Arctium lappa is a new Natural Host of Tomato Spotted Wilt Virus in Korea.

Burdock (Arctium lappa L., belongs to the family Asteraceae), is an edible plant and an oriental medicinal herb in Korea (Han and Koo, 1993). In July 2023, burdocks showing chlorotic ringspots and yellowing on the leaves were observed in nine of approximately 4,000 plants in a greenhouse in Daegu, South Korea. To determine the causal virus species, nine symptomatic leaves from each individual plant were collected and tested using commercially available immunostrips (Agdia, Elkhart, USA) for cucumber mosaic virus (CMV) and tomato spotted wilt virus (TSWV). Seven out of nine samples tested positive for TSWV only. TSWV in South Korea was first reported on sweet pepper from Yesan in 2004 (Kim et al., 2004) and has since spread to various crops. The first report of TSWV infecting burdock plants in the world was from Hawaii in 1995 (Bautista et al., 1995), but TSWV-infected burdock has not been reported in Korea. To further confirm the presence of TSWV, total RNA was extracted from TSWV-positive burdock leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and used in reverse transcription-polymerase chain reaction (RT-PCR) assays with a specific primer set that amplifies 777 bp of nucleocapsid gene (N gene) of TSWV (Yoon et al., 2014). To obtain the complete genome sequence of this TSWV in the burdock plant, named TSWV-DG, fragments of L, M, and S segments were amplified and sequenced. The complete genome sequences of the L (8914 nt), M (4773 nt), and S (2946 nt) segments were obtained by overlapping RT-PCR amplicons. RT-PCR products were cloned into the pGEM-T Easy vector, and selected DNA clones were sequenced using Sanger method (Bioneer, Korea). The complete genome sequences were deposited to GenBank (LC790665, LC790666, and LC790667, respectively). BLASTn analysis showed that sequences of each TSWV-DG segment had maximum nucleotide identities of 99.5%, 99.5%, and 99.5% with TSWV-L, TSWV-M, and TSWV-S (OM154971, OM154970, and OM154969, respectively), which were isolated from water dropwort (Oenanthe crocata) in China (Qiu et al., 2023). To assess the biological activity of TSWV-DG, A. lappa and Nicotiana benthamiana were inoculated mechanically with sap from infected burdock leaves and maintained for visual inspection of virus symptoms at 25 ℃ at 3 weeks. TSWV-DG produced symptoms on the systemic leaves of A. lappa, that included chlorotic spots and yellowing, and on the leaves of N. benthamiana, that included chlorotic spots and mosaic patterns from 14 days-post-inoculation. Meanwhile, mock-inoculated A.lappa and N.benthamiana remained symptomless. The presence of TSWV on the inoculated leaves was subsequently confirmed through Immunostrip and RT-PCR analyses. TSWV may pose a significant threat to the production of A. lappa, which is cultivated as both leafy greens and root vegetables in Korea. Furthermore, A. lappa may not only be at risk of damage from TSWV infection but also act as a potential source of TSWV infection, thereby posing a risk of transmission to other key crops in Korea, such as pepper or potato (Yoon et al., 2014). This is the first report TSWV infecting burdock in South Korea.

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent.

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum 'Xanthi') plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.

The Secret Life of the Inhibitor of Virus Replication.

The inhibitor of virus replication (IVR) is an inducible protein that is not virus-target-specific and can be induced by several viruses. The GenBank was interrogated for sequences closely related to the tobacco IVR. Various RNA fragments from tobacco, tomato, and potato and their genomic DNA contained IVR-like sequences. However, IVRs were part of larger proteins encoded by these genomic DNA sequences, which were identified in Arabidopsis as being related to the cyclosome protein designated anaphase-promoting complex 7 (APC7). Sequence analysis of the putative APC7s of nine plant species showed proteins of 558-561 amino acids highly conserved in sequence containing at least six protein-binding elements of 34 amino acids called tetratricopeptide repeats (TPRs), which form helix-turn-helix structures. The structures of Arabidopsis APC7 and the tobacco IVR proteins were modeled using the AlphaFold program and superimposed, showing that IVR had the same structure as the C-terminal 34% of APC7, indicating that IVR was a product of the APC7 gene. Based on the presence of various transcription factor binding sites in the APC7 sequences upstream of the IVR coding sequences, we propose that IVR could be expressed by these APC7 gene sequences involving the transcription factor SHE1.

The Virus-Induced Transcription Factor SHE1 Interacts with and Regulates Expression of the Inhibitor of Virus Replication (IVR) in N Gene Tobacco.

