Nicotiana Benthamiana Research Articles

Abstract Viruses encode a limited number of proteins and often expropriate host factors for their successful infection and propagation. The molecular composition of the complex used for synthesis of the viral subgenomic (sg) RNAs remains largely unknown. In this study, an RNA motif (45 nt), equivalent to the sequence coding for the N-terminal portion of the tobamovirus coat protein (CP), known to be an enhancer-like element (tentatively designated as EL1) of the CP sg promoter, was used as a bait to capture bound proteins. Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1), a host defense factor with redox activity, was identified and its enzymatic active site was determined to be covered by EL1, thus blocking its reductase activity. In addition, the virus replicase Hel domain interacted with NbTRXh1, resulting in a decrease in NbTRXh1 protein accumulation in N. benthamiana . Thus, the viral RNA element and viral protein integrated their suppression of NbTRXh1 to facilitate viral CP sgRNA synthesis. Understanding this multiplexed regulatory mechanism between tobamovirus EL1 and associated proteins will assist in designing strategies for virus resistance and for optimization of the production of exogenous proteins under the control of the viral sg promoter.

The pathogen Pseudomonas syringae pv. tomato DC3000 suppresses plant immunity via type III effectors (T3Es). The majority of current research has focused on specific effectors targeting a single molecular pattern-triggered immune pathway, leaving a knowledge gap regarding a systems-level immune signaling network in plants. We employed the effectorless polymutant D36E, along with a modular library expressing 13 key T3Es individually, and performed an extensive transcriptomic analysis, integrated with genetic analysis and immune response assays, to identify D36E-induced and repressed by effector (DIRE) genes and associated pathways in Nicotiana benthamiana. The results revealed that the key T3Es collectively perturbed 19% of the N. benthamiana genome. DIRE genes were enriched for immune components, including pattern recognition receptors, kinase cascades, and transcription factors. Of 10 DIRE genes validated by qPCR, all were upregulated by D36E but suppressed by various pathogen effectors. Six of ten DIRE genes were unresponsive to flg22, elf18, or csp22, indicating that DIRE activation bypasses canonical molecular patterns. Silencing seven individual DIRE genes significantly boosted the virulence of D29E+8, a polymutant carrying minimal functional T3E repertoire. This work unveils a set of effector-targeted hubs beyond canonical pattern recognition, providing insights into layered plant defense and complex plant-pathogen signaling dynamics.

Natural variation in the promoter of TaHY5 enhances anthocyanin biosynthesis via upregulated transcription of TaF3H and TaANS in purple grain wheat (Triticum aestivum L.).

miRNA rco-MIR395a-p5_1ss8TC suppresses CsOMT957 expression leading to reduction in cannflavin A accumulation in Cannabis sativa L.

Carotenoids and anthocyanins are the two major classes of pigments in plants and are bioactive compounds with nutritional value in fruit crops. The color diversity in citrus (Citrus spp.) is attributed to the varying levels and composition of carotenoids and anthocyanins. In blood orange (Citrus sinensis) fruits, which accumulate both carotenoids and anthocyanins, these pigments exhibit coordinated changes during ripening; however, the mechanisms underlying this accumulation remain unclear. Here, we reveal that B-BOX DOMAIN PROTEIN 24 (CsBBX24) regulates both carotenoid and anthocyanin biosynthesis during the ripening of blood orange fruits. CsBBX24 was co-expressed with the carotenoid biosynthesis gene PHYTOENE SYNTHASE 1 (CsPSY1) and the anthocyanin regulatory gene CsRuby1, which encodes a MYB-domain protein during fruit ripening. Ectopic expression of CsBBX24 in Hongkong kumquat (C. hindsii), tomato (Solanum lycopersicum), and apple (Malus domestica) calli promoted the accumulation of carotenoids and anthocyanins. An electrophoretic mobility shift assay and transient dual-luciferase assays in Nicotiana benthamiana leaves demonstrated that CsBBX24 can bind to and activate the CsPSY1 promoter for carotenoid biosynthesis and the CsRuby1 promoter for regulation of anthocyanin biosynthesis. Our findings have elucidated the role of CsBBX24 in promoting pigment accumulation during fruit ripening in blood orange, providing a valuable target gene for molecular breeding to improve citrus coloration quality.

