High-quality chromosome-level genome assembly of Nicotiana benthamiana.

Nicotiana benthamiana is a fundamental model organism in plant research. Recent advancements in genomic sequencing have revealed significant intraspecific genetic variations. This study addresses the pressing need for a precise genome sequence specific to its geographic origin by presenting a comprehensive genome assembly of the N. benthamiana LAB strain from the Republic of Korea (NbKLAB). We compare this assembly with the widely used NbLAB360 strain, shedding light on essential genomic differences between them. The outcome is a high-quality, chromosome-level genome assembly comprising 19 chromosomes, spanning 2,762 Mb, with an N50 of 142.6 Mb. Comparative analyses revealed notable variations, including 46,215 protein-coding genes, with an impressive 99.5% BUSCO completeness score. Furthermore, the NbKLAB assembly substantially improved the QV from 33% for NbLAB360 to 49%. This refined chromosomal genome assembly for N. benthamiana, in conjunction with comparative insights, provides a valuable resource for genomics research and molecular biology. This accomplishment forms a strong foundation for in-depth exploration into the intricacies of plant genetics and genomics, improved precision, and a comparative framework.

Two long read-based genome assembly and annotation of polyploidy woody plants, Hibiscus syriacus L. using PacBio and Nanopore platforms.

Improvements in long read DNA sequencing and related techniques facilitated the generation of complex eukaryotic genomes. Despite these advances, the quality of constructed plant reference genomes remains relatively poor due to the large size of genomes, high content of repetitive sequences, and wide variety of ploidy. Here, we developed the de novo sequencing and assembly of high polyploid plant genome, Hibiscus syriacus, a flowering plant species of the Malvaceae family, using the Oxford Nanopore Technologies and Pacific Biosciences Sequel sequencing platforms. We investigated an efficient combination of high-quality and high-molecular-weight DNA isolation procedure and suitable assembler to achieve optimal results using long read sequencing data. We found that abundant ultra-long reads allow for large and complex polyploid plant genome assemblies with great recovery of repetitive sequences and error correction even at relatively low depth Nanopore sequencing data and polishing compared to previous studies. Collectively, our combination provides cost effective methods to improve genome continuity and quality compared to the previously reported reference genome by accessing highly repetitive regions. The application of this combination may enable genetic research and breeding of polyploid crops, thus leading to improvements in crop production.

Complete genome sequence and genome characterization of a novel potyvirus from Lamprocapnos spectabilis.

The genome of a new potyvirus from a Lamprocapnos spectabilis plant in South Korea was sequenced by high-throughput sequencing and confirmed by Sanger sequencing. The new potyvirus was tentatively named "lamprocapnos virus A" (LaVA); its complete genome contains 9,745 nucleotides, excluding the 3'-terminal poly(A) tail. The LaVA genome structure is similar to that of members of the genus Potyvirus and contains an open reading frame encoding a large putative polyprotein of 3,120 amino acids (aa) with conserved motifs. The complete genome shared 48%-56% nucleotide sequence identity and the polyprotein shared 41%-52% aa sequence identity with those of other potyviruses. These values are below the standard thresholds for potyvirus species demarcation. Phylogenetic analysis based on polyprotein sequences showed that LaVA belongs to the genus Potyvirus. To our knowledge, this is the first report of the complete genome sequence and genome characterization of a potyvirus infecting Lamprocapnos spectabilis.

Complete genome sequence of a putative novel alphaendornavirus isolated from Fagopyrum esculentum in South Korea.

The complete genome sequence of a putative new virus isolate, provisionally named "Fagopyrum esculentum endornavirus 2" (FeEV2), is 15,706 nucleotides long with a single, large open reading frame and a typical endornavirus genome organization. FeEV2 shares 19.4%-22.1% nucleotide sequence identity with other known endornavirus genome sequences. The putative polyprotein, RNA-dependent RNA polymerase (RdRp), helicase, and glycosyltransferase (GT) share 10.6%-24.3%, 30.4%-66.1%, 16.3%-45.7%, and 10.1%-21.6% amino acid sequence identity, respectively, with the homologous sequenced proteins from known endornaviruses. This suggests that it is a member of a new, distinct species. Phylogenetic analysis of RdRp sequences places FeEV2 with other Alphaendornavirus genus members (family Endornaviridae). This is the first report of the complete genome sequence of FeEV2, which was isolated from Fagopyrum esculentum in South Korea.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Complete genome sequence and genome organization of scorzonera virus A (SCoVA), a novel member of the genus Potyvirus.