The transcription factor SHE1 was induced by tobacco mosaic virus (TMV) infection in tobacco cv. Samsun NN (SNN) and SHE1 inhibited TMV accumulation when expressed constitutively. To better understand the role of SHE1 in virus infection, transgenic SNN tobacco plants generated to over-express SHE1 (OEx-SHE1) or silence expression of SHE1 (si-SHE1) were infected with TMV. OEx-SHE1 affected the local lesion resistance response to TMV, whereas si-SHE1 did not. However, si-SHE1 allowed a slow systemic infection to occur in SNN tobacco. An inhibitor of virus replication (IVR) was known to reduce the accumulation of TMV in SNN tobacco. Analysis of SHE1 and IVR mRNA levels in OEx-SHE1 plants showed constitutive expression of both mRNAs, whereas both mRNAs were less expressed in si-SHE1 plants, even after TMV infection, indicating that SHE1 and IVR were associated with a common signaling pathway. SHE1 and IVR interacted with each other in four different assay systems. The yeast two-hybrid assay also delimited sequences required for the interaction of these two proteins to the SHE1 central 58-79% region and the IVR C-terminal 50% of the protein sequences. This suggests that SHE is a transcription factor involved in the induction of IVR and that IVR binds to SHE1 to regulate its own synthesis.

First report of Colombian datura virus in Brugmansia suaveolens in Korea.

Brugmansia suaveolens, known as angel's trumpet, is a perennial ornamental shrub in the Solanaceae with large fragrant flowers. In June 2018, a leaf sample of B. suaveolens that showed virus-like symptoms including chlorotic spots, yellowing and mottle on leaves was collected from a greenhouse in Seongnam, South Korea for disease diagnosis (Supplementary Figure S1a, b). Disease incidence in the greenhouse was greater than 80% for about 2,000 B. suaveolens plants. To identify a causal virus, transmission electron microscopy (TEM) was used to analyze symptomatic leaf samples using leaf dips and thin section methods. Filamentous virus particles and pinwheel structures were observed, indicating the presence of a potyvirus (Supplementary Figure S1c, d). To confirm the TEM results, a symptomatic leaf sample was further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using species-specific detection primers for three potyviruses that infect Brugmansia spp.: Colombian datura virus (CDV), Brugmansia mosaic virus (BruMV), and Brugmansia suaveolens mottle virus (BsMoV) (Lucinda et al, 2008; Park et al., 2014; Verma et al., 2014). The sample was positive only for CDV. CDV is transmitted by aphids in a nonpersistent manner and mechanical inoculation and can infect plants in the Solanaceae family including tomato and tobacco (Kahn and Bartels 1968; Schubert et al. 2006; Verhoeven et al. 1996) and has been designated a quarantine virus in Korea. Additional analysis of 13 symptomatic B. suaveolens plants from the infected greenhouse found that all samples except one were infected with CDV. To isolate CDV from B. suaveolens, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Nicotiana tabacum cv. BY via three single-lesion passages followed by propagation in N. benthamiana. For the bioassay of the CDV isolate (CDV-AT-Kr), sap from infected N. benthamiana was mechanically inoculated on 31 indicator plants, including B. suaveolens (Supplementary Table S2). CDV-AT-Kr induced chlorotic local lesions, necrotic local lesions, mottle, and/or mosaic systemically in 10 Nicotiana spp., and mottle and yellowing in tomato. On inoculated B. suaveolens, te mild mottle symptom was reproduced. No symptoms were observed in pepper or Datura stramonium. These results were confirmed by RT-PCR. To characterize CDV-AT-Kr genetically, the complete genome sequence of CDV-AT-Kr was obtained by RT-PCR using specific primers (Supplementary Table S3) and deposited in GenBank (accession no. MW075268). The CDV-AT-Kr RNA consists of 9,620 nt, encoding a polyprotein of 3,076 aa. BLASTn analysis showed that CDV-AT had maximum nucleotide identities of 98.9% at the complete genome level with a CDV isolate (accession no. JQ801448) from N. tabacum in the UK. To our knowledge, this is the first report of CDV infection in B. suaveolens in Korea and the second report in the world of the complete genome sequence. As B. suaveolens is cultivated by vegetative propagation, production and maintenance of virus-free, healthy B. suaveolens is needed. In addition, as new CDV hosts have been repeatedly reported (Pacifico et al., 2016; Salamon et al., 2015; Tomitaka et al., 2014; Verma et al., 2014), we are monitoring nationwide occurrence to prevent the spread of the virus to other crops.

First report of Tomato spotted wilt virus in Petasites japonicus in Korea.