Carya illinoinensis is a forestry species with significant economic and ecological importance. However, climate change may increase the frequency and severity of drought stress and natural disturbances, which could influence C. illinoinensis yields. Enhancing its drought resistance is therefore crucial for sustainable cultivation. The plant homeodomain (PHD)-finger gene family, widely identified across multiple species, plays crucial regulatory roles in stress responses. Notably, the Alfin-like (AL) subfamily has been demonstrated to participate in plant stress resistance. In this study, 62 CiPHDs were identified, of which 8 belonged to the AL subfamily. Comprehensive bioinformatics analyses were performed, and integrative analyses of transcriptome data and qRT-PCR confirmed that CiPHD14 and CiPHD61 exhibited upregulation in response to drought. Subcellular localization confirmed the nuclear positioning of CiPHD14 and CiPHD61, and yeast assays revealed no independent transcriptional activation. Dual-luciferase assay (Dual-LUC) and electrophoretic mobility shift assay (EMSA) revealed the binding of CiPHD14 and CiPHD61 to the G-rich element (5'-GTGGAG-3'). Transient overexpression of CiPHD14 in Nicotiana benthamiana significantly upregulated drought-responsive genes compared to controls. In addition, transgenic Arabidopsis thaliana lines with CiPHD14 overexpression showed enhanced drought resistance compared with wild-type (WT). This work elucidates CiPHD structures and functions while providing gene resources for breeding drought-tolerant C. illinoinensis.

TuNHL1, an NDR1/HIN1 like gene, is essential for YrU1-mediated stripe rust resistance and enhances powdery mildew resistance in plants.

The nuclear transcription factor NFYA3_0 promotes MTA-mediated m6A modification of Potato virus Y genomic RNA to confer antiviral resistance in Nicotiana benthamiana.

Zanthoxylum armatum HD-ZIP II Transcription Factor ZaHB15 Induces Flowering and Negatively Regulates Plant Growth and Drought Tolerance.

This article provides a comprehensive analysis of Virus-Induced Gene Silencing (VIGS), which is an effective tool for studying the functional genomics of organisms that are poorly amenable to genomic editing. The VIGS method is grounded in the plant’s post-transcriptional gene silencing (PTGS) machinery and utilizes recombinant viral vectors to trigger systemic suppression of endogenous plant gene expression, leading to visible phenotypic changes that enable gene function characterization. This article details the application of VIGS in model organisms (Arabidopsis thaliana, Nicotiana benthamiana) and a wide range of crops, with a special focus on the Solanaceae family, particularly pepper (Capsicum annuum L.). This review analyzes the design and structural elements of viral vectors used for VIGS, such as Tobacco Rattle Virus (TRV), Broad Bean Wilt Virus 2 (BBWV2), Cucumber Mosaic Virus (CMV), geminiviruses (CLCrV, ACMV), and satellite virus-based systems. It also critically examines the key factors that determine silencing efficiency. These factors encompass insert design, agroinfiltration methodology, plant developmental stage, agroinoculum concentration, plant genotype, and environmental factors (temperature, humidity, photoperiod). Particular attention is given to optimization strategies, such as the use of viral suppressors of RNA silencing (VSRs). This article concludes with the achievements in using VIGS to identify pepper genes governing fruit quality (color, biochemical composition, pungency), resistance to biotic (bacteria, oomycetes, insects) and abiotic (temperature, salt, osmotic stress) factors, as well as genes regulating plant architecture and development. The results obtained demonstrate the advantages and limitations of VIGS, alongside future perspectives for its integration with multi-omics technologies to accelerate breeding and advance functional genomics studies in pepper.

Fusarium proliferatum causes banana crown rot and produces fumonisins, posing a threat to environmental safety and public health. Here, we report the 43.6-Mb complete genome sequence of F. proliferatum, containing 3026 genes encoding putative small secreted protein (SSP) candidates including 206 effectors. A list of pathogenesis-related genes was identified based on the transcriptome of F. proliferatum during infecting banana fruit. Furthermore, 10 putative SSPs were validated as secreted proteins using a yeast secretion system. Among these, Fp_G08764 and Fp_G03587 triggered cell death, while Fp_G13932 inhibited cell death in Nicotiana benthamiana leaves. Other candidates (Fp_G11318, Fp_G11726, Fp_G09727, Fp_G13547, Fp_G09042, Fp_G08494, and Fp_G11459) exhibited no significant effect on cell death. Additionally, Fp_G11318, Fp_G08764, and Fp_G13932-deletion mutants exhibited reduced pathogenicity on banana peel and N. benthamiana, indicating their essential roles in full virulence. Our results shed more light on the infection mechanism of F. proliferatum, contributing to develop effective techniques to control fungal disease.