The complete genomic sequence of scorzonera virus A (SCoVA) from a Scorzonera austriaca Willd. plant in South Korea was determined by high-throughput sequencing and confirmed by Sanger sequencing. The SCoVA genome contains 9867 nucleotides, excluding the 3'-terminal poly(A) tail. The SCoVA genome structure is typical of potyviruses and contains a single open reading frame encoding a large putative polyprotein of 3168 amino acids. Pairwise comparison analysis of the complete genome and polyprotein sequences of SCoVA with those of other potyviruses showed that they shared the highest nucleotide and amino acid sequences identity (54.47% and 49.57%, respectively) with those of lettuce mosaic virus (GenBank accession number KJ161185). Phylogenetic analysis of the amino acid sequence of the polyprotein confirmed that SCoVA belongs to the genus Potyvirus. These findings suggest that SCoVA should be considered a novel member of the genus Potyvirus, family Potyviridae.

Genome-Wide Comparative Analysis of Flowering-Time Genes; Insights on the Gene Family Expansion and Evolutionary Perspective.

In polyploids, whole genome duplication (WGD) played a significant role in genome expansion, evolution and diversification. Many gene families are expanded following polyploidization, with the duplicated genes functionally diversified by neofunctionalization or subfunctionalization. These mechanisms may support adaptation and have likely contributed plant survival during evolution. Flowering time is an important trait in plants, which affects critical features, such as crop yields. The flowering-time gene family is one of the largest expanded gene families in plants, with its members playing various roles in plant development. Here, we performed genome-wide identification and comparative analysis of flowering-time genes in three palnt families i.e., Malvaceae, Brassicaceae, and Solanaceae, which indicate these genes were expanded following the event/s of polyploidization. Duplicated genes have been retained during evolution, although genome reorganization occurred in their flanking regions. Further investigation of sequence conservation and similarity network analyses provide evidence for functional diversification of duplicated genes during evolution. These functionally diversified genes play important roles in plant development and provide advantages to plants for adaptation and survival in response to environmental changes encountered during evolution. Collectively, we show that flowering-time genes were expanded following polyploidization and retained as large gene family by providing advantages from functional diversification during evolution.

Complete genome sequence and genome organization of achyranthes virus A, a novel member of the genus Potyvirus.

The complete genomic sequence of achyranthes virus A (AcVA), from an Achyranthes bidentata Blume plant in South Korea, was determined. The genomic RNA has 9491 nucleotides (nt), excluding the 3'-terminal poly(A) tail and contains an open reading frame typical of members of the genus Potyvirus, family Potyviridae, encoding a large putative polyprotein of 3103 amino acids (aa). Pairwise comparisons showed that the AcVA sequence shares 47.81-57.78% nt sequence identity at the complete genome level, 41.89-56.41% aa sequence identity at the polyprotein level, and 50-63.8% aa sequence identity at the coat protein level with other members of genus Potyvirus. These pairwise comparison values are below the species demarcation cutoff for the family Potyviridae. Our results therefore suggest that this virus should be regarded as a novel member of the genus Potyvirus, tentatively named "achyranthes virus A".

Comprehensive analysis of Translationally Controlled Tumor Protein (TCTP) provides insights for lineage-specific evolution and functional divergence.

BACKGROUND: Translationally controlled tumor protein (TCTP) is a conserved, multifunctional protein involved in numerous cellular processes in eukaryotes. Although the functions of TCTP have been investigated sporadically in animals, invertebrates, and plants, few lineage-specific activities of this molecule, have been reported. An exception is in Arabidopsis thaliana, in which TCTP (AtTCTP1) functions in stomatal closuer by regulating microtubule stability. Further, although the development of next-generation sequencing technologies has facilitated the analysis of many eukaryotic genomes in public databases, inter-kingdom comparative analyses using available genome information are comparatively scarce. METHODOLOGY: To carry out inter-kingdom comparative analysis of TCTP, TCTP genes were identified from 377 species. Then phylogenetic analysis, prediction of protein structure, molecular docking simulation and molecular dynamics analysis were performed to investigate the evolution of TCTP genes and their binding proteins. RESULTS: A total of 533 TCTP genes were identified from 377 eukaryotic species, including protozoa, fungi, invertebrates, vertebrates, and plants. Phylogenetic and secondary structure analyses reveal lineage-specific evolution of TCTP, and inter-kingdom comparisons highlight the lineage-specific emergence of, or changes in, secondary structure elements in TCTP proteins from different kingdoms. Furthermore, secondary structure comparisons between TCTP proteins within each kingdom, combined with measurements of the degree of sequence conservation, suggest that TCTP genes have evolved to conserve protein secondary structures in a lineage-specific manner. Additional tertiary structure analysis of TCTP-binding proteins and their interacting partners and docking simulations between these proteins further imply that TCTP gene variation may influence the tertiary structures of TCTP-binding proteins in a lineage-specific manner. CONCLUSIONS: Our analysis suggests that TCTP has undergone lineage-specific evolution and that structural changes in TCTP proteins may correlate with the tertiary structure of TCTP-binding proteins and their binding partners in a lineage-specific manner.

