Rapid Amplification Of cDNA Ends Research Articles

First report of pepper chlorosis-associated virus infecting tobacco (Nicotiana tabacum) plants in Sichuan Province in China.

Tobacco (Nicotiana tabacum) is an economically important crop in China, and more than 30 viruses have been reported to infect tobacco (Yin et al. 2022). In July 2022, we observed interveinal necrosis on tobacco leaves in fields in Sichuan Province (N 27.9172, E 105.6662) (Fig. 1). Total RNA was isolated from multiple leaves of one plant using an RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). Total RNAs were pooled, and a TruSeq Stranded Total RNA with RiboZero Gold Kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA. An RNA-Seq library was constructed using VAHTS Universal V6 RNA-seq Library Prep (Nanjing Vazyme, China). High-throughput sequencing was performed on the Illumina DNBseq platform (BGI-ShenZhen, China), which yielded 20,102,087 reads with an average length of 150 nt (total size >6 Gb). Unaligned reads were assembled de novo using SPAdes (Bankevich et al. 2012). Contigs with length ≥200 nt were subjected to local BLASTn and BLASTx analyses against the GenBank nt and nr databases, respectively (Wang et al. 2022). A total of 23 contigs were identified through BLASTx (e-value cut-off = 10 -3), ranging from 631 to 1555 bp long, with 82% to 96% coverage to partial genomic sequences of pepper chlorosis-associated virus (PepCaV-Higashitsuno_2021; Accessions: LC719619 to LC719621) and one contig (6459 bp) with 99% similarity to tobacco mosaic virus (Accession: OP525281) isolate DSMZ PV-0109 from Germany. The complete genome sequence of PepCaV was obtained using primers based on the assembled contigs. The 5'- and 3'-terminal regions of the RNA genome were obtained by 5'- and 3'-rapid amplification of cDNA ends. These amplicons were cloned using the pEASY-Blunt Zero Cloning Kit (TRANSGEN, Nangjing, China) and sequenced by Sanger sequencing. Complete genome sequences of tripartite PepCaV from tobacco samples were 7697, 1808, and 1557 nucleotides long (Accession: OR451987 to OR451989) and showed genome organization typical of the genus Ophiovirus in the family Aspiviridae. The complete sequences of RNA1, RNA2 and RNA3 genome segments shared 92.36%, 90.43%, and 95.24%, nucleotide sequence identities, respectively, with the isolate PepCaV-Higashitsuno_2021 pepper isolate (Accession: LC719619 to LC719621) (Shimomoto et al. 2023), but PepCaV has not been reported to infect N. tabacum. In June 2023, 10 plants collected from each place of Macheng (N 27.9094, E 105.6740), Xiangyang (N 28.0936, E 105.6249) and Moni (N 27.8899, E 105.5936) showing interveinal necrosis symptoms were tested using RT-PCR using PepCaV-MP610-F (5'-TGTTCTCTGCTATGCGGTTG -3') and PepCaV-MP610-R (5'-AGCAATCTCGCACCTGAAGT-3') to product 610bp amplicon. Twenty-five tobacco plants were positive for PepCaV. Single sequence from each location was submitted to GenBank (Accession: PP728631 to PP728633). Sap extracts from the original field leaf samples collected from Sichuan Province were used to mechanically inoculate tobacco plants (10 plants) at the four-leaf stage. After 7 days, leaf samples were tested using RT-PCR assay specific to PepCaV and TMV while samples were positive only for TMV but failed to transmit PepCaV by mechanical inoculation. According to previous literature, ophioviruses may be transmitted though soilborne fungus (Jeong et al. 2014). Further research is needed to understand the transmission, epidemiology, and pathological properties of the PepCaV. To our knowledge, this is the first report documenting natural PepCaV infection of tobacco plants in China, providing a scientific basis for PepCaV infection control in tobacco plantations.

The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa

Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5′-rapid amplification of cDNA ends (5′-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa.

Circumvention of Topoisomerase IIα Intron 19 Intronic Polyadenylation (IPA) in Acquired Etoposide Resistant Human Leukemia K562 Cells.

