cDNA Library Research Articles

High-Throughput Oxford Nanopore Sequencing Unveils Complex Viral Population in Kansas Wheat: Implications for Sustainable Virus Management

Wheat viruses are major yield-reducing factors, with mixed infections causing substantial economic losses. Determining field virus populations is crucial for effective management and developing virus-resistant cultivars. This study utilized the high-throughput Oxford Nanopore sequencing technique (ONT) to characterize wheat viral populations in major wheat-growing counties of Kansas from 2019 to 2021. Wheat leaves exhibiting virus-like symptoms were collected, total RNA was extracted, and cDNA libraries were prepared using a PCR-cDNA barcoding kit, then loaded onto ONT MinION flow cells. Sequencing reads aligned with cereal virus references identified eight wheat virus species. Tritimovirus tritici (wheat streak mosaic virus, WSMV), Poacevirus tritici (Triticum mosaic virus TriMV), Bromovirus BMV (brome mosaic virus BMV), as well as Emaravirus tritici, Luteovirus pavhordei, L. sgvhordei, Bymovirus tritici, and Furovirus tritici. Mixed infections involving two to five viruses in a single sample were common, with the most prevalent being WSMV + TriMV at 16.7% and WSMV + TriMV + BMV at 11.9%. Phylogenetic analysis revealed a wide distribution of WSMV isolates, including European and recombinant variants. A phylogenetic analysis of Emaravirus tritici based on RNA 3A and 3B segments and whole-genome characterization of Furovirus tritici were also conducted. These findings advance understanding of genetic variability, phylogenetics, and viral co-infections, supporting the development of sustainable management practices through host genetic resistance.

Multiple myeloma-associated non-crystalline proximal tubulopathy and crystalline cast nephropathy: Biochemical and structural features of disease-causing monoclonal kappa light chains.

Various tubular diseases in patients with multiple myeloma (MM) are caused by monoclonal immunoglobulin light chains (LCs). However, the physicochemical characteristics of the disease-causing LCs contributing to the onset of MM-associated tubular diseases remain unclear. We herein report a rare case of MM-associated combined tubulopathies: non-crystalline light chain proximal tubulopathy (LCPT) and crystalline light chain cast nephropathy (LCCN). The patient's urinary κ-LC (Bence-Jones proteins, BJP-κ PT-CN) was detected through immunofixation. Renal biopsy revealed cytoplasmic vacuoles in swollen proximal tubular cells and distal tubular casts. Immunohistochemistry showed proximal tubular reabsorption granules and distal tubular casts positively stained with an anti-κ-LC antibody. Electron microscopy identified vacuolation and an increased number of lysosomes in proximal tubular epithelial cells without crystalline structures. Distal tubular casts comprised numerous crystals with both rod-shaped and needle-like configurations and tube-shaped materials. To elucidate the molecular mechanisms underlying tubular toxicity, we performed the following physicochemical analyses of BJP-κ PT-CN: N-terminal amino acid sequencing, cDNA cloning, size-exclusion chromatography, thermal shift assays, and X-ray crystallography. The variable segment of BJP-κ PT-CN was derived from the IGKV1-39 gene. The characteristic features of BJP-κ PT-CN were a positively charged surface patch, concentration-dependent monomer-dimer equilibrium, and the R61G mutation. This is the first biochemical and structural characterization of disease-causing BJPs in MM-associated LCPT and crystalline LCCN. The results obtained suggest that these characteristic features enhance protein binding to negatively charged sites on brush-border membranes in proximal tubules and promote the formation of organized casts in distal tubular lumens.

Genomic, Evolutionary, and Pathogenic Characterization of a New Polerovirus in Traditional Chinese Medicine Viola philippica

Viola philippica, a medicinal herbaceous plant documented in the Chinese Pharmacopoeia, is a promising candidate for research into plant-derived pharmaceuticals. However, the study of newly emerging viruses that threaten the cultivation of V. philippica remains limited. In this study, V. philippica plants exhibiting symptoms such as leaf yellowing, mottled leaves, and vein chlorosis were collected and subjected to RNA sequencing to identify potential viral pathogens. A novel polerovirus, named Viola Philippica Polerovirus (VPPV), was identified in V. philippica. VPPV possesses a linear, positive-sense, single-stranded RNA genome consisting of 5535 nucleotides (nt) and encodes seven highly overlapping open reading frames (ORFs). Two potential recombination events were identified within ORF2, ORF3a, and ORF3, providing insights into the genetic diversity and evolution history of this novel polerovirus. An infectious cDNA clone of VPPV was successfully constructed and shown to infect Nicotiana benthamiana. Using a PVX-based heterologous expression system, the VPPV P0 protein was shown to trigger a systemic hypersensitive response (HR)-like reaction in N. benthamiana, indicating that P0 functions as the main pathogenicity determinant. These findings contributed to the detection and understanding of pathogenic mechanisms and control strategies for VPPV in V. philippica.

