First report of citrus leaf blotch virus infecting Viburnum lentago in South Korea.

Viburnum lentago (family Adoxaceae) is a perennial plant species native to northeastern United States and southern Canada. Globally, V. lentago is a popular garden plant due to its abundant flowers and beautiful autumnal color. V. lentago is also commercially cultivated for medicinal purposes because its roots and fruits can be used in herbal preparations (Jiao et al. 2021). In June 2022, virus-like symptoms of vein chlorosis and yellowing were observed in the leaves of many V. lentago trees planted in a public park in Wonju, South Korea. Leaf samples were collected from five symptomatic V. lentago trees. To identify the causal agent(s) of the virus-like symptoms, total RNA was isolated from one sample using PureLink® RNA Mini Kit (Invitrogen, USA) and subjected to library construction using Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, Inc., USA). RNA-Seq was performed using an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo assembly of 118,878,556 quality-filtered reads was performed using the Trinity pipeline (Kwon et al. 2018), yielding 296,109 contigs. BLASTn and BLASTx analyses of the contigs against the GenBank viral reference database identified only one large contig (8,816 nt) containing a 26-nt poly(A) tail of viral origin. This contig had a maximum nucleotide identity of 85.53 % (with 99 % coverage) with isolate HZ (accession No. MH427034) of citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae), suggesting that the collected sample was infected with CLBV. All collected V. lentago samples were tested using RT-PCR with CLBV-specific primers (CLBV-Det-Fw 5'-AACGAGGCCAATTCTGCTAT-3' and CLBV-Det-Rv 5'-GACTGCTTGACTAACAC-CCA-3'). All samples were positive for CLBV. For biological indexing, sap from the symptomatic V. lentago leaves was mechanically inoculated to indicator plants, including Nicotiana benthamiana, N. occidentalis, N. tabacum, Datura stramonium, Chenopodium quinoa, Vigna unguiculata, and V. lentago. Three months later, only V. lentago developed the same vein chlorosis symptoms observed in the collected samples, and no other tested plants exhibited obvious symptoms. Further, only V. lentago sample tested positive for CLBV using RT-PCR analysis. To determine the complete genome sequence of the CLBV V. lentago isolate, the contig sequence was confirmed by de novo sequencing of the RT-PCR products amplified using CLBV-specific primers. The 5' terminal sequence of the contig was determined using the 5' rapid amplification of cDNA ends method (Seo et al. 2015). The full-length sequence of CLBV isolated from V. lentago was 8,795 nt in length (excluding poly(A) tail), and deposited in GenBank under the accession number OP751940. Although numerous isolates of CLBV have been identified in various plant species, including citrus, kiwi, and lemon plants (Cao et al. 2017), the V. lentago isolate is likely a distinct variant because its CP gene has a maximum nucleotide identity of 85.53 % with that of a kiwi isolate (MH339916). With little information available on viral diseases infecting V. lentago, this is the first identified and completely sequenced CLBV infecting V. lentago. Significantly, V. lentago plants infected with CLBV did not flower throughout the summer period, reducing their value as an ornamental plant. Furthermore, V. lentago might have acted as an intermediate host to transfer CLBV to other crops such as citrus. To the best of our knowledge, this is the first report of CLBV infecting V. lentago in South Korea and the world.

A multiplex RT-PCR assay for detection of emergent pepper Tsw resistance-breaking variants of tomato spotted wilt virus in South Korea.

Tomato spotted wilt virus (TSWV) is a highly destructive virus for pepper. Introgression of the resistance gene Tsw in pepper is used to manage TSWV worldwide; however, the occurrence of Tsw resistance-breaking (RB) variants threatens the pepper industry. Here, we developed a multiplex reverse-transcription PCR assay for detection of recently emerged Tsw RB variants in South Korea with high specificity and sensitivity.

SlHair2 Regulates the Initiation and Elongation of Type I Trichomes on Tomato Leaves and Stems.

