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.

Visual tracking of viral infection dynamics reveals the synergistic interactions between cucumber mosaic virus and broad bean wilt virus 2.

Cucumber mosaic virus (CMV) is one of the most prevalent plant viruses in the world, and causes severe damage to various crops. CMV has been studied as a model RNA virus to better understand viral replication, gene functions, evolution, virion structure, and pathogenicity. However, CMV infection and movement dynamics remain unexplored due to the lack of a stable recombinant virus tagged with a reporter gene. In this study, we generated a CMV infectious cDNA construct tagged with a variant of the flavin-binding LOV photoreceptor (iLOV). The iLOV gene was stably maintained in the CMV genome after more than four weeks of three serial passages between plants. Using the iLOV-tagged recombinant CMV, we visualized CMV infection and movement dynamics in living plants in a time course manner. We also examined whether CMV infection dynamics is influenced by co-infection with broad bean wilt virus 2 (BBWV2). Our results revealed that no spatial interference occurred between CMV and BBWV2. Specifically, BBWV2 facilitated the cell-to-cell movement of CMV in the upper young leaves. In addition, the BBWV2 accumulation level increased after co-infection with CMV.

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.

Ethylene emitted by viral pathogen-infected pepper (Capsicum annuum L.) plants is a volatile chemical cue that attracts aphid vectors.

Plant viruses are obligate intracellular pathogens, and most depend on insect vectors for transmission between plants. Viral infection causes various physiological and metabolic changes in host traits, which subsequently influence the behavior and fitness of the insect vectors. Cucumber mosaic virus (CMV), one of the most widespread pathogens in pepper (Capsicum annuum L.), is transmitted by aphid vectors in a non-persistent manner. Here, we examined whether CMV infection in pepper affects the behavior of aphid vectors (Myzus persicae and Aphis glycines) in pepper. Aphid preference test revealed that significantly more aphids were attracted to CMV-infected pepper plants than to healthy plants. Comparative transcriptome analysis revealed a significant activation of the ethylene biosynthesis pathway in CMV-infected pepper plants. Indeed, gas chromatography analysis demonstrated that ethylene emission was significantly increased by CMV infection in pepper plants. Elevated ethylene emission in ethephon-treated healthy pepper increased their attractiveness to aphids. In contrast, aphid preference decreased after chemical inhibition of ethylene biosynthesis in CMV-infected pepper plants. Our results suggest that the ethylene emitted by CMV infection is a volatile cue that regulates the attractiveness of pepper plants to M. persicae and A. glycines.

Viral Strain-Specific Activation of Pathogen-Associated Molecular Pattern-Triggered Immunity Enhances Symptom Severity in Broad Bean Wilt Virus 2 Infection.

Broad bean wilt virus 2 (BBWV2) is an emerging virus in various economically important crops, especially pepper (Capsicum annuum L.), worldwide. Recently, the emergence of various BBWV2 strains that induce severe symptoms has increased damage to pepper crops. While the symptomatic variations among virus strains should be associated with differences in the transcriptomic reprogramming of host plants upon infection, underlying molecular mechanisms and associated genes are largely unknown. In the present study, we employed transcriptome analysis to identify responsible host factors for symptom enhancement in the BBWV2-pepper pathosystem using two distinct BBWV2 strains, PAP1 (a severe strain) and RP1 (a mild strain). Comparative analysis of the differentially expressed genes (DEGs) revealed that various genes associated with pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and ethylene signaling were significantly upregulated upon infection with the severe PAP1 strain, but not with the mild RP1 strain. Indeed, hormone analysis revealed that ethylene emission was significantly increased in pepper plants infected with PAP1. These observations imply that the activation of the PTI-associated defense responses reinforce symptom formation during BBWV2 infection in a virus strain-specific manner.

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.

Genetic plasticity in RNA2 is associated with pathogenic diversification of broad bean wilt virus 2.

Broad bean wilt virus 2 (BBWV2) is an evolutionarily successful RNA virus with an extensive host range and worldwide distribution that causes severe damage to crops. While numerous BBWV2 isolates from various plant species have been identified and their genome sequences determined, little information is available on the virulence and symptomatic characteristics corresponding to the genomic sequences. In this study, we provide integrated information on the molecular and pathogenic characteristics of three genetically distant BBWV2 isolates: BBWV2-PC, -LS2, and P3 obtained from Gentiana scabra, Leonurus sibiricus, and Pisum sativum, respectively. Phylogenetic and diversity analyses of the BBWV2 population included 42 isolates from various host species and revealed that RNA2 has higher genetic plasticity than RNA1 and may have evolved under host-imposed constraints. In addition, we generated an infectious cDNA clone of BBWV2-PC RNA2 (pBBWV2-PC-R2). Pseudo-recombination analysis of pBBWV2-PC-R2 further demonstrated that RNA2 determines the pathogenic characteristics of the PC isolate.

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.