The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

Genetic diversity assessment and genome-wide association study reveal candidate genes associated with component traits in sweet potato (Ipomoea batatas (L.) Lam).

The Korean sweet potatoes were bred by various cultivars introduced from Japanese, American, Porto Rico, China, and Burundi. This issue enriched their genetic diversity but also resulted in a mixture of cultivars. For genotyping, we collected and sequenced 66 sweet potato germplasms from different localities around Korea, including 36 modern cultivars, 5 local cultivars, and 25 foreign cultivars. This identified 447.6 million trimmed reads and 324.8 million mapping reads and provided 39,424 single nucleotide polymorphisms (SNPs) markers. Phylogenetic clustering and population structure analysis distinctly classified these germplasms into 5 genetic groups, group 1, group 2, group 3, group 4, and group 5, containing 20, 15, 10, 7, and 14 accessions, respectively. Sixty-three significant SNPs were selected by genome-wide association for sugar composition-related traits (fructose, glucose, and total sugars), total starch, amylose content, and total carotenoid of the storage root. A total of 37 candidate genes encompassing these significant SNPs were identified, among which, 7 genes were annotated to involve in sugar and starch metabolism, including galactose metabolism (itf04g30630), starch and sucrose metabolism (itf03g13270, itf15g09320), carbohydrate metabolism (itf14g10250), carbohydrate and amino acid metabolism (itf12g19270), and amino sugar and nucleotide sugar metabolism (itf03g21950, itf15g04880). This results indicated that sugar and starch are important characteristics to determine the genetic diversity of sweet potatoes. These findings not only illustrate the importance of component traits to genotyping sweet potatoes but also explain an important reason resulting in genetic diversity of sweet potato.

Genome sequence and characterization of a novel fabavirus infecting Cirsium setidens (gondre) in South Korea.

The complete nucleotide sequence of a novel gondre (Cirsium setidens)-infecting virus, provisionally named "cirsium virus A" (CiVA), was determined by high-throughput and Sanger sequencing, revealing a genome organization typical of fabaviruses. RNA1 and RNA2 are 5,828 and 3,478 nucleotides long, excluding the 3'-terminal poly(A) tails, each containing a single open reading frame. The highest sequence identity values for the CiVA coat protein and proteinase-polymerase, compared with known fabavirus sequences, were 59.09% and 69.68%, respectively, falling below the current thresholds for Fabavirus species demarcation. Our findings support classifying CiVA as a novel putative member of the genus Fabavirus, subfamily Comovirinae, family Secoviridae.

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.

Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds.

BACKGROUND: Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available. RESULTS: We identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters. CONCLUSIONS: This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

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.

Complete genome sequence of Plantago asiatica virus A, a novel putative member of the genus Polerovirus.

Here, we report the complete genome sequence of a novel polerovirus, "Plantago asiatica virus A" (PlaVA), detected in Plantago asiatica using high-throughput RNA sequencing and validated by Sanger sequencing. The complete PlaVA genome contains 5,881 nucleotides and has seven open reading frames (ORF0-5 and ORF3a) encoding putative proteins (P0-5 and P3a, respectively) in an arrangement that is similar to that of typical Polerovirus members. Pairwise sequence comparisons revealed that P0 to P5 encoded by PlaVA had the highest sequence identity (25.48%-79.21%) to the corresponding proteins of previously reported poleroviruses. A phylogenetic analysis using the PlaVA P1-2 and P3 amino acid sequences and those of members of the family Solemoviridae (formerly Luteoviridae) indicated that although PlaVA belongs to the genus Polerovirus, it does not represent a known species. Consequently, PlaVA should be considered a member of a new species within the genus Polerovirus.

Identification and molecular characterization of a novel kudzu-infecting virus of the family Betaflexiviridae.

A new virus, tentatively named "kudzu virus D" (KuVD) was discovered in kudzu (Pueraria montana var. lobata) in South Korea. Its complete genome comprises 7,922 nucleotides, excluding the poly(A) tail, and contains five open reading frames (ORFs) encoding, from 5' to 3', a replicase (ORF1), three triple gene block proteins TGB1-3 (ORF2-ORF4), and a coat protein (ORF5). This genome organization is typical of members of the subfamily Quinvirinae of the family Betaflexiviridae. Pairwise alignment analysis revealed that the nucleotide sequences of the replicase and coat protein of KuVD were 12.13-54.46% and 24.03-50.67% identical, respectively, to those of other members of the family Betaflexiviridae. These values are far below the current species ICTV demarcation threshold. Consequently, KuVD should be considered a member of a new species in the subfamily Quinvirinae.