Trichomes are hair-like structures that are essential for abiotic and biotic stress responses. Tomato Hair (H), encoding a C2H2 zinc finger protein, was found to regulate the multicellular trichomes on stems. Here, we characterized Solyc10g078990 (hereafter Hair2, H2), its closest homolog, to examine whether it was involved in trichome development. The H2 gene was highly expressed in the leaves, and its protein contained a single C2H2 domain and was localized to the nucleus. The number and length of type I trichomes on the leaves and stems of knock-out h2 plants were reduced when compared to the wild-type, while overexpression increased their number and length. An auto-activation test with various truncated forms of H2 using yeast two-hybrid (Y2H) suggested that H2 acts as a transcriptional regulator or co-activator and that its N-terminal region is important for auto-activation. Y2H and pull-down analyses showed that H2 interacts with Woolly (Wo), which regulates the development of type I trichomes in tomato. Luciferase complementation imaging assays confirmed that they had direct interactions, implying that H2 and Wo function together to regulate the development of trichomes. These results suggest that H2 has a role in the initiation and elongation of type I trichomes in tomato.

A one-step reverse-transcription loop-mediated isothermal amplification assay optimized for the direct detection of cucumber green mottle mosaic virus in cucurbit seeds.

Cucumber green mottle mosaic virus (CGMMV) is a seed-borne virus that causes significant economic losses in farms cultivating cucurbit plants. With the increase in global trade of cucurbit seeds, it is essential to develop a rapid, reliable, and convenient diagnostic method for the direct detection of CGMMV in these seeds for prevention and management of the disease. Here, we developed a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the direct detection of CGMMV in cucurbit seeds. To improve the efficiency of the one-step RT-LAMP assay, six primers were designed to target the most conserved regions of the gene encoding the movement protein of CGMMV. Our one-step RT-LAMP assay was optimized to improve specificity and sensitivity for CGMMV detection in individual seeds. A comparison of the detection sensitivity revealed that our one-step RT-LAMP assay was 100-fold more sensitive than the current reverse transcription-polymerase chain reaction assay used for CGMMV quarantine in Korea. Collectively, the one-step RT-LAMP assay developed in the present study is appropriate for the direct detection of CGMMV in individual cucurbit seeds.

Resistance-Breaking Tomato Spotted Wilt Virus Variant that Recently Occurred in Pepper in South Korea is a Genetic Reassortant.

Tomato spotted wilt virus (TSWV) is a destructive viral pathogen in various crops, including pepper. Although the single dominant gene Tsw has been utilized in pepper breeding to confer resistance to TSWV, the occurrence of TSWV variants that overcome Tsw-mediated resistance has been reported in various countries after several years of growing resistant cultivars. In this study, we determined the complete genome sequence of a resistance-breaking TSWV variant (TSWV-YI) that recently emerged in pepper in South Korea. TSWV-YI infected all of the resistant pepper cultivars tested. The phylogenetic and recombination analyses of the complete TSWV-YI genome sequence showed that it is a reassortant that acquired its L and M RNA segments from the existing South Korean TSWV population and its S RNA in an isolate from another country. Given that TSWV-YI is a resistance-breaking variant, it appears that reassortment of the S RNA led to the emergence of this variant that breaks the Tsw gene in pepper grown in South Korea. Our results suggest that resistance-breaking TSWV variants are a potential threat to pepper production in South Korea and that strategies to manage these variants should be developed to ensure sustainable pepper production.

A Plant Virus-Based Vector System for Gene Function Studies in Pepper.