DNA topoisomerase IIα (TOP2α, 170kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is a significant target for DNA damage stabilizing anti-cancer agents such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90) which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-Rapid Amplification of cDNA Ends (3'-RACE), we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was utilized. We hypothesized that resultant intronic polyadenylation (IPA) can would be attenuated by blocking or mutating the I19 PAS thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide (AMO) was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/Cas9 homology-directed repair (HDR) was used to mutate the cryptic I19 PAS (AATAAA-->ACCCAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs. Significance Statement Results presented here indicate that CRISPR/Cas9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide (AMO) targeting the PAS also partially circumvented resistance. Results demonstrate the importance of intronic polyadenylation (IPA) in acquired drug resistance and points to tractable strategies to overcome this form of resistance to TOP2-targeted agents.

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Characterisation of a Novel Insect-Specific Virus Discovered in Rice Thrips, Haplothrips aculeatus

Insects constitute the largest proportion of animals on Earth and act as significant reservoirs and vectors in disease transmission. Rice thrips (Haplothrips aculeatus, family Phlaeothripidae) are one of the most common pests in agriculture. In this study, the full genome sequence of a novel Ollusvirus, provisionally named “Rice thrips ollusvirus 1” (RTOV1), was elucidated using transcriptome sequencing and the rapid amplification of cDNA ends (RACE). A homology search and phylogenetic tree analysis revealed that the newly identified virus is a member of the family Aliusviridae (order Jingchuvirales). The genome of RTOV1 contains four predicted open reading frames (ORFs), including a polymerase protein (L, 7590 nt), a glycoprotein (G, 4206 nt), a nucleocapsid protein (N, 2415 nt) and a small protein of unknown function (291 nt). All of the ORFs are encoded by the complementary genome, suggesting that the virus is a negative-stranded RNA virus. Phylogenetic analysis using polymerase sequences suggested that RTOV1 was closely related to ollusvirus 1. Deep small RNA sequencing analysis reveals a significant accumulation of small RNAs derived from RTOV1, indicating that the virus replicated in the insect. According to our understanding, this is the first report of an Ollusvirus identified in a member of the insect family Phlaeothripidae. The characterisation and discovery of RTOV1 is a significant contribution to the understanding of Ollusvirus diversity in insects.

Differential Analysis of Immunoglobulin Gene Expression Pattern in Chickens of Distinct Breeds and Developmental Periods.

Immunoglobulin is an essential component of the body's defense against pathogens, aiding in the recognition and clearance of foreign antigens. Research concerning immunoglobulin gene and its diversity of expression across different breeds within the same species is relatively scarce. In this study, we employed RACE (Rapid Amplification of cDNA Ends) technology, prepared DNA libraries, performed high-throughput sequencing, and conducted related bioinformatics analysis to analyze the differences in immunoglobulin gene diversity and expression at different periods in Hy-line brown hens, Lueyang black-bone chickens, and Beijing-You chickens. The study found that the composition of chicken immunoglobulin genes is relatively simple, with both the light chain and heavy chain having a functional V gene. Additionally, the mechanisms of immunoglobulin diversity generation tended to be consistent among different breeds and periods of chickens, primarily relying on abundant junctional diversity, somatic hypermutation (SHM), and gene conversion (GCV) to compensate for the limitations of low-level V(D)J recombination. As the age increased, the junctional diversity of IgH and IgL tended to diversify and showed similar expression patterns among different breeds. In the three chicken breeds, the predominant types of mutations observed in IGHV and IGLV SHM were A to G and G to A transitions. Specifically, IGLV exhibited a preference for A to G mutations, whereas IGHV displayed a bias towards G to A mutations. The regions at the junctions between framework regions (FR) and complementarity-determining regions (CDR) and within the CDR regions themselves are typically prone to mutations. The locations of GCV events in IGLV and IGHV do not show significant differences, and replacement segments are concentrated in the central regions of FR1, CDR, and FR2. Importantly, gene conversion events are not random occurrences. Additionally, our investigation revealed that CDRH3 in chickens of diverse breeds and periods the potential for diversification through the incorporation of cysteine.This study demonstrates that the diversity of immunoglobulin expression tends to converge among Hy-line brown hens, Lueyang black-bone chickens, and Beijing-You chickens, indicating that the immunoglobulin gene expression mechanisms in different breeds of chickens do not exhibit significant differences due to selective breeding.

Deep mining of the Sequence Read Archive reveals major genetic innovations in coronaviruses and other nidoviruses of aquatic vertebrates.

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.

Characterization of Kiss/Kissr system and expression profiling through developmental stages indicate kiss1 to be the active isotype in Clarias magur.

