Search Results (1,054)

Soybean mosaic virus (SMV) is a major viral pathogen that causes significant yield losses and a reduction in seed quality in susceptible soybean cultivars. Resistance breeding is the most effective, economical, and eco-friendly strategy for prevention of SMV-induced damage. Accurate and convenient assessment of SMV resistance is an essential prerequisite for resistance breeding. In this study, we constructed a green fluorescent protein (GFP)-tagged SMV recombinant virus (SMV-GFP) by yeast homologous recombination technology. It was proved that the recombinant virus can not only be used to track the viral infection process in Nicotiana benthamiana and soybean, but also to quantify the viral load based on relative fluorescence area (RFA) value. Using this recombinant virus, the resistance of 286 soybean germplasms from Northeast China to SMV was evaluated. A genome-wide association study (GWAS) was conducted using the RFA values of the 286 soybean accessions to find possible SMV-resistance genes. The results revealed 72 single nucleotide polymorphism (SNP) loci on chromosome 13 closely associated with SMV resistance, and a total of 40 genes were discovered within the candidate regions. By integrating the results of gene functional annotation and haplotype analysis, Glyma.13g176600 encoding a membrane attack complex/perforin (MACPF) domain-containing protein and Glyma.13g177000 encoding a DUF761-containing protein were identified as the most probable candidate genes associated with SMV resistance. Overall, the GFP-based rapid evaluation system developed in this study will facilitate breeding for resistance to SMV in soybean. Full article

Calcium-dependent protein kinases (CDPKs) are unique serine/threonine kinases that play significant roles in response to environmental stresses in plants. In this study, we comprehensively characterized the CDPK gene family in the apple cultivar ‘Hanfu’ at the genome-wide level, and 38 MdCDPKs were identified. They were unevenly distributed across 14 chromosomes. Based on phylogenetic analysis, the MdCDPKs were classified into four subfamilies. Conserved domain analysis indicated that MdCDPKs contain the catalytic kinase domain and the Ca²⁺ binding domain. During Colletotrichum gloeosporioides infection, the expression level of MdCDPK24 was significantly upregulated. Subsequently, MdCDPK24 was fused to GFP to generate the MdCDPK24-GFP construct, and confocal microscopy imaging confirmed its cytoplasmic localization in Nicotiana benthamiana leaves. Using agrobacterium-mediated transformation, we generated the overexpression of MdCDPK24 transgenic calli. MdCDPK24-overexpressing calli demonstrated significantly reduced disease severity against C. gloeosporioides infection, indicating its positive role in apple bitter rot resistance. The analysis of the CDPK gene family in the apple cultivar ‘Hanfu’ provides a new insight into the identification of CDPK genes involved in biotic stress. MdCDPK24 represents a promising candidate for genetic manipulation to enhance apple bitter rot resistance. Full article

N′ resistance is intrinsically broken by tobacco mosaic virus but is still effective against pepper mild mottle virus (PMMoV), including those breaking L resistance in peppers. To evaluate the durability of N′ resistance to PMMoV, we performed random mutagenesis of the coat protein (CP) gene of PMMoV. We isolated 11 CP mutants with two to six amino acid changes that escaped the N′-mediated resistance response in Nicotiana sylvestris. Some mutants and their derivatives, which had minimal mutations to escape N′-mediated resistance, exhibited reduced accumulation in inoculated leaves and loss of systemic infectivity in a susceptible pepper (Capsicum annuum) cultivar, as determined by RT-PCR analysis. Although the mutant CPs also escaped recognition by L³ and L⁴ resistance proteins from pepper in transient expression assays, the loss of systemic infectivity suggests that the mutants are unlikely to overcome L-mediated resistance. In Nicotiana benthamiana, a highly susceptible systemic host of PMMoV, ELISA and RT-qPCR indicated that the mutants consistently infected the host systemically, albeit with attenuated virulence and reduced virus accumulation, especially in younger leaves. The results collectively suggest that the reduced virus accumulation enabled the mutant PMMoV to escape N′-mediated resistance, and as a trade-off, compromised its virulence. The results also suggest that PMMoV CP modulates the systemic symptoms. Full article