Butterbur (Petasites japonicus [Siebold & Zucc.] Maxim.) is a perennial herb of the Asteraceae family that is cultivated for medicinal and nutritional purposes. Due to long-term vegetative propagation of virus-infected native species, the yield and quality of butterbur plants have deteriorated. Five viruses have been reported to infect this species: alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), butterbur mosaic virus (ButMV), broad bean wilt virus 2 (BBWV-2), and cucumber mosaic virus (CMV) (Ham et al. 2016; Tochihara and Tamura 1976). From 2018 to 2019, butterbur plants in four greenhouses in Nonsan, South Korea (Supplementary Figure S1a, b) were found to show virus-like symptoms such as chlorotic and necrotic ring spots, necrosis, and mild mosaic on the leaves. Disease incidence was greater than 80% in one greenhouse (~1,000 m2). To identify the causal virus, we collected 17 symptomatic butterbur leaf samples from these greenhouses and performed reverse-transcription polymerase chain reaction (RT-PCR) analysis using species-specific detection primers for the five reported viruses and tomato spotted wilt virus (TSWV) (Supplementary Table S2). RT-PCR results showed that 12 samples from three greenhouses showing necrotic ring spots and mosaic symptoms were infected with a mixture of TSWV and ButMV, whereas 5 samples from one greenhouse showing mild mosaic symptoms were infected only with ButMV. TSWV (genus Orthotospovirus, family Tospoviridae) is transmitted by thrips and causes serious damage to a wide range of economically important plants (Pappu et al. 2009). ButMV (genus Carlavirus, family Betaflexiviridae) is transmitted by aphids, as well as infected vegetative propagation material (Hashimoto et al. 2009) and is the most predominant virus in butterbur in Korea (Ham et al. 2016). To isolate TSWV from butterbur, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Chenopodium quinoa, via three single-lesion passages followed by propagation in Nicotiana tabacum cv. Samsun. Thirty different indicator plant species were used for the bioassay of the TSWV isolate (TSWV-NS-BB20) by mechanical inoculation method (Supplementary Table S3). RT-PCR analysis confirmed that TSWV-NS-BB20 induced necrotic local lesions and mosaic on Nicotiana species and ring spots and mosaic on tomatoes and peppers. Notably, TSWV-NS-BB20 reproduced necrotic local lesions and mild mosaic symptoms on butterbur plants which were infected with ButMV with no obvious symptoms. To characterize TSWV-NS-BB20 genetically, the complete genome sequences of L (8914 nt), M (4751 nt), and S (2917 nt) RNA segments were obtained by RT-PCR using specific primers for TSWV as described previously (Kwak et al., 2020). The obtained sequences were deposited in GenBank under accession nos. MT643236, MT842841, and MN854654, respectively. BLASTn analysis showed that sequences of each segment had maximum nucleotide identities of 99.0, 98.9, and 98.6% to TSWV-L, M, and S (KP008128, FM163373, and KP008129) of TSWV-LL-N.05 isolate from tomato in Spain. Since 2018, TSWV outbreaks on butterbur are observed every year and thus may act as a potential source of TSWV infection for other crops of importance to Korea, such as pepper. Owing to the butterbur vegetative propagation, the identification of TSWV infection in butterbur will be helpful for future virus management to generate virus-free materials. To our knowledge, this is the first report of TSWV infection of butterbur.

The Use of a Tobacco mosaic virus-Based Expression Vector System in Chrysanthemum.

Chrysanthemums (Chrysanthemum morifolium) are susceptible to tobacco mosaic virus (TMV). TMV-based expression vectors have been used in high-throughput experiments for production of foreign protein in plants and also expressing green fluorescent protein (GFP) to allow visualization of TMV movement. Here, we used TMV expressing the GFP to examine the infection of chrysanthemum by a TMV-based expression vector. Viral replication, movement and GFP expression by TMV-GFP were verified in upper leaves of chrysanthemums up to 73 days post inoculation (dpi) by RT-PCR. Neither wild-type TMV nor TMV-GFP induced symptoms. GFP fluorescence was seen in the larger veins of the inoculated leaf, in the stem above the inoculation site and in petioles of upper leaves, although there was no consistent detection of GFP fluorescence in the lamina of upper leaves under UV. Thus, a TMV-based expression vector can infect chrysanthemum and can be used for the in vivo study of gene functions.

Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > ß-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) < PP2A < GAPDH. For local infection by TMV, the most stable genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH < PP2A < UCE. Using two of the most stable and the two least stable validated reference genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus.

Differentially expressed genes of Chenopodium amaranticolor in response to cymbidium mosaic virus infection.

Cymbidium mosaic virus (CymMV)-induced expressed sequence tag (EST) clones from Chenopodium amaranticolor were identified. CymMV was mechanically inoculated onto C. amaranticolor, and local lesion symptoms were observed. Inoculated leaves were collected on serial days post inoculation (dpi) to identify activated or suppressed genes. mRNA isolation and suppression subtractive hybridization (SSH) were then performed to identify differentially expressed genes related to the local lesion response. Fifty-three ESTs, including genes related to defense and stress responses (e.g., lipoxygenase, jasmonate-induced protein, and heat shock protein), were generated. In addition, a large proportion of the ESTs were found to be involved in photosynthesis, as determined by their functional categories. Expression levels of several EST genes were observed using quantitative real-time reverse transcription-polymerase chain reaction, and the evaluated genes showed varying levels of expression during the experimental period. In this study, differentially expressed sequences via SSH were identified from CymMV-infected C. amaranticolor, and profiling and annotation were carried out to determine the expression pattern of CymMV and its interaction with C. amaranticolor.