Kisspeptin (Kiss) and kisspeptin receptor (Kissr) system is a key regulator of GnRH expression in several vertebrates. The Indian catfish, Clarias magur, is popular in the Indian sub-continent, and a neo-type of the Asian catfish, C. batrachus. Catfish breeding is constrained as males do not release milt captivity with/without stimulation. Magur Kiss/Kissr system comprising of kiss1, kiss2, kissr1, and kissr2 genes was characterized for the first time. Full-length mRNA was sequenced using RACE PCR. Neighbor-joining tree of predicted proteins shows one clade of teleost orthologs. Magur whole genome (NCBI GenBank) has single copies of each gene, though yet unannotated/misannotated. Anomalies in the nomenclature of earlier sequences in GenBank were noted. Relative gene expression was profiled during various ontogenic stages, in six tissues including brain and gonads at maturity, and also in brains and gonads of premature and spent fish. Expression of gnrh1, gnrhr1, and gnrhr2 was estimated concomitantly. The kiss1 was the first to be twofold upregulated (P < 0.05) at 12h post fertilization. Kiss/Kissr genes expressed primarily in the brain, ovary, and testis. Though kiss2 was 10 times higher than kiss1, only kiss1 showed significant modulation across stages and appears to be the active isotype that regulates GnRH in magur.

Effects of Fat and Carnitine on the Expression of Carnitine Acetyltransferase and Enoyl-CoA Hydratase Short-Chain 1 in the Liver of Juvenile GIFT (Oreochromis niloticus).

Carnitine acetyltransferase (CAT) and Enoyl-CoA hydratase short-chain 1 (ECHS1) are considered key enzymes that regulate the β-oxidation of fatty acids. However, very few studies have investigated their full length and expression in genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China. Here, we cloned CAT and ECHS1 full-length cDNA via the rapid amplification of cDNA ends, and the expressions of CAT and ECHS1 in the liver of juvenile GIFT were detected in different fat and carnitine diets, as were the changes in the lipometabolic enzymes and serum biochemical indexes of juvenile GIFT in diets with different fat and carnitine levels. CAT cDNA possesses an open reading frame (ORF) of 2167 bp and encodes 461 amino acids, and the ECHS1 cDNA sequence is 1354 bp in full length, the ORF of which encodes a peptide of 391 amino acids. We found that juvenile GIFT had higher lipometabolic enzyme activity and lower blood CHOL, TG, HDL-C, and LDL-C contents when the dietary fat level was 2% or 6% and when the carnitine level was 500 mg/kg. We also found that the expression of ECHS1 and CAT genes in the liver of juvenile GIFT can be promoted by a 500 mg/kg carnitine level and 6% fat level feeding. These results suggested that CAT and ECHS1 may participate in regulating lipid metabolism, and when 2% or 6% fat and 500 mg/kg carnitine are added to the feed, it is the most beneficial to the liver and lipid metabolism of juvenile GIFT. Our results may provide a theoretical basis for GIFT feeding and treating fatty liver disease.

Complete genome analysis of a novel narnavirus in sweet viburnum (Viburnum odoratissimum).

Trees and shrubs provide important ecological services. However, few studies have surveyed the virome in trees and shrubs. In this study, we discovered a new positive-sense RNA virus originating from Viburnum odoratissimum, which we named "Vo narna-like virus". The complete genome of Vo narna-like virus is 3,451 nt in length with an open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRP) protein. Phylogenetic analysis placed this virus within the betanarnavirus clade, sharing 53.63% amino acid sequence identity with its closest relative, Qingdao RNA virus 2. The complete sequence of the virus was confirmed by rapid amplification of cDNA ends (RACE) and Sanger sequencing. Small interfering RNA (siRNA) analysis indicated that this virus interacts with the RNA interference (RNAi) pathway of V. odoratissimum. This isthe first report of a narnavirus in V. odoratissimum.

Molecular Characterization, Expression and In Situ Hybridization Analysis of a Pedal Peptide/Orcokinin-type Neuropeptide in Cuttlefish Sepiella japonica.