Plant protein phosphatase 2C (PP2C) is recognized as one of the most critical protein family in plants and plays a pivotal role in disease resistance responses. However, the involvement of tomato PP2C family members in resistance to bacterial wilt caused by Ralstonia solanacearum remains poorly understood. In this study, we found that silencing SlPP2C28 increased tomato susceptibility to R. solanacearum; then, we introduced the tomato gene SlPP2C28, which exhibits a strong response to R. solanacearum, into the Nicotiana benthamiana genome via Agrobacterium-mediated transformation, generating high-expression transgenic lines OE-2 and OE-3. Following inoculation with R. solanacearum, the transgenic tobacco plants displayed reduced wilting symptoms, delayed disease onset, lower disease index, reduced stem cross-section damage, decreased internal bacterial colonization, diminished accumulation of reactive oxygen species in leaves, enhanced expression of SlPP2C28, up-regulated expression of defense-related genes NbSOD, NbPOD, and NbPAL along with an increase in the activities of their corresponding enzymes, and elevated expression levels of pathogenesis-related genes NbPR1a, NbPR2, NbPR4, and NbPR10. Collectively, these findings demonstrate that the SlPP2C28 gene has the function of enhancing resistance to bacterial wilt disease. Full article

Pinus massoniana Lamb. is an economically important conifer native to China. However, it is highly susceptible to the pine wood nematode (Bursaphelenchus xylophilus, PWN), the causal agent of pine wilt disease (PWD), resulting in substantial ecological and economic losses. To elucidate potential molecular defense mechanisms, 50 NAC (NAM, ATAF1/2, and CUC2) transcription factors (PmNACs) were identified in the P. massoniana genome. Phylogenetic analysis divided these PmNACs into seven subfamilies, and motif analysis identified ten conserved motifs associated with stress responses. Twenty-three genes were selected for expression analysis in various tissues and under exogenous salicylic acid (SA), methyl jasmonate (MeJA), and PWN infection. Six genes (PmNAC1, PmNAC8, PmNAC9, PmNAC17, PmNAC18, and PmNAC20) were significantly up-regulated by both hormonal treatment and PWN infection, implying their involvement in JA/SA-mediated immune pathways. Functional characterization showed PmNAC8 is a nuclear-localized transcription factor with autoactivation activity. Furthermore, transient overexpression of PmNAC8 in Nicotiana benthamiana induced reactive oxygen species (ROS) accumulation and necrotic lesions. Collectively, these results elucidate NAC-mediated defense responses to PWN infection in P. massoniana and identify candidate genes for developing PWD-resistant pine varieties. Full article

Oxybenzone (OBZ), an organic ultraviolet filter, is an emerging contaminant posing severe threats to ecosystem health. Using tobacco (Nicotiana tabacum) as a model plant, this study investigated the alleviation mechanisms of exogenous silicon (Na₂SiO₃, Si) and bamboo-based biochar (Bc) under OBZ stress. We systematically analyzed physiological and biochemical responses, including phenotypic parameters, reactive oxygen species metabolism, photosynthetic function, chlorophyll synthesis, and endogenous hormone levels. Results reveal that OBZ significantly inhibited tobacco growth and triggered a reactive oxygen species (ROS) burst. Additionally, OBZ disrupted antioxidant enzyme activities and hormonal balance. Exogenous Bc mitigated OBZ toxicity by adsorbing OBZ, directly scavenging ROS, and restoring the ascorbate-glutathione (AsA-GSH) cycle, thereby enhancing photosynthetic efficiency, while Si alleviated stress via cell wall silicification, preferential regulation of root development and hormonal signaling, and repair of chlorophyll biosynthesis precursor metabolism and PSII function. The mechanisms of the two stress mitigators were complementary, Bc primarily relied on physical adsorption and ROS scavenging, whereas Si emphasized metabolic regulation and structural reinforcement. These findings provide practical strategies for simultaneously mitigating organic UV filter pollution and enhancing plant resilience in contaminated soils. Full article

