Search Results (584)

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

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

Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1–4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293–993 nt) and rep (1561–2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species. Full article

AT-hook motif nuclear-localized (AHL) genes play critical roles in chromatin remodeling and gene transcription regulation, profoundly influencing plant growth, development, and stress responses. While AHL genes have been extensively characterized in multiple plant species, their biological functions in pepper (Capsicum annuum L.) remain largely uncharacterized. In this study, we identified 45 CaAHL genes in the pepper genome through bioinformatics approaches. Comprehensive analyses were conducted to examine their chromosomal distribution, phylogenetic relationships, and the structural and functional features of their encoded proteins. Phylogenetic clustering classified the CaAHL proteins into six distinct subgroups. Transcriptome profiling revealed widespread expression of CaAHL genes across diverse tissues—including roots, stems, leaves, flowers, seeds, pericarp, placenta, and fruits—at various developmental stages. Quantitative real-time PCR further demonstrated that CaAHL1, CaAHL33, and CaAHL23 exhibited consistently high expression throughout flower bud development, whereas CaAHL36 showed preferential upregulation at early bud development stages. Expression profiling under hormone treatments and abiotic stresses indicated that CaAHL36 and CaAHL23 are auxin-inducible but are repressed by ABA, cold, heat, salt, and drought stress. Subcellular localization assays in Nicotiana benthamiana leaf epidermal cells showed that both CaAHL36 and CaAHL23 were predominantly localized in the nucleus, with faint expression also detected in the cytoplasm. Collectively, this study provides foundational insights into the CaAHL gene family, laying the groundwork for future functional investigations of these genes in pepper. Full article

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses. It often exhibits combined infection with white clover mosaic virus (WCMV) and occurs with a synergistic effect at 3:1 (AMV: WCMV). This study sought to clarify whether this synergistic effect is related to the molecular mutation of the coat protein (CP) sequences of the two viruses and their interactions, as well as the effect of the WCMV CP concentration on infection with AMV. This study identified and analyzed the CP sequences of two viruses after the co-infection of AMV and WCMV in Nicotiana benthamiana and found that the CP sequences of the two viruses mutated after co-infection with AMV and WCMV compared with the sequences from separate single infections with each virus. The mutation rate of the nucleotide bases was 7.66% and 3.37% in the Co-AMV CP and Co-WCMV CP, respectively, and 9.05% and 5.77% in the amino acid, respectively. The effect of WCMV CP and AMV at different proportions antagonistically affected infection with AMV when the proportion of WCMV CP: AMV was 3:1, 2:1, and 1:1. These proportions of treatment alleviated the symptoms caused by infection with N. benthamiana and reduced the relative expression of the AMV CP by 0.56, 0.47, and 0.76-fold, respectively, compared with single infection by AMV. Thus, the CP sequences of both viruses mutated after the co-infection of AMV and WCMV, and a proportion of WCMV CP: AMV of 3:1, 2:1, and 1:1 inhibited infection by AMV. Full article

Plant species harbor diverse rhizospheric bacteria within their resilient root zones, serving as a valuable reservoir of bioactive microorganisms with strong potential for natural antifungal and plant growth-promoting applications. This study aimed to investigate the antagonistic potential of Bacillus zhangzhouensis, isolated from Zygophyllum oxianum in the Aral Sea region, Uzbekistan, against the fungal pathogen Cytospora mali. Due to its strong antifungal activity, B. zhangzhouensis was selected for bioactive compound profiling. Methanolic extracts were fractionated via silica and Sephadex gel chromatography, followed by antifungal screening using the agar diffusion method. A highly active fraction (dichloromethane/methanol, 9:1) underwent further purification, yielding twelve antifungal sub-fractions. Mass spectrometry analysis across positive and negative ion modes identified 2475 metabolites, with polar solvents—particularly methanol—enhancing compound recovery. Refinement using Bacillus-specific references identified six known antibiotics. Four pure compounds were isolated and structurally characterized using NMR: oleanolic acid, ursolic acid, cyclo-(Pro-Ser), and uracil. Their growth regulatory activity was assessed on Amaranthus retroflexus, Nicotiana benthamiana, triticale, and Triticum aestivum at concentrations of 5, 20, 100, and 500 mg L−1. All compounds negatively affected root growth in a concentration-dependent manner, especially in monocots. Interestingly, some treatments enhanced stem growth, particularly in N. benthamiana. These findings indicate that B. zhangzhouensis produces diverse bioactive compounds with dual antifungal and plant growth-modulatory effects, highlighting its potential as a biocontrol agent and a source of natural bioactive compounds. Full article