Neuropeptide pedal peptide (PP) and orcokinin (OK), which are structurally related active peptides, have been widely discovered in invertebrates and constitute the PP/OK neuropeptide family. They have complex structures and play myriad roles in physiological processes. To date, there have been no related reports of PP/OK-type neuropeptide in cephalopods, which possess a highly differentiated multi-lobular brain. Rapid Amplification of cDNA Ends (RACE) was employed to obtain the open reading frame (ORF) of PP/OK-type neuropeptide in Sepiella japonica (termed as Sj-PP/OK). Various software were used for sequence analysis. Semi-quantitative PCR was applied to analyze the tissue distribution profile, quantitative real-time PCR (qRT-PCR) was used to study spatio-temporal expression throughout the entire growth and development period, and in situ hybridization (ISH) was employed to observe the tissue location of Sj-PP/OK. in the present study, we identified the ORF of Sj-PP/OK. The putative precursor of Sj-PP/ OK encodes 22 mature peptides, of which only tridecapeptides could undergo post-translationally amidated at C-terminus. Each of these tridecapeptides possesses the most conserved and frequent N-terminus Asp-Ser-Ile (DSI). Sequence analysis revealed that Sj-PP/OK shared comparatively low identity with other invertebrates PP or OK. The tissue distribution profile showed differences in the expression level of Sj-PP/OK between male and female. qRT-PCR data demonstrated that Sj-PP/OK was widely distributed in various tissues, with its expression level increasing continuously in the brain, optic lobe, liver, and nidamental gland throughout the entire growth and development stages until gonad maturation. ISH detected that Sj-PP/OK positive signals existed in almost all regions of the optic lobe except the plexiform zone, the outer edge of all functional lobes in the brain, epithelial cells and the outer membrane layer of the accessory nidamental gland. These findings suggest that Sj-PP/OK might play a role in the regulation of reproduction, such as vitellogenin synthesis, restoration, and ova encapsulation. The study indicated that Sj-PP/OK may be involved in the neuroendocrine regulation in cephalopods, providing primary theoretical basis for further studies of its regulation role in reproduction.

Hepatocyte growth factor (HGF) gene: molecular characterisation of complete coding sequence and expression profile in Tarim red deer (Cervus hanglu yarkandensis) antlers

Context The cDNA sequence of hepatocyte growth factor (HGF) gene in Tarim red deer has not been reported yet. Aims This study aims to obtain the full-length cDNA sequence of HGF and analyse its expression in different parts of developing antler tissues. The result provides foundational data for understanding the potential role of HGF in regulating antler growth and development. Methods Rapid amplification of cDNA ends was used to obtain the full-length cDNA sequence of Tarim red deer HGF. The pET28a (+) vector was constructed for prokaryotic expression of the recombinant protein in E. coli BL21 (DE3). The expression of HGF in different antler tissues was detected using real-time quantitative polymerase chain reaction and immunohistochemistry. Key results The full-length cDNA of Tarim red deer HGF was found to consist of a 156 bp 5’untranslated region (UTR), a 112 bp 3’UTR, and a 2193 bp open reading frame encoding a protein of 730 amino acids. The recombinant HGF protein expressed in prokaryotic cells formed inclusion bodies. HGF and its receptor c-Met were expressed in all four different antler tissues, with the highest expression level in velvet skin, followed by bone, cartilage, and the lowest in the mesenchyme. Conclusions The study successfully obtained the full-length cDNA sequence of Tarim red deer HGF and identified the expression profile of HGF and c-Met in different antler tissues. HGF is a candidate gene that may play a role in regulating the growth and development of deer antler. Implications These findings provide fundamental data for further investigations into the role of HGF in antler development. Understanding the function of HGF in antler development could have implications for elucidating the mechanism of antler regeneration.

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Identification and Genome Characterization of a Novel Virus within the Genus Totivirus from Chinese Bayberry (Myrica rubra).

Chinese bayberry (Myrica rubra) is an economically significant fruit tree native to eastern Asia and widely planted in south-central China. However, studies about the viruses infecting M. rubra remain largely lacking. In the present study, we employed the metatranscriptomic method to identify viruses in M. rubra leaves exhibiting yellowing and irregular margin symptoms collected in Fuzhou, a city located in China's Fujian province in the year 2022. As a consequence, a novel member of the genus Totivirus was identified and tentatively named "Myrica rubra associated totivirus 1" (MRaTV1). The genome sequencing of MRaTV1 was determined by overlapping reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The two deduced proteins encoded by MRaTV1 have the highest amino acid (aa) sequence identity to the coat protein (CP) and RNA-dependent RNA polymerase (RdRP) of Panax notoginseng virus A (PNVA), a member of the genus Totivirus within the family Totiviridae, at 49.7% and 61.7%, respectively. According to the results of the phylogenetic tree and the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV) for the genus Totivirus, MRaTV1 is considered a new member of the genus Totivirus.