Anthocyanins, crucial water-soluble pigments in plants, determine coloration in floral and fruit tissues, while fulfilling essential physiological roles in terms of plant growth, development, and stress adaptation. The biosynthesis of anthocyanins is transcriptionally regulated by WRKY factors, one of the largest plant-specific transcription factor families. Euscaphis japonica is an East Asian species, prized for its exceptionally persistent butterfly-shaped fruits that undergo pericarp dehiscence, overturning, and a color transition to scarlet red. This species represents an ideal system for studying anthocyanin regulation. However, the mechanisms by which WRKY transcription factors orchestrate anthocyanin accumulation during this process remain unknown. In this study, we identified 87 WRKY genes (EjaWRKYs) from the E. japonica genome. Phylogenetic analysis was used to classify these genes into three primary groups, with five subgroups, revealing conserved gene structures and motif compositions, supported by collinearity and comparative synteny analyses. Crucially, ten EjaWRKYs exhibited peak expression during the mature fruit stages, showing positive correlations with key anthocyanin biosynthesis genes. Functional validation through the use of transient transactivation assays in Nicotiana benthamiana confirmed that the five selected EjaWRKYs bind W-box elements and strongly activate reporter gene expression. Our results reveal EjaWRKYs’ regulation of anthocyanin accumulation in E. japonica fruit, provide the first comprehensive WRKY family characterization of this species, and establish a foundation for manipulating ornamental traits in horticultural breeding. Full article

The GRAS gene family not only performs a variety of regulatory functions in plant growth and development but also plays a key role in the defense mechanisms of plants in response to environmental stresses. Although GRASs have been identified in many species, research on them in Nicotiana benthamiana remains relatively limited until now. In this study, we comprehensively analyzed the GRAS gene family in N. benthamiana plants. Phylogenetic analysis displayed that all identified NbGRASs were classified into eight different subfamilies. Gene duplication analysis revealed that segmental duplication was the main driving force for the expansion of the NbGRAS gene family, with a total of 40 segmental duplication pairs identified. NbGRASs were unevenly distributed across the 19 chromosomes. Additionally, both gene families exhibited a relatively weak codon usage bias, a pattern shaped by mutational and selective pressures. Expression analysis showed that NbGRASs had tissue-specific expression patterns, with relatively high expression levels being observed in leaves and roots. The expression of NbGRASs was significantly changed under tomato yellow leaf curl virus or bamboo mosaic virus infection, suggesting that these NbGRASs can be involved in the plant’s antiviral response. These findings provide new perspectives for in-depth understanding of the evolution and functions of the GRAS gene family in N. benthamiana. Full article

Plant subtilases, as hydrolytic enzymes, contribute to certain plant stress response pathways by cleaving precursor proteins into active peptides or through other less well-characterized mechanisms. Phytaspases represent a specific subgroup of subtilases, and their participation in rapid stress responses, particularly to herbivory attacks and drought, is already well established, in contrast to their poorly understood role in long-term responses. This study investigated the involvement of phytaspase NbSBT1.9-2 in the long-term stress responses of Nicotiana benthamiana. Plants were subjected to either mild to severe mechanical wounding or drought stress, followed by the detection of phytaspase activity and gene expression in the leaf tissue. The results revealed a distinct involvement of phytaspase in the wounding response, showing increased activity and upregulated expression correlated with the extent and recurrence of wounding. In contrast, no significant change in phytaspase activity was observed in the leaves under drought, alongside salinity and heat stress conditions. Consequently, phytaspase association with the long-term response to mechanical injury was demonstrated using N. benthamiana as a model organism. Full article

Uncaria rhynchophylla, a medicinal plant extensively used in traditional Chinese medicine, is an important plant source of terpenoid indole alkaloids (TIAs), but the mechanism of TIA biosynthesis at molecular level remains unclear. Geraniol synthase (GES) serves as a crucial enzyme in catalyzing the formation of geraniol from geranyl pyrophosphate (GPP) in various plants, but the functional characterization of the GES gene in U. rhynchophylla has not been investigated. In this study, a GES was identified and characterized through genome mining and bioinformatic analysis. Functional validation was performed via a protein catalysis experiment, transient expression in Nicotiana benthamiana, and methyl jasmonate (MeJA) induction experiments. The full-length UrGES gene was 1761 bp, encoding a protein product of 586 amino acids with an estimated 67.5 kDa molecular weight. Multiple sequence alignments and phylogenetic analysis placed UrGES within the terpene synthase g (TPS-g) subfamily, showing high similarity to known GESs from other plants. Enzymatic assays confirmed that recombinant UrGES catalyzed GPP conversion to a single product of geraniol. The transient expression of UrGES resulted in geraniol accumulation in N. benthamiana, further confirming its function in vivo. UrGES expression was observed in leaves, stems, and roots, where leaves had the highest transcript levels. Moreover, MeJA treatment significantly upregulated UrGES expression, which positively correlated with an increase in alkaloid content. This study functionally characterizes UrGES as a geraniol synthase in U. rhynchophylla, contributing to the current knowledge of the TIA biosynthetic pathway. These findings may offer insights for future metabolic engineering aiming to enhance TIA yields for pharmaceutical and industrial applications. Full article