Introduction Sugarcane mosaic virus (SCMV, Potyvirus ) causes mosaic diseases and seriously threatens sugarcane production. Potyviral 6K2 protein plays a key role in viral infections. We previously screened a tetraspanin (TET)-like protein that interacts with SCMV-6K2 from a sugarcane cDNA yeast library. Although TETs have been extensively studied in response to viral infections in animals, the TET gene family in sugarcane and its role in SCMV infections remain largely unknown. This study aimed to identify the TET genes in sugarcane and determine their response to SCMV infection. Methods We employed genome-wide identification, phylogenetic analysis, real-time quantitative PCR (RT-qPCR), subcellular localization, and multiple protein–protein interaction assays to characterize TETs and their interactions with viral 6K2 proteins. Results We identified 35, 113, 73, and 17 TETs in the genomes of Saccharum sp ontaneum , sugarcane cultivar R570, sugarcane cultivar Xintaitang 22 (XTT22), and Nicotiana benthamiana , respectively. Phylogenetic tree analysis classified the TETs into nine distinct groups. Nine TET genes were cloned from XTT22 and designated ScTET2 , ScTET8 , ScTET13 , ScTET23 , ScTET34 , ScTET55 , ScTET67 , ScTET78 , and ScTET96 . RT-qPCR demonstrated the differential expression of these genes following SCMV infection. Furthermore, subcellular localization assays revealed that they were mainly localized to the plasma membrane (PM), except for ScTET2 and ScTET8, which were localized in the cytoplasm and formed irregular spherical structures of different sizes. Yeast two-hybrid (Y2H), bimolecular fluorescent complementation, and luciferase complementation assays revealed extensive interactions between the ScTETs and SCMV-6K2, primarily in the PM. Y2H assays also showed that TETs of Arabidopsis and N. benthamiana extensively interacted with the 6K2 protein of turnip mosaic virus. Discussion This study reveals a potential mechanism by which potyviruses employ 6K2 to interact with TETs to establish infection in host plants, thus highlighting potential molecular targets for engineering sugarcane resistance against SCMV.

Modification of proteins by ubiquitin is a dynamic and reversible process. It is unclear whether rice stripe virus (RSV) can modulate the plant deubiquitination pathway. In this study, we found that RSV infection can specifically upregulate the expression of the deubiquitinase NbUBP16. Further analysis revealed that NbUBP16 stabilizes serine hydroxymethyltrasferase (SHMT1) by binding to NbSHMT1 and removing its polyubiquitination modification mediated by E3 ligase MEL, which inhibits downstream SHMT1-mediated ROS accumulation and thereby facilitates RSV infection. Our findings provide new insights into the molecular arms race between pathogens and plants, demonstrating how a plant virus can undermine plant defenses by hijacking host deubiquitination pathways.Supplementary InformationThe online version contains supplementary material available at 10.1007/s44154-025-00265-2.

The brown planthopper (Nilaparvata lugens, BPH) is a notorious sap-sucking pest on rice (Oryza sativa) causing substantial yield losses. During BPH oviposition, its egg-associated secretions trigger rice defense responses; however, the role of their specific components in plant immune modulation remains poorly understood. This study aimed to investigate the effect of the oviduct-secreted protein 1 (NlOdsp1) from BPH egg-associated secretions on plant defense. NlOdsp1, a planthopper-specific gene, is predominantly expressed in the female adults and oviduct of BPH. NlOdsp1 activates cell death and reactive oxygen species production in Nicotiana benthamiana and impairs the performance of the whitefly and aphid. Moreover, its 39 amino acid peptide is recognized by plants as a minimal immunogenic epitope. NlOdsp1 significantly induces H2O2 accumulation in rice and enhances rice resistance to BPH. NlOdsp1 from BPH egg-associated secretions serves as a critical and unavoidable elicitor, triggering plant resistance to various insects. The essential role of this protein for BPH reproduction may preclude evolutionary adaptations to evade detection by rice. © 2025 Society of Chemical Industry.

BackgroundTerpenoids constitute a diverse group of primary and secondary metabolites that are extensively distributed in living organisms and play key roles in growth, development, and environmental adaptation. Terpenoids are derived from two isomeric precursors that are interconverted by isopentenyl-diphosphate delta-isomerase (IDI), in both the plastids and cytoplasm of plants. The plastidial pathway supplies precursors for diterpenoids and carotenoids, whereas the cytoplasmic pathway provides precursors for sesquiterpenoids and triterpenoids. A family of terpene synthases (TPSs) produce most terpenoids such as sesquiterpenes, hemiterpenes, monoterpenes, diterpenes and sesterterpenes, which in allotetraploid peanut (Arachis hypogaea L.) have been relatively underexplored.ResultsIn this study, 77 AhTPS genes were identified in the peanut genome and phylogenetically classified into five subfamilies. These AhTPSs are organized in clusters across chromosomes and exhibit conserved gene structures and motifs within each subfamily. AhTPSs in the TPS-c and -e/f subfamilies, specifically copalyl diphosphate synthase (CPS) and kaurene synthase (KS), were localized to plastids through transient expression in Nicotiana benthamiana leaves. The expression of AhCPS3 was detected a significant increase in response to abscisic acid (ABA) and methyl jasmonate (MeJA), which was notably distinct from the expression patterns of other AhCPSs and AhKS. Furthermore, 12 terpenoids were identified during seed development. Module-trait correlation analysis disclosed that the expression levels of genes encoding AhCPSs (involved in diterpene biosynthesis) and phytoene synthases (AhPSYs, involved in carotenoid biosynthesis) were significantly correlated with the abundances of soyasaponins (triterpenoids) during seed development in peanut. Additionally, the gene encoding β-amyrin synthase (AhβAS), which produces the backbone of triterpenoids, was identified in a significant module and was also induced by ABA and MeJA. Protein-protein interaction networks indicated AhCPSs, AhPSYs, and AhβAS shared a common interacting protein, AhIDI.ConclusionsThese findings provide valuable insights into the potential cross-talk in terpenoid biosynthesis across different cellular compartments.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12013-x.