Effect of Guanylate Cyclase-22-like on Ovarian Development of Orius nagaii (Hemiptera: Anthocoridae).

This study identified and characterized the gene encoding recep tor-type guanylate cyclase-22-like (GCY-22; OnGCY) from the pirate bug Orius nagaii, an important biological control agent. The full-length cDNA of the GCY of O. nagaii was obtained by rapid amplification of cDNA ends (RACE); it had a total length of 4888 base pairs (bp), of which the open reading frame (ORF) was 3750 bp, encoding a polypeptide of 1249 amino acid residues. The physicochemical properties of OnGCY were predicted and analyzed by using relevant ExPASy software, revealing a molecular formula of C6502H10122N1698O1869S57, molecular weight of ~143,811.57 kDa, isoelectric point of 6.55, and fat index of 90.04. The resulting protein was also shown to have a signal peptide, two transmembrane regions, and a conserved tyrosine kinase (tyrkc). Silencing OnGCY by RNA interference significantly inhibited ovarian development and decreased fertility in female O. nagaii in the treated versus the control group. Additionally, OnGCY silencing significantly decreased the expression levels of other GCY and Vg genes. Thus, these results clarify the structure and biological function of OnGCY, which has an important role in insect fecundity. The results also provide a reference for agricultural pest control and future large-scale breeding of biological control agents.

First report of Kalanchoe Latent Virus naturally infecting common bean (Phaseolus vulgaris L.) in South Korea.

The common bean (Phaseolus vulgaris; family: Fabaceae) is an economically and nutritionally important food crop worldwide (Ganesan et al. 2017). In 2021, several plants collected from different provinces in South Korea had symptoms of viral infections (e.g., mild yellow-greenish speckling, stunting, crinkling, and deformed leaves). To identify the causal pathogens, total RNA was isolated from pooled leaf tissues from all samples (n = 29) for paired-end high-throughput sequencing (HTS). The cDNA library was constructed after eliminating ribosomal RNA using the TruSeq RNA Sample Prep Kit and then sequenced using the Illumina NovaSeq 6000 platform (Macrogen, Korea). The 297,868,156 paired-end clean reads (150 nt) were de novo assembled using Trinity with default parameters. BLASTx was used for the contig analysis, which revealed the pooled samples were infected with several plant viruses (e.g., turnip mosaic virus, zucchini yellow mosaic virus, cucumber mosaic virus, lily mottle virus). Notably, the assembled contigs included a single viral contig (8,472 nt) comprising the nearly complete KLV genome (HTS mean coverage: 39.46%). Kalanchoe latent virus (KLV; genus: Carlavirus; family: Betaflexiviridae) has been detected in Kalanchoë blossfeldiana (Hearon 1982), Chenopodium quinoa (Dinesen et al. 2009), and Graptopetalum paraguayense (Sorrentino et al. 2017). The sequence was most similar (96.28% nucleotide identity; 99% query coverage) to KLV isolate DSMZ PV-0290 (GenBank: OP525283) from Denmark. The contig sequence was validated via reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from the 29 individually stored samples and nine primer sets specific for the KLV contig. All nine contig-specific overlapping fragments were amplified from only a P. vulgaris plant with mild yellowing mosaic symptoms collected on July 6, 2021, in Jeongseon County, South Korea. Additionally, 5' and 3' rapid amplification of cDNA ends (RACE)-specific primers were designed for the KLV contig sequence to determine the terminal ends of the genome of the South Korean KLV isolate using the 5'/3' RACE System (Invitrogen, Carlsbad, CA, USA). All of the amplified and overlapping fragments were cloned into the RBC T&A Cloning Vector (RBC Bioscience, Taipei, Taiwan) and sequenced using the Sanger method. The obtained full-length genomic sequence of the KLV isolate (KLV-SK22) was 8,517 nt long and was deposited in GenBank OQ718816. According to the BLASTn analysis, KLV-SK22 was highly similar (96.30% sequence identity; 100% query coverage) to the DSMZ PV-0290 isolate. Phylogenetic trees constructed on the basis of coat protein and RNA-dependent RNA polymerase amino acid sequences revealed that KLV-SK22 is closely related to the DSMZ PV-0290 and PV-0290B isolates from Denmark, respectively. At the genome and gene levels, the individual sequence identities between the carlaviruses and other KLV isolates were 96.29% to 100% (Adams et al. 2004). Additionally, an RT-PCR analysis using detection primers specific for KLV-SK22 did not detect KLV in 15 samples (P. vulgaris = 3, Glycine max = 8, Pueraria montana = 2, Trifolium repens = 1, and Vigna angularis = 1) randomly collected from different regions in South Korea. Based on these results, KLV infection may not be widespread at this time in South Korea. To the best of our knowledge, this is the first report of KLV in P. vulgaris in South Korea or elsewhere. Our findings will aid future research on the epidemiology and long-term management of KLV-related diseases.