Tomato yellow leaf curl virus (TYLCV) is a highly destructive pathogen of global tomato crops. The open reading frame (ORF) of TYLCV V2 contains two initiation codons (ATG1/V2-1 and ATG2/V2-2), producing distinct protein isoforms. Using custom antibodies, we confirmed V2-1 and V2-2 expression in infected Nicotiana benthamiana and tomato plants. Deletion mutants revealed their specialized roles: V2-1 was indispensable for viral replication and systemic spread—its loss severely reduced pathogenicity and genome accumulation. V2-2 acted as an auxiliary factor, and its deletion attenuated symptoms but kept the virus infection. Host-specific effects were observed—V2-1 deletion led to lower viral DNA/coat protein levels in N. benthamiana than in tomato, suggesting host-dependent regulation. Mutant viruses declined progressively in tomato, indicating host defense clearance. Heterologous co-expression of both isoforms via potato virus X induced systemic necrosis in N. benthamiana, demonstrating functional synergy between isoforms. Both initiation codons were essential for V2-mediated suppression of transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). This study uncovers the mechanistic divergence of V2 isoforms in TYLCV infection, highlighting their collaborative roles in virulence and host manipulation. The findings advance understanding of geminivirus coding complexity and offer potential targets for resistance strategies. Full article

Background/Objectives: Beak and feather disease virus (BFDV) is the causative agent of psittacine beak and feather disease (PBFD), affecting psittacine birds. There is currently no commercial vaccine or treatment for this disease. This study developed a novel BFDV coat protein mRNA vaccine encapsidated by TMV coat protein to form pseudovirions (PsVs) and tested its immunogenicity alongside BFDV coat protein (CP) subunit and DNA vaccine candidates. Methods: mRNA and BFDV CP subunit vaccine candidates were produced in Nicotiana benthamiana and subsequently purified using PEG precipitation and gradient ultracentrifugation, respectively. The DNA vaccine candidate was produced in E. coli cells harbouring a plasmid with a BFDV1.1mer pseudogenome. Immunogenicity of the vaccine candidates was evaluated in African grey parrot chicks. Results: Successful purification of TMV PsVs harbouring the mRNA vaccine, and of the BFDV-CP subunit vaccine, was confirmed by SDS-PAGE and western blot analysis. TEM analyses confirmed formation of TMV PsVs, while RT-PCR and RT-qPCR cDNA amplification confirmed encapsidation of the mRNA vaccine candidate within TMV particles. Restriction digests verified presence of the BFDV1.1mer genome in the plasmid. Four groups of 5 ten-week-old African grey parrot (Psittacus erithacus) chicks were vaccinated and received two boost vaccinations 2 weeks apart. Blood samples were collected from all four groups on day 14, 28 and 42, and sera were analysed using indirect ELISA, which showed that all vaccine candidates successfully elicited specific anti-BFDV-CP immune responses. The subunit vaccine candidate showed the strongest immune response, indicated by higher binding titres (>6400), followed by the mRNA and DNA vaccine candidates. Conclusions: The candidate vaccines present an important milestone in the search for a protective vaccine against PBFD, and their inexpensive manufacture could considerably aid commercial vaccine development. Full article