Genomic detection and molecular characterization of two distinct isolates of cycas necrotic stunt virus from Paeonia suffruticosa and Daphne odora.

Complete genome sequences of two cycas necrotic stunt virus (CNSV) isolates from Paeonia suffruticosa and Daphne odora were determined. Phylogenetic trees and pairwise comparisons using complete RNA1- and RNA2-encoded polyproteins showed that the two CNSV isolates are divergent (83.19%-89.42% in polyprotein 1 and 73.61%-85.78% in polyprotein 2). A comparative analysis based on taxonomic criteria for the species demarcation of nepoviruses confirmed that they are not new species but distinct variants. This is the first report of the complete genome sequences of CNSV detected in P. suffruticosa and D. odora, and the first report of CNSV infecting P. suffruticosa.

Complete genome sequence of a tentative new member of the genus Badnavirus identified in Codonopsis lanceolata.

High-throughput sequencing revealed a tentative new badnavirus infecting Codonopsis lanceolata, provisionally named Codonopsis vein clearing virus (CoVCV). The complete 8,112-nt CoVCV genomic DNA sequence (GenBank accession: MK044821) comprises three open reading frames (ORFs) encoding conserved domains, with typical features of badnaviruses. Additionally, BLASTn searches indicated the CoVCV genome sequence is most similar to the grapevine vein clearing virus (GVCV) genome (72% identity and 46% query coverage). Moreover, the polyprotein encoded in CoVCV ORF3 is most similar to the corresponding protein of GVCV, with 60% amino acid sequence identity (89% query coverage). These results suggest that CoVCV is a new member of the genus Badnavirus in the family Caulimoviridae.

Genome analysis of Hibiscus syriacus provides insights of polyploidization and indeterminate flowering in woody plants.

Hibiscus syriacus (L.) (rose of Sharon) is one of the most widespread garden shrubs in the world. We report a draft of the H. syriacus genome comprised of a 1.75 Gb assembly that covers 92% of the genome with only 1.7% (33 Mb) gap sequences. Predicted gene modeling detected 87,603 genes, mostly supported by deep RNA sequencing data. To define gene family distribution among relatives of H. syriacus, orthologous gene sets containing 164,660 genes in 21,472 clusters were identified by OrthoMCL analysis of five plant species, including H. syriacus, Arabidopsis thaliana, Gossypium raimondii, Theobroma cacao and Amborella trichopoda. We inferred their evolutionary relationships based on divergence times among Malvaceae plant genes and found that gene families involved in flowering regulation and disease resistance were more highly divergent and expanded in H. syriacus than in its close relatives, G. raimondii (DD) and T. cacao. Clustered gene families and gene collinearity analysis revealed that two recent rounds of whole-genome duplication were followed by diploidization of the H. syriacus genome after speciation. Copy number variation and phylogenetic divergence indicates that WGDs and subsequent diploidization led to unequal duplication and deletion of flowering-related genes in H. syriacus and may affect its unique floral morphology.

The first complete sequence and genome structure of daphne virus Y.

From Daphne odora Thunb., an ornamental shrub in the Republic of Korea, a potyvirus was identified that has an RNA genome of 9,448 nucleotides (excluding the 3'-terminal poly(A) tail) encoding a polyprotein of 3,065 amino acids, with nine putative protease cleavage sites producing ten proteins. Since this potyvirus shared the highest nucleotide sequence identity (91 %; query coverage 5 %) with the available partial sequence of daphne virus Y (DVY) from New Zealand (EU179854), it was considered a Korean isolate of DVY. This is the first molecular characterization of the complete genome sequence of a DVY isolate.

Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants.

We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR.