Single cell transcriptomics in blood of patients with chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Single-cell RNA sequencing (scRNA-seq) provides gene expression profiles at the single-cell level. Hence, we evaluated gene expression in the peripheral blood of patients with COPD.MethodsPeripheral blood samples from seven healthy controls and eight patients with COPD were obtained in this study. The 10X Genomics Chromium Instrument and cDNA synthesis kit were utilized to generate a barcoded cDNA library for single cell RNA-sequencing. We compared the scRNA-seq data between the COPD and control groups using computational analysis. Functional analyses were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses.ResultsscRNA-seq was used to analyze the transcriptome of peripheral blood mononuclear cells from seven normal controls and eight patients with COPD. We found an increased number of monocyte/macrophages in the COPD group compared to the normal control group. Among the differentially expressed genes (DEGs) in monocyte/macrophages, we identified 15 upregulated genes (EGR1, NR4A1, CCL3, CXCL8, PTGS2, CD83, BCL2A1, SGK1, IL1B, BTG2, NFKBIZ, DUSP2, MAFB, PLAUR and CCL3L1) and 7 downregulated genes (FOLR3, RPS4Y1, HLA-DRB5, NAMPT, CD52, TMEM176A and TMEM176B) in the COPD group compared to the normal control group.ConclusionsUsing scRNA-seq, we found differences in cell type distribution, especially in monocyte/ macrophages. Several upregulated and downregulated genes were found in the monocyte/macrophages of the COPD group.

ZjMAPKK4 Interacted With ZjNAC78 Regulates Cold Tolerance Response in Jujube.

Jujube (Ziziphus ujuba Mill.) holds great importance as a fruit tree in China, with strong tolerance to drought and saline stress, but its growth is limited by vulnerability to cold stress. Consequently, the role of MAPK cascades in mediating jujube cold stress response remains unclear, with the specific function of ZjMAPKK4 in this context yet to be fully elucidated. Thus, in the current study, it was found that ZjMAPKK4 was significantly upregulated compared with other ZjMAPK cascade genes after cold treatment. Heterologous transformation of ZjMAPKK4 in Arabidopsis, VIGS-induced ZjMAPKK4 transiently silencing and overexpression of ZjMAPKK4 in jujube callus assays demonstrated that ZjMAPKK4 positively regulated the cold resistance of jujube. Furthermore, to elucidate the molecular regulation mechanism behind ZjMAPKK4 under cold stress, 25 key DEGs were screened out by transcriptome analysis. Yeast screening cDNA library, yeast two-hybrid, LCA and Co-IP analysis showed ZjMAPKK4 interacted with ZjNAC78 and VIGS-induced ZjNAC78 silenced sour jujube plants showed cold sensitivity and the expression level of cold response genes were downregulated after cold stress. All the results demonstrated that ZjMAPKK4 could interact with ZjNAC78 to regulate the downstream ZjICE-ZjCBF genes to regulate the cold tolerance of jujube.

Molecular and biological characterization of infectious full-length cDNA clones of two viruses in Paris yunnanensis, including a novel potyvirus