First Report of Clover yellow vein virus Infecting Senna septemtrionalis (Arsenic bush).

Clover yellow vein virus (ClYVV) is a member of the genus Potyvirus, family Potyviridae and was reported to infect many plant species, such as Ammi majus L., Phaseolus vulgaris L., Vicia faba L., Lens culinaris L., Borago officinalis L., Cicer arietinum L., Gladiolous gandavensis L., Glycine max L., Trifolium repens L., and Dendrobium sp. (Irey et al. 2006; Ortiz et al. 2009; Park et al. 2014; Yoon et al. 2022). Senna septemtrionalis (Viv.) H.S.Irwin & Barneby (arsenic bush), a species in the subfamily Caesalpinioideae, is widely distributed in tropical and subtropical regions (Datiles et al. 2022). In June 2021, virus-like symptoms of mosaic, chlorosis, and leaf-curling were observed in arsenic bush in Kunming, Yunnan province, China. Symptomatic leaves were collected from four arsenic bush (SS1-4), and asymptomatic leaves were collected from 3 additional arsenic bush plants (SS5-7) (eXtra S1). To identify the putative causal virus, sap of symptomatic leaves (SS1) was stained with 1 % phosphotungstic acid and observed under a transmission electron microscope (TEM). Potyvirus-like particles (about 750-800 nm  13 nm) were observed from the sample (eXtra S1). Total RNA was extracted from sample SS1 using TRIzol Reagent (Invitrogen, USA) and subjected to the Illumina NovaSeq platform for RNA-Seq. After trimming and quality control of raw data, 24,125,963 high-quality clean reads were assembled into 72,835 Unigenes using Trinity software. BLAST searches indicated that the nucleotide sequence of Unigene c29731 (10,893 nt) and its deduced amino acid sequence shared 82.62% to 96.45% and 92.60% to 99.19% identity with several ClYVV isolates, respectively. Unigene c29731 had the highest coverage ratio (88%) and the highest nucleotide sequence identity (96.45%) with ClYVV isolate IA-2016 (GenBank accession No. MK292120.1). The complete genome of ClYVV SS1 isolate (ClYVV-SS, GenBank accession No. OP868578) was determined using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) (Chen et al. 2001). A total of 224,936 out of 24,125,963 reads were mapped to the ClYVV-SS1 genome, yielding an average depth of coverage of 3,056.823 (min=1, max=7,859) at nucleotides from 1 to 9,324 of ClYVV genome (eXtra S2). BLASTN results indicated that the complete genome of ClYVV-SS1 shared 96.45% nucleotide sequence identity and 99% coverage ratio with ClYVV isolate IA-2016 genome. Phylogenetic analysis showed that ClYVV-SS1 and other ClYVV isolates clustered together (eXtra S2). RT-PCR was performed on samples (SS2-7) using a pair of primers of the coat protein gene (5'- TCCGACAAAGATAAGTTGAATGCTGGTG-3' and 5'-GAATCGTGCTCCAGCAATGTGA-3') designed from multiple sequences alignment (MSA). Using SS1 sample as positive control, amplicons of ~813 bp were obtained from three symptomatic samples (SS2-4) but not the asymptomatic ones (SS5-7). A total of 17 of 20 arsenic bushes developed symptoms of mosaic and leaf-curling approximately two weeks after mechanical inoculation with arsenic bush (SS1) sap, with 10 uninoculated plants used as control (eXtra S1). RT-PCR was performed for all tested plants. 17 symptomatic arsenic bushes tested positive for ClYVV, while all other samples tested negative. This confirmed that the symptomatic arsenic bushes were infected with ClYVV. To our knowledge, this is the first report of ClYVV infecting arsenic bush.