While pepper (Capsicum annuum) is a highly recalcitrant species for genetic transformation studies, plant virus-based vectors can provide alternative and powerful tools for transient regulation and functional analysis of genes of interest in pepper. In this study, we established an effective virus-based vector system applicable for transient gain- and loss-of-function studies in pepper using Broad bean wilt virus2 (BBWV2). We engineered BBWV2 as a dual gene expression vector for simultaneous expression of two recombinant proteins in pepper cells. In addition, we established enhanced and stable expression of recombinant proteins from the BBWV2-based dual vector via coexpression of a heterologous viral suppressor of RNA silencing. We also developed a BBWV2-based virus-induced gene silencing (VIGS) vector, and we successfully silenced the phytoene desaturase gene (PDS) using the BBWV2-based VIGS vector in various pepper cultivars. Additionally, we optimized the BBWV2-based VIGS system in pepper by testing the efficiency of PDS gene silencing under different conditions. This BBWV2-based vector system represents a convenient approach for rapid and simple analysis of gene functions in pepper.

Silencing of AtRAP, a target gene of a bacteria-induced small RNA, triggers antibacterial defense responses through activation of LSU2 and down-regulation of GLK1.

Plants fine-tune their sophisticated immunity systems in response to pathogen infections. We previously showed that AtlsiRNA-1, a bacteria-induced plant endogenous small interfering RNA, silences the AtRAP gene, which encodes a putative RNA binding protein. In this study, we demonstrate that AtRAP functions as a negative regulator in plant immunity by characterizing molecular and biological responses of the knockout mutant and overexpression lines of AtRAP upon bacterial infection. AtRAP is localized in chloroplasts and physically interacts with Low Sulfur Upregulated 2 (LSU2), which positively regulates plant defense. Our results suggest that AtRAP negatively regulates defense responses by suppressing LSU2 through physical interaction. We also detected downregulation of the transcription factor GOLDEN2-LIKE 1 (GLK1) in atrap-1 using microarray analysis. The glk1 glk2 double mutant showed enhanced resistance to Pseudomonas syringae pv. tomato, which is consistent with a previous study showing enhanced resistance of a glk1 glk2 double mutant to Hyaloperonospora arabidopsidis. Taken together, our data suggest that silencing of AtRAP by AtlsiRNA-1 upon bacterial infection triggers defense responses through regulation of LSU2 and GLK1.

The complete genome sequence of a novel virus, bellflower veinal mottle virus, suggests the existence of a new genus within the family Potyviridae.

A new virus was isolated from a bellflower (Campanula takesimana) plant showing veinal mottle symptoms, and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 8,259 ribonucleotides. Electron microscopic observation revealed that the viral genome is packaged as a filamentous particle with an average length of approximately 760 nm. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 34.1% (with 95% coverage) to that of the isolate AD of Chinese yam necrotic mosaic virus (CYNMV; genus Macluravirus). Phylogenetic analysis and comparison of the encoded amino acid sequences with those of other viruses demonstrated that the identified virus shows minimal sequence similarity to known viruses and should therefore be considered a member of a new genus in the family Potyviridae. The name bellflower veinal mottle virus (BVMoV) is proposed for this new virus.

Complete genome sequence of longan witches' broom-associated virus, a novel member of the family Potyviridae.

The complete genome sequence of a new virus isolated from a longan (Dimocarpus longan Lour.) plant showing witches' broom syndrome was determined. The viral genome is composed of a monopartite single-stranded RNA of 9,428 nucleotides excluding the 3' poly(A) tail and contains one large single open reading frame encoding a polyprotein of 3086 amino acids. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 35% (with 85% coverage) to that of the isolate Minnesota of rose yellow mosaic virus (RoYMV; family Potyviridae; genus not assigned). Molecular and phylogenetic analysis of the genome and encoded protein sequences showed that the identified virus has the general features that are characteristic of members of the family Potyviridae although it has extremely low sequence similarity to known members of the family Potyviridae. The name longan witches' broom-associated virus (LWBaV) is proposed for this new virus, which may be considered a member of a new genus in the family Potyviridae.

Complete genome sequence of yacon necrotic mottle virus, a novel putative member of the genus Badnavirus.