Paris yunnanensis, also named as Rhizoma Paridis in the Chinese Pharmacopeia, is a perennial Chinese medicinal herb commonly grown in Southwest China. However, several viruses have been found infecting this plant in recent years. Using high-throughput sequencing (HTS) and Sanger sequencing, this study obtained the complete genome sequences of three capillovirus isolates and one potyvirus isolate. Genomic and phylogenetic analyses revealed that these three capillovirus isolates are the same virus as the newly reported capillovirus, Paris polyphylla chlorotic mottle virus. The newly found potyvirus isolate shares 52.4–68.9% nucleotide sequence identity with other known potyviruses and thus, is grouped into the bean common mosaic virus subgroup. Based on the nucleotide sequence identity, we consider this virus a novel potyvirus species and propose ‘Paris potyvirus 5’ (ParPV-5) as its common name, and ‘Potyvirus shilinense’ as its species name. To characterize their biological features, two infectious clones, representing the two viruses, have been constructed through homologous recombination or yeast homologous recombination, and inoculated to several species plants, respectively. The results showed both of the viruses can infect P. yunnanensis and Nicotiana benthamiana. In addition, Paris polyphylla chlorotic mottle virus (PpCMV) can infect N. tabacum var. Xanthi nc, Cucurbita moschata, and Capsicum annuum, and ParPV-5 can infect Cucumis sativus L. and Bidens pilosa L. However, except mild leaf deformation exhibited on the PpCMV-inoculated C. moschata plants, no obvious symptom were observed in these plants including P. yunnanensis. A total of 179 field P. yunnanensis leaf samples from four counties in 2020–2021, and all 640 P. yunnanensis plants from a whole study plot of Lijiang in 2024, were tested using RT-PCR and specific primers, the results showed that PpCMV is a potential preponderant species in some regions, and ParPV-5 has the possible transmission from the original site to other regions.

A brief history of the cortical thick ascending limb: a systems-biology perspective.

Here, we review key events in the accrual of knowledge about the cortical thick ascending limb (CTAL) of the kidney, starting with its initial characterization by Maurice Burg in 1973. Burg's work showed that the CTAL actively reabsorbs NaCl and that, because its water permeability is virtually zero, it can lower the luminal NaCl concentration to a "static head" level well below blood levels. This process is central to the kidney's ability to excrete dilute urine in states of high water intake. Following Burg's original observations, Greger and Schlatter, working in the 1980s, identified the membrane transport processes responsible for transepithelial NaCl transport in the CTAL. In the 1990s, several investigators identified the key transporter genes and proteins at a molecular level by cDNA cloning. The successful completion of human and mouse genome sequencing projects at the turn of the century led to the development of transcriptomic and proteomic methodologies that allowed the identification of complete transcriptomes and proteomes of CTAL cells. Knowledge accrual was enhanced by the development of differential equation-based models of transport in the CTAL in the 2010s. Here, we used a simplified mathematical model of NaCl ("salt"), urea, and water transport in the CTAL to address three key questions about CTAL function: 1) What is the mechanism of Burg's "static head" phenomenon? 2) How does the kidney compensate for the very short length of the CTALs of juxtamedullary nephrons? 3) Which of the three isoforms of the apical Na-K-2Cl cotransporter (NKCC2) dominates functionally in the CTAL?NEW & NOTEWORTHY Here, we review key events in the accrual of knowledge about the cortical thick ascending limb (CTAL) of the kidney, starting with its initial characterization by Maurice Burg in 1973, and culminating with the application of systems biology techniques including mathematical modeling.

Pinus radiata cDNA Library for the Screening of Phytophthora Effector Protein Targets in Yeast.

Yeast two-hybrid library screening enables the discovery of novel protein-protein interactions. Identifying cytosolic host proteins targeted by host-translocated Phytophthora effector proteins relies on the mRNA amount, quality, and composition used to prepare the yeast two-hybrid cDNA library. Here we describe the steps required for the preparation of a Pinus radiata cDNA library optimized for Phytophthora effector target screening in yeast. This method may also be applied to other host plants and pathogen species.

In silico antiviral effect assessment of some venom gland peptides from Odontobuthus doriae scorpion against SARS-CoV-2.

SARS-CoV-2 is from the enveloped virus family responsible for the COVID-19 pandemic. No efficient drugs are currently available to treat infection explicitly caused by this virus. Therefore, searching for effective treatments for severe illness caused by SARS-CoV-2 is crucial. Scorpion venoms are significant sources of peptides with pharmaceutical potential, including antivirals. Although some studies have determined the antiviral effects of some scorpion peptides on other members of the Coronaviridae family, a few anti-SARS-CoV-2 effects of these peptides have been reported until now. This study assessed the antiviral effects of five predicted antimicrobial peptides with potential for antiviral activities from the Iranian yellow scorpion "Odontobuthus doriae" by computational methods. These peptides were selected from the cDNA library that our research team constructed. A 3D model of peptides was designed with I-TASSER. The models were refined using a 200 ns Molecular Dynamics (MD) simulation using Gromacs 2021.2 software. Refined models were Docked with the RBD domain of SARS-CoV-2 spike protein using HADDOCK software. The docking of human ACE2 peptide with the RBD domain was also assessed. The docked complexes (RBD-peptide and RBD-ACE2) were refined again by a 100 ns MD simulation and then analyzed. The results from molecular docking after molecular dynamics simulation showed that ODAMP2 and ODAMP5 after stabilizing analysis and according to MMPBSA results (with -59.24 kcal/mol and -51.82 kcal/mol, respectively) have a strong binding affinity to the RBD domain of COVID-19 spike protein compared to human ACE2 and some other studied components. Therefore, this peptide can be an excellent candidate for use as an agent to inhibit the RBD domain of SARS-COV2 virus in clinical studies for medicinal purposes after in vitro and in vivo laboratory evaluations.