Complete genome sequence of polygonatum kingianum mottle virus infecting Polygonatum kingianum Coll. et Hemsl in Yunnan, China.

The complete genome sequence of a putative novel potyvirus, tentatively named "polygonatum kingianum mottle virus" (PKgMV; GenBank accession no. ON428226), infecting Polygonatum kingianum in China, was obtained by next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE). PKgMV exhibits the typical genome organization and characteristics of members of the genus Potyvirus, with a length of 10,002 nucleotides (nt) and a large open reading frame (nt 108 to 9,746) encoding a polyprotein of 3,212 amino acids (aa) (363.68 kDa). Pairwise comparisons revealed that the PKgMV polyprotein shares 50.5-68.6% nt and 43.1-72.2% aa sequence identity with reported members of the genus Potyvirus. Moreover, phylogenetic analysis indicated that PKgMV is closely related to polygonatum kingianum virus 1 (PKgV1; accession no. MK427056). These results suggest that the PKgMV is a novel member of the genus Potyvirus of the family Potyviridae.

Identification and expression of strigolactone biosynthesis and signaling genes and the in vitro effects of strigolactones in olive (Olea europaea L.).

Strigolactones (SLs), synthesized in plant roots, play a dual role in modulating plant growth and development, and in inducing the germination of parasitic plant seeds and arbuscular mycorrhizal fungi in the rhizosphere. As phytohormones, SLs are crucial in regulating branching and shaping plant architecture. Despite the significant impact of branching strategies on the yield performance of fruit crops, limited research has been conducted on SLs in these crops. In our study, we identified the transcript sequences of SL biosynthesis and signaling genes in olive (Olea europaea L.) using rapid amplification of cDNA ends. We predicted the corresponding protein sequences, analyzed their characteristics, and conducted molecular docking with bioinformatics tools. Furthermore, we quantified the expression levels of these genes in various tissues using quantitative real-time PCR. Our findings demonstrate the predominant expression of SL biosynthesis and signaling genes (OeD27, OeMAX3, OeMAX4, OeMAX1, OeD14, and OeMAX2) in roots and lateral buds, highlighting their importance in branching. Treatment with rac-GR24, an SL analog, enhanced the germination frequency of olive seeds in vitro compared with untreated embryos. Conversely, inhibition of SL biosynthesis with TIS108 increased lateral bud formation in a hard-to-root cultivar, underscoring the role of SLs as phytohormones in olives. These results suggest that modifying SL biosynthesis and signaling pathways could offer novel approaches for olive breeding, with potential applicability to other fruit crops.

Phototropin2 3’UTR overlaps with the AT5G58150 gene encoding an inactive RLK kinase

BackgroundThis study examines the biological implications of an overlap between two sequences in the Arabidopsis genome, the 3’UTR of the PHOT2 gene and a putative AT5G58150 gene, encoded on the complementary strand. AT5G58150 is a probably inactive protein kinase that belongs to the transmembrane, leucine-rich repeat receptor-like kinase family. Phot2 is a membrane-bound UV/blue light photoreceptor kinase. Thus, both proteins share their cellular localization, on top of the proximity of their loci.ResultsThe extent of the overlap between 3’UTR regions of AT5G58150 and PHOT2 was found to be 66 bp, using RACE PCR. Both the at5g58150 T-DNA SALK_093781C (with insertion in the promoter region) and 35S::AT5G58150-GFP lines overexpress the AT5G58150 gene. A detailed analysis did not reveal any substantial impact of PHOT2 or AT5G58150 on their mutual expression levels in different light and osmotic stress conditions. AT5G58150 is a plasma membrane protein, with no apparent kinase activity, as tested on several potential substrates. It appears not to form homodimers and it does not interact with PHOT2. Lines that overexpress AT5G58150 exhibit a greater reduction in lateral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may participate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identified proteins with ATPase activity, which are involved in proton transport and cell elongation, as putative interactors of AT5G58150. Membrane kinases, including other members of the LRR RLK family and BSK kinases (positive regulators of brassinosteroid signalling), can also act as partners for AT5G58150.ConclusionsAT5G58150 is a membrane protein that does not exhibit measurable kinase activity, but is involved in signalling through interactions with other proteins. Based on the interactome and root architecture analysis, AT5G58150 may be involved in plant response to salt and osmotic stress and the formation of roots in Arabidopsis.