The complete genome sequence of a previously undescribed virus isolated from a yacon plant exhibiting necrotic mottle, chlorosis, stunting, and leaf malformation symptoms in Gyeongju, Korea, was determined. The genome of this virus consists of one circular double-stranded DNA of 7661 bp in size. The genome contained four open reading frames (ORFs 1 to 4) on the plus strand that potentially encode proteins of 26, 32, 234, and 25 kDa. Protein BLAST analysis showed that ORF3, which is the largest ORF, has 45 % amino acid sequence identity (with 89 % coverage) to the ORF3 of fig badnavirus 1 (FBV-1), a recently identified badnavirus. Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Badnavirus. The name yacon necrotic mottle virus (YNMoV) is proposed for this new virus.

Complete genome sequence of bellflower vein chlorosis virus, a novel putative member of the genus Waikavirus.

The complete genome sequence of a new virus isolated from a bellflower (Campanula takesimana) plant was determined. The genome of this virus is composed of monopartite single-stranded RNA of 11,649 nucleotides in length. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 42% (with 99% coverage) to the polyprotein of the isolate Orissa of rice tungro spherical virus (RTSV; genus Waikavirus). Phylogenetic analysis strongly supports that the identified virus is a member of a new species of the genus Waikavirus. The name bellflower vein chlorosis virus (BVCV) is proposed for this new virus.

Complete genome sequence of motherwort yellow mottle virus, a novel putative member of the genus Torradovirus.

The complete genome sequence of a new virus isolated from a motherwort plant exhibiting yellow mottle, mild mosaic, and stunting symptoms in Andong, Korea, was determined. The genome of this virus is composed of two single-stranded RNAs (7068 and 4963 nucleotides in length, respectively) carrying poly(A) tails. RNA1 contains one large open reading frame (RNA1-ORF1), while two potential ORFs (RNA2-ORF1 and RNA2-ORF2) were found in RNA2. BLAST searches of protein databases showed that RNA1-ORF1 and RNA2-ORF2 have maximum amino acid sequence identities of 53 % and 57 % to the RNA1-ORF1 and RNA2-ORF2, respectively, of lettuce necrotic leaf curl virus (LNLCV, a recently identified torradovirus). Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Torradovirus. The name "motherwort yellow mottle virus" (MYMoV) is proposed for this new virus.

Mutations in the capsid protein of Brome mosaic virus affecting encapsidation eliminate vesicle induction in planta: implications for virus cell-to-cell spread.

Positive-strand RNA viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation, or egress. Our previous transmission electron microscopic (TEM) analysis showed that ectopic expression of wild-type (wt) capsid protein (CP) of Brome mosaic virus (BMV) has an intrinsic property of modifying the endoplasmic reticulum (ER) to induce vesicles similar to those present in wt BMV infection. In this study, we evaluated the functional significance of CP-mediated vesicle induction to the BMV infection cycle in planta. Consequently, the cytopathologic changes induced by wt CP or its mutants defective in virion assembly due to mutations engineered in either N- or C-proximal domains were comparatively analyzed by TEM in two susceptible (Nicotiana benthamiana and Chenopodium quinoa) and one nonhost (N. clevelandii) plant species. The results showed that in susceptible hosts, CP-mediated ER-derived vesicle induction is contingent on the expression of encapsidation-competent CP. In contrast, unlike in N. benthamiana and C. quinoa, transient expression of wt CP in nonhost N. clevelandii plants eliminated vesicle induction. Additionally, comparative source-to-sink analysis of virus spread in leaves of N. benthamiana and N. clevelandii coexpressing wt BMV and Cucumber mosaic virus (CMV) showed that despite trans-encapsidation, CMV failed to complement the defective cell-to-cell movement of BMV. The significance and relation of CP-mediated vesicle induction to virus cell-to-cell movement are discussed.

Molecular genetic analysis of cucumber mosaic virus populations infecting pepper suggests unique patterns of evolution in Korea.