Replication-competent recombinant porcine reproductive and respiratory syndrome (PRRS) virus expressing antiviral cytokine interferon-ω5 as a modified live virus vaccine.

Porcine reproductive and respiratory syndrome (PRRS), caused by the highly variable PRRS virus (PRRSV), presents a significant challenge to the swine industry due to its pathogenic and economic burden. The virus evades host immune responses, particularly interferon (IFN) signaling, through various viral mechanisms. Traditional vaccines have shown variable efficacy in the field, prompting the exploration of novel vaccination strategies. This study investigates a reverse genetics approach to develop a modified live virus (MLV) vaccine expressing the potent antiviral cytokine interferon-ω5 (IFN-ω5) to combat PRRSV. The study utilizes an infectious cDNA clone of PRRSV, incorporating genetic modifications for IFN-ω5 expression. A comparative evaluation, including in vitro and particularly in vivo assessments here, was conducted to determine the vaccine's efficacy. Results indicate that pigs vaccinated with the IFN-ω5 MLV exhibited significant differences compared to the mock group in terms of body temperature, weight gain, antibody response, viral load, cytokine profile, and lung lesions following PRRSV challenge. This study underscores the potential of reverse genetics and IFN-ω5 expression as a promising strategy for developing effective PRRSV vaccines. The findings provide valuable insights into the mechanisms of immune response and viral pathogenesis, highlighting the importance of early immune activation in combating PRRSV infection.

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Screening, validation and functional characterization of genes encoding proteins that interact with sensory neuron membrane protein 1b (SNMP1b) from Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Sensory neuron membrane proteins (SNMPs) play critical roles in insect olfactory system. However, functional studies outside of Drosophila remain limited, especially in Coleoptera species. In our previous study, a SNMP1 (CbuqSNMP1b) was identified from Cyrtotrachelus buqueti (Coleoptera: Curculionidae), an insect pest that seriously influence the development of the bamboo industry. Here in a membrane protein yeast two-hybrid system, protein interactions between CbuqSNMP1b as a bait protein and a cDNA library of antenna of male C. buqueti adults as prey protein were assessed. Of 29 proteins identified as putative interactors, the Minus-C odorant-binding protein (CbuqOBP1) was selected for further analysis. The interaction between CbuqSNMP1b and CbuqOBP1 was further confirmed by both the in vivo yeast spotting analysis and the in vitro glutathione-S-transferase pull-down assay. Fluorescence binding assays indicated that the interaction between CbuqSNMP1b and CbuqOBP1 could enhance the binding abilities of CbuqOBP1 to four adult C. buqueti biologically active volatiles. The knockdown of CbuqSNMP1b + CbuqOBP1 expression by RNA interference significantly reduced the behavior responses of male adults to ethyl hexanoate and trans,trans-2,4-Nonadienal. These results increase our understanding of insect SNMP1 and will aid in exploring the underlying mechanisms of CbuqSNMP1b functions in the future.

ELISA-assay for Pig Taenia solium Cysticercosis by using T18 recombinant antigen

Cysticercosis is caused by infection with the larval stage of tapeworm parasite Taenia solium. This zoonose is a major public health issue with high prevalence in Madagascar, around 21% and 7% to 20% respectively in swine and human. ELISA and Enzyme-linked Immuno-electro Transfer Blot (EITB) remained the main laboratory serological diagnostic tools available in the island. Four native glycoproteins are recognized by antibodies in a majority of sera from patients with cysticercosis and a similar pattern seems to be present in infected pigs. For a better management of this disease in Madagascar, the aim of this study is to set-up and validate the one majority glycoprotein antigen T18 (18KDa) for diagnosis test of pig cysticercosis, produced as recombinant protein. Using a cDNA library prepared from T. solium cysticerci obtained from different regions of Madagascar, we first analyzed the polymorphism of the T18 gene. Then, gene was cloned into the pMAL-c2X plasmid for expression as an N-terminal maltose-binding protein (MBP) fusion protein and C-terminal hexa-His tagged protein. Product protein was then validated by ELISA test of pig sera. Preliminary results show a strong conservation of T18 gene. And indeed larger amounts of soluble recombinant protein were obtained from these antigen. T18 recombinant-ELISA present 70% of sensitivity and 100% of specificity.

