Search Results (261)

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

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

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

Plant Molecular Farming (PMF) capitalizes on the unique properties of plants as bioreactors to efficiently produce valuable proteins, pharmaceuticals, and enzymes. This review emphasizes the critical role of transient expression systems, particularly in Nicotiana benthamiana, due to its susceptibility to various pathogens. Viral vector-based transient expression has proven essential during health emergencies like COVID-19, enabling rapid recombinant protein production. The review also evaluates different transient expression platforms and highlights their applications in biopharmaceutical production, education, synthetic biology, and gene editing. Advances in viral vector modification, hydroponics, and Controlled Environment Agriculture (CEA) are presented as transformative innovations enhancing scalability and regulatory compliance. Furthermore, glycoengineering advancements broaden the range of producible biopharmaceuticals, improving global medication access. By exploring these advancements, this review underscores the vast potential of transient expression systems to meet dynamic scientific and market demands, positioning PMF as a vital component in modern biotechnology. Full article

Tomato brown rugose fruit virus (ToBRFV) is an important emerging virus that poses a serious threat to the global agricultural economy. Ubiquitination is one of the key post-translational protein modification types in plant responses to biotic stress, but the extent to which ToBRFV infection alters the overall ubiquitination status has not been reported. This study conducted integrated ubiquitome and proteome analyses of Nicotiana benthamiana leaves infected with ToBRFV and identified differentially ubiquitinated proteins. A total of 346 lysine sites on 302 identified proteins were found to be affected, with 260 sites exhibiting upregulated ubiquitination levels in 224 proteins and 86 sites showing downregulated ubiquitination levels in 80 proteins. The differentially ubiquitinated proteins were primarily localized in the cytoplasm (29%), nucleus (18%), plasma membrane (8.9%), mitochondria (5.1%), and chloroplasts (4.6%). Fourteen conserved ubiquitination motifs, including ENNNK, ENNK, SK, and KNG, were identified. Furthermore, enrichment analysis indicated that ToBRFV infection induces an increase in the ubiquitination levels of proteins associated with ion transport, MAPK signaling pathways, and plant hormone signal transduction, while the ubiquitination levels of proteins related to carbon metabolism and secondary metabolite synthesis decreased. Functional analysis of the three differentially ubiquitinated proteins revealed that a RING/U-box superfamily protein negatively regulates ToBRFV infection. Our work provides the first systematic analysis of the ubiquitination profile in N. benthamiana leaves following ToBRFV infection, providing important resources for further studies on the regulatory mechanisms of ubiquitination in plant responses to ToBRFV. Full article

Marssonina coronaria is the causal agent of apple blotch, which poses a significant threat to apple production worldwide. Here, Illumina and Oxford Nanopore sequencing were combined to generate a high-quality M. coronaria YL1 genome assembly (54.5 Mb, 23 contigs). Based on genome annotation, 97 candidate effector proteins (CEPs) were identified, and 61 CEPs were successfully cloned for functional analysis. Transient expression assays in Nicotiana benthamiana revealed that eight CEPs significantly suppressed BAX-induced cell death, with McCEP12, McCEP23, McCEP24, and McCEP52 concurrently inhibiting flg22-triggered reactive oxygen species bursts. Two signal peptide-dependent cell death-inducing effectors were identified: McNLP1, containing an NPP1 domain, and McCEP3. McCEP3 exhibited evolutionary conservation within Ascomycota, with its homologous gene VmMcCEP3 from Valsa mali inducing cell death in N. benthamiana. McEP03-triggered cell death was independent of BAK1/SOBIR1 receptor kinases. This study provides a high-quality genomic resource for M. coronaria and sheds light on the mechanisms by which its CEPs modulate host immunity, offering new insights into the molecular interactions between the pathogen and its host. Full article

Jujube witches’ broom (JWB) disease, caused by Candidatus Phytoplasma ziziphi (Ca. P. ziziphi), severely threatens the production of Chinese jujube (Ziziphus jujuba Mill.). Emerging evidence highlights the critical role of phytoplasma-secreted effectors in pathogenesis, though few have been functionally characterized. Here, we identified a Sec-dependent effector, JWB790, from Ca. P. ziziphi, which was shown to suppress plant immunity. Through transient expression assays in Nicotiana benthamiana, pathogen inoculation assays, the generation of transgenic Arabidopsis thaliana plants, and RNA-seq-based transcriptomic profiling, we systematically investigated the virulence function of JWB790. Our findings revealed that JWB790 is highly expressed in JWB-infected tissues. The transient expression of JWB790 in N. benthamiana suppressed BAX-induced cell death and H₂O₂ accumulation. Furthermore, the stable overexpression of JWB790 in A. thaliana compromised disease resistance, accompanied by reduced H₂O₂ accumulation and callose deposition triggered by flg22. Additionally, the RNA-seq analysis of JWB790 transgenic Arabidopsis plants indicated that the overexpression of JWB790 altered the expression of biotic stress-related genes. In summary, JWB790 is a virulence factor that suppresses plant immunity and promotes pathogen proliferation. These results advance our understanding of Ca. P. ziziphi pathogenesis. Full article