Cleavage of Beclin 1 by metacaspase 1 activates antiviral autophagy in plants.

Plants deploy intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) to detect pathogenic effectors and trigger effector-triggered immunity (ETI). Helper NLRs, such as N Required Gene 1 (NRG1), act downstream of sensor NLRs to transduce immune signals. NRG1s have been implicated to oligomerize to form Ca²⁺-permeable channels at the plasma membrane, playing a critical role in TIR-NLR (TNL)-mediated ETI. Although its biochemical function is well understood, the transcriptional regulation of NRG1 remains unclear. Here, we show that ETI activation by various TNLs upregulates NbNRG1 expression in Nicotiana benthamiana. We identify NbWRKY57 as a key transcriptional activator that directly binds the W-box motif in the NbNRG1 promoter. Knockdown or knockout of NbWRKY57 compromises TNLRoq1-mediated hypersensitive response (HR) cell death and pathogen resistance, while its overexpression restores mutant HR phenotypes, underscoring its positive regulatory role in TNL-mediated ETI. Moreover, NbWRKY57 is essential not only for TNLRoq1-mediated ETI but also for ETI mediated by other TNLs, including N and RPP1. We further demonstrate that calcium signaling contributes to NbNRG1 activation and TNL-mediated ETI. Together, these findings uncover a previously unknown regulatory mechanism controlling helper NLR transcription and provide insights into the transcriptional activation of NRG1 during ETI.

Ubiquitin-conjugating enzyme (UBC) plays a significant role in plant hormone signal transduction. In this study, we observed that TuMV infection markedly upregulates UBC mRNA expression, suggesting a close association between UBC and viral infection. Using Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to downregulate UBC expression in Nicotiana benthamiana, we found that UBC-silenced plants exhibited enhanced susceptibility to TuMV compared with control plants. Conversely, transient overexpression of UBC protein suppressed viral propagation. Further analysis by reverse transcription quantitative PCR (RT-qPCR) revealed a substantial downregulation in the expression of SA and JA biosynthetic genes in UBC-silenced plants. Accordingly, liquid chromatography–tandem mass spectrometry (LC-MS/MS) confirmed a marked decrease in the accumulation of the corresponding hormones. Exogenous application of SA or JA partially restored antiviral resistance in UBC-silenced plants, indicating that hormonal deficiency contributes to enhanced viral susceptibility. Collectively, our results demonstrate that UBC positively regulates SA and JA biosynthesis. UBC silencing impairs both SA- and JA-mediated defense pathways, thereby facilitating viral infection in N. benthamiana.

Citrus fruit de-greening, a critical process for quality and marketability, is governed by chlorophyll degradation, yet its regulatory mechanisms remain poorly understood. Here, we identify FcrNAC22, a NAC transcription factor (TF) in kumquat (Fortunella crassifolia), as a pivotal regulator of chlorophyll catabolism activated in response to de-greening cues. FcrNAC22 functions as a transcriptional activator induced by red light, abscisic acid (ABA), and ethephon, with both its mRNA and protein levels peaking at the fruit colour-breaker stage. The overexpression of FcrNAC22 in Nicotiana benthamiana leaves, tomato (Solanum esculentum), and kumquat fruits expedited chlorophyll breakdown and upregulated the expression of chlorophyll catabolic genes (CCGs). In contrast, the interference with FcrNAC22 expression in kumquat fruits impeded chlorophyll degradation and suppressed the transcription of CCGs. Protein-DNA interaction assays verified that FcrNAC22 directly binds to and activates the promoters of chloroplast-localized STAY-GREEN (FcrSGR), chlorophyllase (FcrCLH), pheophytinase (FcrPPH), pheophorbide a oxygenase (FcrPAO), and NON-YELLOW COLORING1 (FcrNYC1), which explains the de-greening phenotypes witnessed in the aforementioned transgenic FcrNAC22 lines. These findings not only reveal FcrNAC22 as a crucial integrator of environmental and developmental signals, but also provide a theoretical basis for manipulating fruit de-greening in citrus and related species.

Light-induced soil legacy alleviates continuous cropping adversity on Nicotiana benthamiana growth