1 virion RNA (0.5 mg/ml). At 4 weeks postinoculation, necrotic symptoms were observed on inoculated leaves of gentian cultivars 'Alta' and 'Albireo' at 18°C but not at 28°C (Figure 2a). To detect SWBV infections, rabbit SWBV-C1 virion-speci c antiserum was prepared by Scrum Inc. (Tokyo, Japan), and press-blot assays [18] using the anti-SWBV-C1 antiserum were performed. At 10 days after inoculation, SWBV-C1 infections in the inoculated leaves of 'Alta' and 'Albireo' were detected at 18°C but not at 28°C (Figure 2b). The low-temperature-preferred multiplication and virulence of SWBV-C1 were observed in not only gentians but also in Arabidopsis (Figure 2c, d).
However, in other experimental plants, including N. benthamiana, Nicotiana tabacum, and C. quinoa, SWBV-C1 e ciently multiplied at both 18°C and 28°C (Table 1), which was consistent with previous ndings for GeVA [9]. In comparison with GeVA, SWBV-C1-induced symptoms in gentians were similar or more severe (Online Resource 2). In particular, SWBV-C1 induced more severe symptoms than GeVA in the inoculated leaves of Col-0 ( Figure 2e). Additionally, SWBV-C1 infections in Col-0 leaves but not gentian leaves were detected by the presence of small dots at 23°C (Figure 2b and 2d), while GeVA infections were not detected at either 23°C or 28°C (Figure 2d), suggesting that SWBV-C1 is more virulent than GeVA in Arabidopsis. Overall, although some differences between SWBV-C1 and GeVA were observed, these data highlight their common host ranges and viral multiplication properties. Thus, we speculated that a common mechanism may underlie the host-speci c preference for low temperature associated with SWBV-C1 and GeVA multiplication. We then focused on further analyzing SWBV-C1.
To elucidate tombusvirus-gentian/Arabidopsis interactions, we established an infectious SWBV-C1 genomic RNA synthesis system to analyze viral factors. The full-length cDNA of SWBV-C1 genomic RNA was ampli ed using the primer pair T7-GtSWBV5′-FW (5′-AAGCTTGCATGCCTGCAGTAATACGACTCACTATAGGAAATTCTCCAGGATTTCTC-3′) and GtSWBV-RV, and the resulting cDNA fragment was inserted into the PstIand XbaI double-digested pUC19 vector using an In-Fusion HD Cloning Kit (TaKaRa) to construct pT7-SWBV-C1 (Figure 2f). The 5′ primer (T7-GtSWBV5′-FW) contained the T7 promoter sequence. On the basis of previous reports of biologically active bromovirus cDNA clones [19,20], an extra G residue was added to the 5′ terminus of the SWBV-C1 genome to enhance the e ciency of in vitro transcription. The 3′ primer (GtSWBV-RV) contained a MluI site for the linearization of the cloned plasmid. The SWBV-C1 genomic sequence in pT7-SWBV-C1 was identical to that determined in the above experiments. pT7-SWBV-C1 was digested with MluI and transcribed by T7 RNA polymerase. The resulting in vitro transcripts contained extra residues at the 5′ and 3′ termini of the SWBV-C1 genomic sequence (Figure 2f). Because the addition of these extra residues did not have any detrimental effects on the infectivity of SWBV in N. benthamiana (Online Resource 3), we used the transcripts for further infection assays. After the Japanese gentian cultivar 'Alta' was inoculated with SWBV-C1 transcripts, severe necrotic symptoms were induced at 18°C but not 28°C (Figure 2g), and the resulting symptoms were similar to those produced by inoculation with SWBV virion RNA.
Additionally, the low-temperature-preferred multiplication was detected in gentian and Arabidopsis leaves inoculated with the SWBV-C1 transcript (Figure 2h). Overall, the infectivity of the SWBV-C1 transcript were identical to those of the SWBV-C1 virion RNA in all the tested plants under the test conditions (Table 1). Thus, these data con rmed that viral factors underlying SWBV-C1 features (e.g., host range, symptoms, and host-speci c low-temperature-preferred multiplication) are encoded in the same genomic RNA sequence. The SWBV factors, especially those involved in SWBV-C1-characteristic features, will be analyzed using this biologically active SWBV cDNA clone in the future.