Squash leaf curl China virus (SLCCNV) belongs to the species Begomovirus cucurbitachinaense in the genus Begomovirus and can infect some Cucurbitaceae crops except for watermelon (Citrullus lanatus). In this study, watermelon plants showing symptoms typical to begomovirus infection in field were observed in Zhejiang Province of China, and SLCCNV presence was identified through PCR and next-generation sequencing (NGS). The pairwise sequence identity of the DNA-A genome shows that SLCCNV watermelon isolate belongs to the SLCCNV/CN strain and shares 96% nucleotide identity with the previously sequenced SLCCNV/CN. An infectious clone of SLCCNV watermelon isolate was constructed using the tandem repeat fragment method. Through agrobacterium-mediated inoculation, the clone could induce systemic infection with typical symptoms in watermelon, melon (Cucumis melo), squash (Cucurbita pepo), pumpkin (Cucurbita maxima), wax gourd (Benicasa hispida), cucumber (Cucumis sativus), and N. benthamiana. It was further demonstrated that the progeny virions derived from the cloned watermelon isolate could be transmitted by whitefly rather than the sap. To the best of our knowledge, this is the first report of a natural infection of SLCCNV on watermelon in China, and the first complete report on the molecular characteristics and pathogenicity of watermelon-infecting SLCCNV in the world. Full article

The role of WRKY transcription factor proteins in plant defense reactions against fungal and bacterial pathogens is well studied, but less information is available about plant–virus interactions. We observed the rapid and strong activation of the transcription factor gene, CaWRKY2, in pepper leaves following inoculation with Obuda pepper virus (ObPV). In contrast, CaWRKY2 was only weakly induced by pepper mild mottle virus (PMMoV) inoculation. To carry out a functional analysis of CaWRKY2, the gene was transiently overexpressed in Nicotiana benthamiana leaves by agroinfiltration. Four days later, CaWRKY2-overexpressing and empty vector control leaves were inoculated with tobacco mosaic virus (TMV). Transiently overexpressing CaWRKY2 did not affect the replication rate of TMV in the inoculated leaves. However, TMV inoculation up-regulated the expression of a pathogenesis-related gene (NbPR-1b) and a lipoxygenase (NbLOX1) gene significantly more strongly in N. benthamiana leaves overexpressing CaWRKY2 than in empty vector control leaves. Intriguingly, CaWRKY2 overexpression delayed (by 3 days) the development of systemic necrosis and plant death caused by TMV in N. benthamiana. These results suggest that CaWRKY2 is able to hinder the spread of TMV from inoculated leaves towards vascular tissues and systemic leaves in N. benthamiana. Full article

Lysozyme is an enzyme that hydrolyzes bacterial cell walls, which is functional for destroying the integrity of bacteria, enhancing the activity of immune cells, participating in immune signal transmission, helping to maintain the micro-ecological balance of the gastrointestinal tract, etc. Egg white lysozyme (EWL), as one of the typical representatives of lysozyme, is the most widely used enzyme in production so far, and is also one of the most complex structures of lysozyme. EWL also helps protect plants from fungal and bacterial diseases. Here, we report the effect of EWL on infections from plant viruses. The EWL gene was cloned and characterized. The EWL protein sequence analysis identified a conserved domain of lysozyme activity and the sharing of a 100% identical EWL protein from the Coturnix japonica lysozyme. Then, the EWL gene was cloned into the plant expression vector pEAQ-HT-DEST3 and transiently expressed in Nicotiana benthamiana (N. benthamiana). We found that EWL expression in N. benthamiana significantly contributed to infections by the turnip mosaic virus (TuMV) but not by the tobacco mosaic virus (TMV). Plants that transiently expressed EWL showed an obvious increase in resistance to Botrytis cinerea (B.cinerea). Our results suggested a new research point for the application of EWL on plant pathogen infections. Full article

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is the most destructive disease of the banana. Effectors play a crucial role in Foc TR4–banana interaction; however, only a few effectors have been functionally characterized. Our previous secretome studies on Foc TR4 highlighted an uncharacterized protein without any conserved domains (named FoUpe9), which was predicted to be a candidate effector. Herein, bioinformatics analysis showed that FoUpe9 was highly conserved among Fusarium species. FoUpe9 was highly induced during the early infection stages in the banana. A yeast signal sequence trap assay showed that FoUpe9 is a secretory protein. FoUpe9 could inhibit cell death and ROS accumulation triggered by BAX through the Agrobacterium-mediated Nicotiana benthamiana expression system. Subcellular location showed that FoUpe9 was located in the nucleus and cytoplasm of N. benthamiana cells. Deletion of the FoUpe9 gene did not affect mycelial growth, conidiation, sensitivity to cell-wall integrity, or osmotic and oxidative stress, but significantly attenuated fungal virulence. FoUpe9 deletion diminished fungal colonization and induced ROS production and expression of SA-related defense genes in banana plants. These results suggest that FoUpe9 enhances Foc TR4 virulence by inhibiting host immune responses and provide new insights into the functions of the uncharacterized proteins, further enhancing our understanding of effector-mediated Foc TR4 pathogenesis. Full article