The Chlamydia pneumoniae inclusion membrane protein Cpn0308 interacts with host protein ACBD3.

Chlamydia pneumoniae is an obligate intracellular bacterium of eukaryotic cells characterized by a unique biphasic life cycle; its biosynthesis and replication must occur within a cytoplasmic vacuole or inclusion. Certain inclusion membrane proteins have been demonstrated to mediate the interactions between intra-inclusion chlamydial organisms and the host cell. It has been demonstrated previously that the C. pneumoniae-encoded Cpn0308 localizes to the inclusion membrane; however, its function remains unknown. In the current study, a yeast two-hybrid assay was conducted to screen Cpn0308 as a bait against a HeLa cell cDNA library, revealing its binding to the host protein acyl-coenzyme A binding domain-containing 3 (ACBD3). The interaction between Cpn0308 and ACBD3 was confirmed through co-immunoprecipitation and GST (Glutathione S-transferase) pull-down assays. The two proteins were also co-localized in HeLa cells co-expressing Cpn0308 and ACBD3, as well as in C. pneumoniae-infected cells, as observed under confocal fluorescence microscopy. Given that ACBD3 plays a crucial role in maintaining host cell lipid homeostasis and its Golgi dynamic domain is responsible for interacting with Cpn0308, we hypothesize that the Cpn0308-ACBD3 interaction may facilitate C. pneumoniae's acquisition of host lipids, thereby benefiting chlamydial survival. This study lays a foundation for further elucidating the mechanisms of Cpn0308-mediated C. pneumoniae pathogenesis.IMPORTANCEThe biosynthesis and replication of Chlamydia pneumoniae (Cpn) must occur within the cytoplasmic vacuoles or inclusions of host cells. Inclusion bodies play a crucial role in mediating the interactions between Cpn and host cells. Cpn0308 is localized to the inclusion membrane; however, its function is unknown. In this study, Cpn0308 was found to bind to host protein acyl-coenzyme A binding domain-containing 3 (ACBD3) through some standard approaches. Co-localization of the two proteins was observed in both original HeLa cells and Cpn-infected HeLa cells. ACBD3 plays a significant role in maintaining lipid homeostasis in host cells; we speculate that the Cpn0308-ACBD3 interaction may facilitate the acquisition of host lipids by C. pneumoniae, thereby enhancing chlamydial survival.

Unveiling the Movement of RanBP1 During the Cell Cycle and Its Interaction with a Cyclin-Dependent Kinase (CDK) in Plants

In the Nicotiana tabacum flower development study, we identified SCI1 (Stigma/style Cell-cycle Inhibitor 1), a regulator of cell proliferation. SCI1 interacts with NtCDKG;2 (N. tabacum Cyclin-Dependent Kinase G;2), a homolog of human CDK11, which is responsible for RanGTP-dependent microtubule stabilization, regulating spindle assembly rate. In a Y2H screening of a cDNA library using NtCDKG;2 as bait, a RanBP1 (Ran-Binding Protein 1) was revealed as its interaction partner. RanBP1 is an essential regulatory protein of the RanGTPase system, contributing to the formation of the Ran gradient, which modulates different important cellular processes. RanBP1 is crucial in the nuclear import/export machinery during interphase and spindle checkpoint formation during cell division. These processes are well studied in animals, but very little is known about them in plants. We confirmed NtCDKG;2 and NtRanBP1 interaction by pairwise Y2H and characterized the localization of both proteins during plant cell division. We demonstrated the presence of NtRanBP1 in the cytoplasm during interphase and its nuclear arrest at mitosis onset. Meanwhile, we showed that NtCDKG;2 is localized in the mitotic spindle during cell division, indicating an analogous function to the human CDK11. We propose that the phosphorylation of the nuclear export signal at RanBP1 by NtCDKG;2 may be responsible for the reported nuclear arrest.