Complete nucleotide sequence of hemisteptia virus A, a polero-like virus.

The complete genomic nucleotide sequence of hemisteptia virus A (HemVA) from a Hemisteptia lyrata Bunge plant in South Korea was identified by high-throughput sequencing. The HemVA genome consists of 6,122 nucleotides and contains seven putative open reading frames, ORF0-5 and ORF3a, encoding the putative proteins P0-P5 and P3a, respectively. Pairwise amino acid sequence analysis shows that the HemVA P1-P5 proteins have the highest sequence identity (23.68%-54.15%) to the corresponding proteins of members of the families Solemoviridae and Tombusviridae. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences indicated that HemVA should be classified as a member of a distinct species in the genus Polerovirus.

First Report of Cotton Leafroll Dwarf Virus Infecting Hibiscus syriacus in South Korea.

Three cotton leafroll dwarf virus (CLRDV; genus Polerovirus, family Solemoviridae) genotypes have recently been identified (Tabassum et al., 2021; Ramos-Sobrinho et al., 2021). This virus is widespread in the United States (Thiessen et al., 2020; Aboughanem-Sabanadzovic et al., 2019; Tabassum et al., 2020) and has also been reported to infect chickpea (Cicer arietinum) in Uzbekistan (Kumari et al., 2020). As well, CLRDV was detected from 23 weed species (16 families), including Hibiscus sabdariffa (Sedhain et al., 2021, Hagan et al., 2019). From June to September 2019, virus-like symptoms, including mild leaf stunting, crinkling, and deformation, were observed in multiple plants (n=14) in several provinces of South Korea (e-Xtra Table. 1). To characterize the associated viruses, pooled leaf tissues from all 14 samples were used for total RNA isolation, followed by paired-end high-throughput sequencing (HTS) on the Illumina NovaSeq 6000 platform (Macrogen, South Korea). A total of 614,424,952 trimmed and high-quality reads were assembled into 506,024 contigs using Trinity de novo transcriptome assembly. The resulting contigs were compared with viral sequences in the GenBank database using BLASTx analysis. Several viral contigs were identified, including cucumber mosaic virus, apple stem pitting virus, apple stem grooving virus, cherry virus A, and CLRDV. The CLRDV contig of 5,800 nucleotides (nt) with an average coverage of 307x shared 92.1% identity (query coverage: 99%) with the CLRDV isolate CN-S5 (KX588248). To confirm CLRDV infection and to obtain its complete genome sequence, total RNA was extracted from each of the 14 samples and used for reverse transcription (RT)-PCR with six overlapping sets of primers designed from the HTS contig (e-Xtra Table. 2). The expected product sizes were obtained only for the Hibiscus syriacus L. (family: Malvaceae) sample showing foliar mild vein clearing symptoms on the leaves (e-Xtra Fig.1). All RT-PCR products were cloned using the RBC TA Cloning vector (Taipei, Taiwan) and at least five positive clones per cloned DNA fragment were sequenced. The 5 and 3 termini sequences were determined as described previously (Zhao et al. 2016). The complete genome of CLRDV isolate SK (OK073299) was determined to be 5,862 nt and it shared 89-91% complete genome identity with 12 other CLRDV isolates based on pairwise comparisons (e-Xtra Table. 3). Maximum likelihood phylogenetic analysis based on the complete genome and P3-CP aa sequences showed that CLRDV-SK is more closely related CN-S5 (e-Xtra Fig. 2). In the fall of 2021, additional H. syriacus samples (n=18) with mild chlorosis, blistering and crinkling symptoms were collected from 2 provinces of South Korea and tested by RT-PCR using the primers: CLRDV-SK-101-120 For & CLRDV-SK-1021-1040 Rev targeting a region of the ORF0. Two of 18 samples (11.1%) tested positive for CLRDV. The 16 negative samples only showed symptoms of mild yellowing. The RT-PCR products were cloned and sequenced. In pairwise comparisons, the obtained sequences (OM339522-23) were 95.85% and 96.06% identical to the corresponding sequences of CLRDV isolate SK. This is the first report of CLRDV occurrence in H. syriacus in South Korea to the best of our knowledge. Our findings will assist further studies on the epidemiology and sustainable management of diseases caused by CLRDV. Acknowledgments This work was supported by IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries; Project No. AGC1762111), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. References Tabassum, A., et al., 2021. PloS One. 16: e0252523 Ramos-Sobrinho, R., et al., 2021. Viruses. 13:2230 Thiessen, L.D., et al. 2020. Plant Dis. 104:3275 Aboughanem-Sabanadzovic, N., et al. 2019. Plant Dis. 103:1798 Tabassum, A., et al. 2020. Microbiol. Res. Announce. 9:e00812-20 Kumari, S.G., et al. 2021. Plant Dis. 104:2532 Sedhain, N.P., et al. 2021. Crop protection 144:105604 Hagan, A., er al. 2019. Alabama Cooperative Extension System. ANR:2539 Zhao, F., et al. 2016. Arch. Virol. 161:2047 Conflict of interest The authors declare that they have no conflict of interest.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