Field trials were carried out over two tobacco cropping seasons (2020 and 2021) to assess the effectiveness of soil (PRE-T) and post-transplant (POST-T (OT)) herbicides in a tobacco crop, depending on rainfall and the type of soil. The effectiveness of PRE-T and POST-T (OT) herbicides alternated according to the presence of weeds, treatments, the region, and years. Unpredictable meteorological conditions throughout the two study years likely influenced the control of weeds. An unusually moist May in 2020 with a precipitation of 29 mm in the first WA PRE-T before the emergence of weeds generated the leaching of the PRE-T herbicide from the surface of the soil, which was likely the most probable reason for the reduced effectiveness of PRE-T-applied herbicides (less than 77%) in comparison to the POST-T (OT) application treatment in 2020 in the Prilep region. Conversely, the restricted rainfall after PRE-T and POST-T (OT) application may have caused the unsatisfactory efficacy of both PRE-T and POST-T (OT) herbicide treatments in the Titov Veles region in 2021 (less than 78 and 80%, respectively) in comparison with 2020. Excessive rain immediately after PRE-T and POST-T (OT) application resulted in the injury of tobacco plants in the Prilep region in 2020 and 2021, which was between 8 and 25%, and 7 and 22%, respectively, after seven DAHAs across both treatments. The injuries caused by pendimethalin and metolachlor were more serious. The yields of tobacco after both PRE-T and POST-T treatment in each region typically reflect the overall effectiveness of weed control and the extent of tobacco crop injury. Full article

The cigarette production from Nicotiana tabacum generates significant amounts of waste, with an estimated 68.31 million tons of pre- and post-harvest waste discarded annually. The pre-harvest waste includes the upper parts of the plant, inflorescences, and bracts, which are removed to help the growth of the lower leaves. This study explores the potential of apical leaves from Nicotiana tabacum var. Virginia, discarded during the budding stage (pre-harvest waste). The leaves were extracted using environmentally friendly solvents (green solvents), including distilled water (DW) and two natural deep eutectic solvents (NaDESs), one consisting of simple sugars, fructose, glucose, and sucrose (FGS) and the other consisting of choline chloride and urea (CU). The anti-inflammatory and anti-aging potential of these green extracts was assessed by the inhibition of key enzymes related to skin aging. The xanthine oxidase and lipoxygenase activities were mostly inhibited by CU extracts with IC₅₀ values of 63.50 and 8.0 μg GAE/mL, respectively. The FGS extract exhibited the greatest hyaluronidase inhibition (49.20%), followed by the CU extract (33.20%) and the DW extract (20.80%). Regarding elastase and collagenase inhibition, the CU extract exhibited the highest elastase inhibition, while all extracts inhibited collagenase activity, with values exceeding 65%. Each extract had a distinct chemical profile determined by LC-ESI-QTOF-MS/MS and spectrophotometric methods, with several shared compounds present in different proportions. CU extract is characterized by high concentrations of rutin, nicotiflorin, and azelaic acid, while FGS and DW extracts share major compounds such as quinic acid, fructosyl pyroglutamate, malic acid, and gluconic acid. Ames test and Caenorhabditis elegans assay demonstrated that at the concentrations at which the green tobacco extracts exhibit biological activities, they did not show toxicity. The results support the potential of N. tabacum extracts obtained with NaDESs as antiaging and suggest their promising applications in the cosmetic and cosmeceutical industries. Full article

Dryopteris fragrans (L.) Schott synthesizes volatile sesquiterpenes through the mevalonate pathway (MVA), in which 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) serves as the key rate-limiting enzyme. Although HMGR plays a crucial role in terpenoid biosynthesis, its functional characteristics in D. fragrans and its involvement in stress responses remain unclear. This study identified three HMGR genes (DfHMGR1/2/3) from the transcriptome data of D. fragrans. Bioinformatics analysis revealed that the encoded proteins are localized to the endoplasmic reticulum and share high sequence similarity with fern homologs. Under abiotic stress conditions, DfHMGRs exhibited differential expression patterns, with marked upregulation under salt and drought stress. To validate the functions of these genes, we generated transgenic Nicotiana tabacum L. plants overexpressing DfHMGRs. Compared with wild-type controls, the transgenic lines showed enhanced tolerance to drought and heat stress. Promoter analysis identified functional regulatory regions controlling DfHMGR expression, and co-expression network analysis predicted 21 potential transcriptional regulators. This study validates the function of D. fragrans HMGRs in a heterologous system and provides candidate genes for improving stress resistance in plants. Full article