Generation of effective and specific human TCRs against tumor/testis antigen NY-ESO-1 in mice with humanized T cell recognition system.

Generation of high avidity T cell receptors (TCRs) reactive to tumor-associated antigens (TAA) is impaired by tolerance mechanisms, which is an obstacle to effective T cell therapies for cancer treatment. NY-ESO-1, a human cancer-testis antigen, represents an attractive target for such therapies due to its broad expression in different cancer types and the restricted expression in normal tissues. Utilizing transgenic mice with a diverse human TCR repertoire, we isolated effective TCRs against NY-ESO-1157-165 restricted to HLA-A*02:01. We compared the functions of the murine-derived TCR with human-derived TCRs and an affinity matured TCR, using in vitro co-culture and in vivo adoptive T cell transfer in tumor-bearing mice. Alanine scan, x-scan, LCL assay were employed to address the cross-reactivity of the NY-ESO-1157-165 specific TCRs. We also used human tissue cDNA library and human primary cells to assess the safety of adoptive T cell therapies targeting NY-ESO-1 antigen in the clinic. One of the murine-derived human TCRs, TCR-ESO, exhibited higher functional avidity compared to human-derived NY-ESO-1157-165 specific TCRs. TCR-ESO appeared to have similar efficiency in antigen recognition as an in vitro affinity-matured TCR, TCR 1G4-α95LY, which was applied in clinical trials. TCR-ESO showed little cross-reactivity, in contrast to TCR 1G4-α95LY. Our data indicate that highly effective TCRs against NY-ESO-1 are likely deleted in humans due to tolerance mechanisms, and that the TCR gene loci transgenic mice represent a reliable source to isolate effective and highly-specific TCRs for adoptive T cell therapies.

Construction and characterization of an infectious cDNA clone of turtle grass virus X from a naturally infected Thalassia testudinum plant.

Seagrasses are a polyphyletic group of marine flowering plants that play crucial roles in nearshore ecology, yet their interactions with viruses remain largely unexplored. This study presents the construction and characterization of an infectious cDNA clone of the potexvirus turtle grass virus X (TGVX). The complete genome of this positive-sense single-stranded RNA virus was amplified from field samples of Thalassia testudinum and assembled into a pLX-based mini binary vector using a multi-fragment directional cloning strategy, resulting in the infectious clone pLX-TGVX. Agroinfection assays of potexvirus-free T. testudinum plants resulted in systemic infections by TGVX, as confirmed by multiplex RT-PCR experiments and phenotypic changes reflecting virus-induced symptoms. Ultrastructural studies also demonstrated significant cytopathological changes resulting from TGVX infection, including chloroplast swelling, reduced thylakoid grana, and the presence of viral replication organelles and filamentous virus-like particles. The development of the TGVX infectious clone offers a novel tool for investigating the impact of this virus on seagrass health and productivity. This study demonstrates the first successful agroinfection of a marine plant with an infectious clone, creating a new avenue for studying viruses identified through sequence-based surveys and paving the way for exploring the ecological significance of viral infection in these critical marine ecosystems.IMPORTANCEThis study pioneers the construction of an infectious clone of turtle grass virus X and describes its application in the natural marine plant host, Thalassia testudinum. The creation of this infectious clone not only provides a valuable tool for marine plant virology research but also opens new avenues for exploring the influence of viral infections on the health and productivity of seagrass meadows. Given that seagrasses play a crucial role in sediment stabilization, nutrient cycling, and habitat provisioning, understanding the impact of viruses on these ecosystems is essential for their effective conservation and management. This methodological advance enables detailed studies of viral replication, virus-host interactions, and the broader ecological implications of viral infections in marine plants.

A Virulence Factor from Sclerotinia sclerotiorum Targets the Host Chloroplast Proteins to Promote Infection.