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.

Complete genome sequence of platycodon closterovirus 1, a novel putative member of the genus Closterovirus.

A new member of the genus Closterovirus was detected in Platycodon grandiflorus using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named "platycodon closterovirus 1" (PlaCV1), comprises 16,771 nucleotides with nine predicted open reading frames (ORFs) having the typical closterovirus genome organization. PlaCV1 shares 37%-50% nucleotide sequence identity with other known closterovirus genome sequences. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), viral heat shock protein 90-like protein (HSP90h), minor coat protein (CPm), and coat protein (CP) show 47-68%, 39-66%, 24-52%, 21-57%, and 16-35% amino acid sequence identity, respectively, to homologous proteins in previously identified closteroviruses, suggesting that it represents a distinct, new species in the genus. Phylogenetic analysis of HSP70h sequences places PlaCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of PlaCV1 infecting P. grandiflorus in the Republic of Korea.

Complete genome sequence of artemisia virus B, a new polerovirus infecting Artemisia princeps in South Korea.

The complete genome sequence of a new polerovirus found naturally infecting Artemisia princeps, artemisia virus B (ArtVB), was determined using high-throughput sequencing. The ArtVB genome comprises 6,141 nucleotides and contains six putative open reading frames (ORF0 to ORF5) with a genome structure typical of poleroviruses. A multiple sequence alignment showed that the complete ArtVB genome shares 50.98% nucleotide sequence identity with ixeridium yellow mottle virus 1 (IxYMaV-1, GenBank accession no. KT868949). ArtVB shares the highest amino acid sequence identity in P0 and P3-P5 (21.54%-51.69%) with other known poleroviruses. Phylogenetic analysis indicated that ArtVB should be considered a member of a new species within the genus Polerovirus, family Luteoviridae.

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".

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.

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.

Genome-wide Analysis of Alternative Splicing in An Inbred Cabbage (Brassica oleracea L.) Line 'HO' in Response to Heat Stress.

INTRODUCTION: High-throughput RNA sequencing (RNA-Seq) studies demonstrate that Alter-native Splicing (AS) is a widespread mechanism that enhances transcriptome diversity, particularly in plants exposed to environmental stress. In an attempt to determine the transcriptome and AS patterns of cabbage inbred line "HO" under Heat Stress (HS), RNA-Seq was carried out using HS-treated and con-trol samples. Genome-wide analysis indicated that AS is differentially regulated in response to HS. The number of AS events markedly increased in HS-treated samples compared to the control. CONCLUSION: We identified 1,864 genes, including Heat shock transcription factor (Hsf) and heat shock protein (Hsp) genes, that exhibited >4-fold changes in expression upon exposure to HS. The enriched Gene Ontology (GO) terms of the 1,864 genes included 'response to stress/abiotic stimulus/chemical stimulus', among, which the genes most highly induced by HS encode small Hsps and Hsf proteins. The heat-induced genes also showed an increased number of AS events under HS conditions. In addi-tion, the distribution of AS types was altered under HS conditions, as the level of Intron Retention (IR) decreased, whereas other types of AS increased, under these conditions. Severe HS-induced AS was al-so observed in Hsfs and Hsps, which play crucial roles in regulating heat tolerance. Our results support the notion that AS of HS-related genes, such as HsfA2 and HsfB2a, are important for heat stress adapta-tion in cabbage.

Silencing of an α-dioxygenase gene, Ca-DOX, retards growth and suppresses basal disease resistance responses in Capsicum annum.

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the ß-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.