Studying genetic structure and diversity of viruses is important to understand the evolutionary mechanisms that generate and maintain variations in viral populations. Cucumber mosaic virus (CMV) is endemic in most pepper fields in Korea. Currently, no effective methods for control of CMV are available due to many environmental and biological factors such as the extensive evolutionary capacity of CMV. Thus, analyzing the genetic structure of CMV populations may facilitate the development of strategies for the control of CMV. In this study, 252 pepper (Capsicum annuum) samples showing virus symptoms were collected by field surveys performed throughout Korea in 2007. Reverse-transcription polymerase chain reaction analyses revealed that, in total, 165 collected samples were infected with CMV. Forty-five CMV isolates were randomly selected within each regional subpopulation and analyzed by full-genome sequencing. Analyses of genetic diversity showed that the 2b gene of CMV is under weaker purifying selection than the other genes. Based on the phylogenetic analysis of RNA1, the CMV isolates from pepper were divided into three clusters in subgroup I. Our full-genome sequence-based molecular analyses of the CMV Korean population suggest that the subpopulations of CMV have been geographically localized in pepper fields in Korea.

Engineering of stable infectious cDNA constructs of a fluorescently tagged tomato chlorosis virus.

Tomato chlorosis virus (ToCV) is an emerging pathogen that cause severe yellow leaf disorder syndrome in tomato plants. In this study, we aimed to generate a recombinant ToCV tagged with green fluorescent protein (GFP) to enable real-time monitoring of viral infection in living plants. Transformation of the full-length cDNA construct of ToCV RNA1 into Escherichia coli resulted in instability issues, which were successfully overcome by inserting a plant intron into RNA1. Subsequently, a GFP tag was engineered into a cDNA construct of ToCV RNA2. The resulting recombinant ToCV-GFP could systemically infect Nicotiana benthamiana plants, and GFP expression was observed along the major veins. Utilizing ToCV-GFP, we also showed that ToCV engages in antagonistic relationships with two different tomato-infecting viruses in mixed infections in N. benthamiana. This study demonstrates the potential of ToCV-GFP as a valuable tool for the visual tracking of infection and movement of criniviruses in living plants.

The VP53 protein encoded by RNA2 of a fabavirus, broad bean wilt virus 2, is essential for viral systemic infection.

Plant viruses evolves diverse strategies to overcome the limitations of their genomic capacity and express multiple proteins, despite the constraints imposed by the host translation system. Broad bean wilt virus 2 (BBWV2) is a widespread viral pathogen, causing severe damage to economically important crops. It is hypothesized that BBWV2 RNA2 possesses two alternative in-frame translation initiation codons, resulting in the production of two largely overlapping proteins, VP53 and VP37. In this study, we aim to investigate the expression and function of VP53, an N-terminally 128-amino-acid-extended form of the viral movement protein VP37, during BBWV2 infection. By engineering various recombinant and mutant constructs of BBWV2 RNA2, here we demonstrate that VP53 is indeed expressed during BBWV2 infection. We also provide evidence of the translation of the two overlapping proteins through ribosomal leaky scanning. Furthermore, our study highlights the indispensability of VP53 for successful systemic infection of BBWV2, as its removal results in the loss of virus infectivity. These insights into the translation mechanism and functional role of VP53 during BBWV2 infection significantly contribute to our understanding of the infection mechanisms employed by fabaviruses.

Contribution of small RNA pathway components in plant immunity.

Small RNAs regulate a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity, in a sequence-specific manner. Accumulating evidence reveals that host endogenous small RNAs and small RNA pathway components play important roles in plant immune responses against various pathogens, including bacteria, fungi, oomycetes, and viruses. Small-RNA-mediated defense responses are regulated through diverse pathways and the components of these pathways, including Dicer-like proteins, RNA-dependent RNA polymerases, Argonaute proteins, and RNA polymerase IV and V, exhibit functional specificities as well as redundancy. In this review, we summarize the recent insights revealed mainly through the examination of two model plants, Arabidopsis and rice, with a primary focus on our emerging understanding of how these small RNA pathway components contribute to plant immunity.

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

Helper virus-independent transcription and multimerization of a satellite RNA associated with cucumber mosaic virus.

Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.