Fusarium wilt, caused by Fusarium solani, is a devastating disease that leads to significant losses in ginger (Zingiber officinale) crops worldwide. To explore the molecular mechanisms underlying F. solani infection and disease progression, we performed a comparative transcriptome analysis of ginger rhizomes during storage, comparing inoculated and non-inoculated samples. A total of 647 and 6398 DEGs were identified in the 1.5- and 2-day infection groups, respectively. KEGG analysis revealed that most DEGs were enriched in the plant–pathogen interaction pathway, with both PTI and ETI being activated. Six DEGs in this pathway were validated by qRT-PCR at two time points, showing a strong correlation with FPKM values from the transcriptome data. Furthermore, transient expression analysis in Nicotiana benthamiana leaves demonstrated that overexpressing ZoCEBiP1 helped scavenge excess ROS, thereby reducing disease severity. Transcriptional profiling of DEGs in the plant–pathogen interaction pathway revealed significant changes in genes involved in ROS and NO metabolism. In F. solani-infected ginger rhizomes, levels of H₂O₂ and O₂⁻ were elevated, along with increased activities of antioxidant enzymes (POD, CAT, SOD, and APX) and higher NO content and NOS activity. These findings elucidated the early defense response of ginger rhizomes to F. solani infection and provided insights for developing effective strategies to manage fungal diseases. Full article

Soil salinization threatens agriculture by inducing osmotic stress, ion toxicity, and oxidative damage. SnRK2 genes are involved in plant stress responses, but their roles in salt stress response regulation of tobacco remain unclear. Through genome-wide analysis, we identified 54 SnRK2 genes across four Nicotiana species (N. tabacum, N. benthamiana, N. sylvestris, and N. tomentosiformis). Phylogenetic reconstruction clustered these genes into five divergent groups, revealing lineage-specific expansion in diploid progenitors (N. tomentosiformis) versus polyploidy-driven gene loss in N. tabacum. In silico promoter analysis uncovered regulatory networks involving light, hormones, stress, and developmental signals, with prevalent ABA-responsive elements (ABREs) supporting conserved stress-adaptive roles. Structural analysis highlighted functional diversification through variations in intron–exon architecture and conserved kinase motifs. This study provides a genomic atlas of SnRK2 evolution in Nicotiana, offering a foundation for engineering salt-tolerant crops. Full article

LTR retrotransposons are widespread genomic elements that significantly impact genome structure and function. In Arabidopsis thaliana, the EVD LTR retrotransposon encodes a GAG protein essential for retrotransposon particle assembly. Here, we present a comprehensive analysis of the structural features, intracellular localization, and transcriptomic effects of the EVD GAG (evdGAG) protein. Using AlphaFold3, we identified canonical capsid (CA-NTD and CA-CTD) and nucleocapsid (NC) domains, with predicted disordered regions likely facilitating oligomerization. Transient expression of GFP-tagged evdGAG in protoplasts of A. thaliana and distant plant species (Nicotiana benthamiana and Helianthus annuus) revealed the formation of multiple large cytoplasmic aggregates resembling retrosomes, often localized near the nucleus. Stable overexpression of evdGAG in wild-type and ddm1 mutant backgrounds induced significant transcriptomic changes, including up-regulation of stress response and defense-related genes and downregulation of photosynthesis and chloroplast-associated pathways. Importantly, genes linked to stress granule formation were also up-regulated, suggesting a role for evdGAG in modulating cellular stress responses. Our findings provide novel insights into the cellular and molecular properties of plant retrotransposon GAG proteins and their influence on host gene expression. Full article

Therapeutic antibodies with lambda light chains (λ-Abs) are underrepresented compared to kappa light chains (κ-Abs). Here, we evaluated two SARS-CoV-2-specific monoclonal antibodies (mAbs) that exhibit high (P5C3) and low (H4) antigen binding as κ and λ variants. mAbs expressed in glycoengineered Nicotiana benthamiana did not show differences in expression levels, glycosylation, and antigen binding, while κ-Abs exhibited slightly increased thermodynamic stability over λ-Abs. SARS-CoV-2 neutralization and IgG-FcγR immune complex studies revealed increased activities of H4 IgG1κ compared to H4 IgG1λ, with no differences observed between P5C3 variants. Our results indicate that constant light chain variability and Ab specificity contribute to Ab features, a fact that should be considered in engineering therapeutics. Full article