Chloroplasts are not only places for photosynthesis, but also participate in plant immunity and are important targets of pathogens. Pathogens secrete chloroplast-targeted proteins (CTPs) that disrupt host immunity and promote infection. Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus with a broad host range. However, little is known about the pathogenic mechanisms underlying this wide host range. In this study, we investigated the role of Chloroplast-Targeted Protein 1 (SsCTP1) secreted by S. sclerotiorum in pathogenesis, which inhibits plant immunity and promotes pathogen infections. SsCTP1 was highly up-regulated during the early stages of S. sclerotiorum infection in various hosts, and its transient expression in Nicotiana benthamiana revealed that it was predominantly localized within chloroplasts. Mutants with SsCTP1 deletion exhibited a similar growth rate and colony morphology to the wild type, but significantly reduced pathogenicity in various hosts. Moreover, SsCTP1 inhibited chitin-induced callose deposition and defense gene expression, and enhanced sensitivity to S. sclerotiorum in N. benthamiana. Similarly, transgenic Arabidopsis thaliana overexpressing SsCTP1 displayed an increased susceptibility to S. sclerotiorum. Furthermore, two host proteins that interact with SsCTP1, Coproporphyrinogen-III oxidase (GmCPX), and shikimate kinase 2 (GmSKL2) were identified by screening the soybean cDNA library, and these interactions were confirmed in vivo. Importantly, the silencing of NbCPX by virus-induced gene silencing enhanced N. benthamiana resistance to S. sclerotiorum. Our results indicate that SsCTP1 is an important pathogenic factor that contributes to the wide host range of S. sclerotiorum and may inhibit plant immunity by targeting the chloroplast proteins GmCPX and GmSKL2, which are ubiquitous in host plants.

Identification and characterization of tissue-specific genes in response to handling stress in topmouth culter (Culter alburnus) kidney, liver and muscle tissues

Topmouth culter (Culter alburnus) is an important freshwater economic fish in China. However, external stress often triggers strong reactions, resulting in low survival rates and reduced productivity. The absence of stress-related genes has significantly limited our understanding of stress response in this fish. Therefore, 85,846,206 high-quality reads were acquired from kidney, liver and muscle cDNA libraries of topmouth culter using illumina sequencing technology in this study. Comparative analysis revealed that 3,158; 409 and 1,952 unigenes were specifically expressed in the kidney, liver and muscle transcriptome, respectively. Additionally, 83 crucial tissue-specific genes were identified within three stress-related GO terms: response to stimulus (GO:0050896), response to stress (GO:0006950) and response to hypoxia (GO:0001666). From these, 18 tissue-specific genes were further isolated. During the short-term stress experiment (two repeated handling stress, including chasing 2 min and netting out of water for 30s), significant changes were observed in the cortisol levels of both the treatment and recovery groups. Furthermore, notable changes were noted in the expression of LCP2, PTK2b and P-selectin genes in the kidney; FABP1, IGFBP1 and CYP4V2 genes in the liver; and MYH10, Myogenin 2 and Toponin C genes in the muscle of topmouth culter in the treatment and recovery groups (P < 0.05). The tissue-specific transcriptome profiles generated in this study offer valuable insights into the molecular and functional mechanisms associated with stress response in topmouth culter. We characterizated genes related to stress response in tissues such as the kidney, liver and muscle, these findings offer novel insights into stress research in fish. We can further explore the breeding of strains with enhanced stress resistance and promote the healthy development of topmouth culter industry.

Comparative analyses of three grapevine Pinot gris virus cDNA clones reveal insights into the pathological properties of different phylogroups.

Grapevine Pinot gris virus (GPGV) is an emerging grapevine virus associated with grapevine leaf mottling and deformation (GLMD) disease. Being a recently identified virus, the molecular biology, pathological properties, and etiological complexity of GPGV remain poorly studied. Previous research revealed that GPGV comprises genetically different variants, some encoding a larger movement protein (MP) and others a shorter MP due to a C/T polymorphic site in ORF2 encoding MP. Variants that encode the shorter MP are associated with severe disease, whereas variants encoding the longer MP are associated with mild or no symptoms. However, this has yet to be demonstrated experimentally. Here, we report the construction of a wildtype cDNA clone, pGPGV-SY, based on ON93-12, a local isolate from Syrah closely related to the variants encoding the larger MP. Surprisingly, our clone exhibited significantly faster replication and caused more severe disease symptoms than pRI::GPGV-lat, an Italian GPGV clone, with a longer MP and demonstrated similar efficacies with that of pRI::GPGV-vir, another Italian clone with a shorter MP. A single C to T mutation at the polymorphic site of pGPGV-SY resulted in a two-fold higher RNA accumulation in the grapevine. Findings from this work constitute a leap toward the long-standing and complex question pertaining to the relationship between GPGV variant groups and GLMD. Integrating findings from this work and those by others, we propose an updated model to explain the complex relationship between GPGV variants and GLMD.