Rpi-blb2-Mediated Hypersensitive Cell Death Caused by Phytophthora infestans AVRblb2 Requires SGT1, but not EDS1, NDR1, Salicylic Acid-, Jasmonic Acid-, or Ethylene-Mediated Signaling.

Potato Rpi-blb2 encodes a protein with a coiled-coil-nucleotide binding site and leucine-rich repeat (CC-NBS-LRR) motif that recognizes the Phytophthora infestans AVRblb2 effector and triggers hypersensitive cell death (HCD). To better understand the components required for Rpi-blb2-mediated HCD in plants, we used virus-induced gene silencing to repress candidate genes in Rpi-blb2-transgenic Nicotiana benthamiana plants and assayed the plants for AVRblb2 effector. Rpi-blb2 triggers HCD through NbSGT1-mediated pathways, but not NbEDS1- or NbNDR1-mediated pathways. In addition, the role of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) in Rpi-blb2-mediated HCD were analyzed by monitoring of the responses of NbICS1-, NbCOI1-, or NbEIN2-silenced or Rpi-blb2::NahG-transgenic plants. Rpi-blb2-mediated HCD in response to AVRblb2 was not associated with SA accumulation. Thus, SA affects Rpi-blb2-mediated resistance against P. infestans, but not Rpi-blb2-mediated HCD in response to AVRblb2. Additionally, JA and ET signaling were not required for Rpi-blb2-mediated HCD in N. benthamiana. Taken together, these findings suggest that NbSGT1 is a unique positive regulator of Rpi-blb2-mediated HCD in response to AVRblb2, but EDS1, NDR1, SA, JA, and ET are not required.

Cucumber Pti1-L is a cytoplasmic protein kinase involved in defense responses and salt tolerance.

Homologs of the cytoplasmic protein kinase Pti1 are found in diverse plant species. A clear role of Pti1 in plant defense response has not been established. We identified a Pti1 homolog in cucumber (CsPti1-L). CsPti1-L expression was induced when cucumber plants were challenged with the fungal pathogen Sphaerotheca fuliginea or with salt treatment. CsPti1-L expression in cucumber leaves also was induced by methyl jasmonate, salicylic acid, and abscisic acid. CsPti1-L exhibited autophosphorylation activity and was targeted to the cytoplasm. Transgenic Nicotiana benthamiana expressing CsPti1-L exhibited greater cell death and increased ion leakage in response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, resistance to Botrytis cinerea infection, and higher tolerance to salt stress. RT-PCR analysis of transgenic N. benthamiana overexpressing CsPti1-L revealed constitutive upregulation of multiple genes involved in plant-defense and osmotic-stress responses. Our results suggest a functional role for CsPti1-L as a positive regulator of pathogen-defense and salt-stress responses.

Complete genome sequence of a South Korean isolate of Brugmansia mosaic virus.

We determined the complete genome sequence of a South Korean (SK) isolate of Brugmansia mosaic virus (BruMV), which has recently been proposed to be a member of a new species in the genus Potyvirus. The genomic RNA of BruMV isolate SK is 9781 nucleotides in length (excluding the 3'-terminal poly (A)) and shares complete nucleotide and polyprotein amino acid sequence identities of 85.6 % and 93.1 %, respectively, with the type isolate (D-437) of BruMV described in the USA. To our knowledge, this is the first report providing evidence of considerable sequence variation in BruMV.

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene.

Paclitaxel is produced by various species of yew trees and has been extensively used to treat tumors. In our research, a taxadiene synthase (TS) gene from Taxus brevifolia was used to transform the roots of cultured ginseng (Panax ginseng C.A. Meyer) to produce taxadiene, the unique skeletal precursor to taxol. The TS gene was successfully introduced into the ginseng genome, and the de novo formation of taxadiene was identified by mass spectroscopy profiling. Without any change in phenotypes or growth difference in a TS-transgenic ginseng line, the transgenic TSS3-2 line accumulated 9.1 µg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation was 14.6 and 15.9 µg per g of dry weight, respectively. This is the first report of the production of taxadiene by engineering ginseng roots with a taxadiene synthase gene.

Genome sequence of the plant growth-promoting rhizobacterium Bacillus sp. strain JS.

Volatile and nonvolatile compounds emitted from the plant growth-promoting rhizobacterium Bacillus sp. strain JS enhance the growth of tobacco and lettuce. Here, we report the high-quality genome sequence of this bacterium. Its 4.1-Mb genome reveals a number of genes whose products are possibly involved in promotion of plant growth or antibiosis.

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants.

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 µM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 µM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.