Nicotiana Tabacum Cv Research Articles

Transcriptome disclosure of hormones inducing stigma exsertion in Nicotiana tabacum by corolla shortening

BackgroundStigma exsertion is an essential agricultural trait that can promote cross-pollination to improve hybrid seed production efficiency. However, the molecular mechanism controlling stigma exsertion remains unknown.ResultsIn this study, the Nicotiana tabacum cv. K326 and its two homonuclear-heteroplasmic lines, MSK326 (male-sterile) and MSK326SE (male-sterile and stigma exserted), were used to investigate the mechanism of tobacco stigma exsertion. A comparison of the flowers between the three lines showed that the stigma exsertion of MSK326SE was mainly due to corolla shortening. Therefore, the corollas of the three lines were sampled and presented for RNA-seq analysis, which found 338 candidate genes that may cause corolla shortening. These genes were equally expressed in K326 and MSK326, but differentially expressed in MSK326SE. Among these 338 genes, 15 were involved in hormone synthesis or signal transduction pathways. Consistently, the content of auxin, dihydrozeatin, gibberellin, and jasmonic acid was significantly decreased in the MSK326SE corolla, whereas abscisic acid levels were significantly increased. Additionally, seven genes involved in cell division, cell cycle, or cell expansion were identified. Protein-protein interaction network analysis identified 45 nodes and 79 protein interactions, and the largest module contained 20 nodes and 52 protein interactions, mainly involved in the hormone signal transduction and pathogen defensive pathways. Furthermore, a putative hub gene coding a serine/threonine-protein kinase was identified for the network.ConclusionsOur results suggest that hormones may play a key role in regulating tobacco stigma exsertion induced by corolla shortening.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Plant-Produced Therapeutic Crizanlizumab Monoclonal Antibody Binds P-Selectin to Alleviate Vaso-occlusive Pain Crises in Sickle Cell Disease.

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks. Anti-P-selectin antibody Crizanlizumab (Crimab) has been used to treat SCD. In this study, the heavy and light chain (HC and LC) genes of anti-P-Selectin antibody Crimab were cloned into a plant expression binary vector. The HC gene was under control of the duplicated 35S promoter and nopaline synthase (NOS) terminator, whereas the LC gene was under control of the potato proteinase inhibitor II (PIN2) promoter and PIN2 terminator. Agrobacterium tumefaciens LBA4404 was used to transfer the genes into the tobacco (Nicotiana tabacum cv. Xanthi) plant. In plants the genomic PCR and western blot confirmed gene presence and expression of HC and LC Crimab proteins in the plant, respectively. Crimab was successfully purified from transgenic plant leaf using protein A affinity chromatography. In ELISA, plant-derived Crimab (CrimabP) had similar binding activity to P-selectin compared to mammalian-derived Crimab (CrimabM). In surface plasmon resonance, the KD (dissociation binding constant) and response unit values were lower and higher than CrimabP, respectively. Taken together, these results demonstrate that the transgenic plant can be applied to produce biofunctional therapeutic monoclonal antibody.

MdARF3 switches the lateral root elongation to regulate dwarfing in apple plants.

Apple rootstock dwarfing and dense planting are common practices in apple farming. However, the dwarfing mechanisms are not understood. In our study, the expression of MdARF3 in the root system of dwarfing rootstock 'M9' was lower than in the vigorous rootstock from Malus micromalus due to the deletion of the WUSATAg element in the promoter of the 'M9' genotype. Notably, this deletion variation was significantly associated with dwarfing rootstocks. Subsequently, transgenic tobacco (Nicotiana tabacum) cv. Xanthi was generated with the ARF3 promoter from 'M9' and M. micromalus genotypes. The transgenic apple with 35S::MdARF3 was also obtained. The transgenic tobacco and apple with the highly expressed ARF3 had a longer root system and a higher plant height phenotype. Furthermore, the yeast one-hybrid, luciferase, electrophoretic mobility shift assays, and Chip-qPCR identified MdWOX4-1 in apples that interacted with the pMm-ARF3 promoter but not the pM9-ARF3 promoter. Notably, MdWOX4-1 significantly increased the transcriptional activity of MdARF3 and MdLBD16-2. However, MdARF3 significantly decreased the transcriptional activity of MdLBD16-2. Further analysis revealed that MdARF3 and MdLBD16-2 were temporally expressed during different stages of lateral root development. pMdLBD16-2 was mainly expressed during the early stage of lateral root development, which promoted lateral root production. On the contrary, pMmARF3 was expressed during the late stage of lateral root development to promote elongation. The findings in our study will shed light on the genetic causes of apple plant dwarfism and provide strategies for molecular breeding of dwarfing apple rootstocks.

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV)

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

Integrated transmission electron microscopy and proteomic analyses reveal the cytoarchitectural response to cucumber mosaic virus infection in tobacco

Cucumber mosaic virus (CMV) causes huge economic losses to agriculture every year; thus, understanding the mechanism of plant resistance to CMV is imperative. In this study, an integrated analysis of transmission electron microscopy (TEM) observations and proteomic results was used to identify cytoarchitectural differences in Nicotiana tabacum cv. NC82 (susceptible) and cv. Taiyan 8 (T.T.8; resistant) following infection with CMV. The TEM observations showed that the structure of the chloroplasts and mitochondria was severely damaged at the late stage of infection in NC82. Moreover, the chloroplast stroma and mitochondrial cristae were reduced and disaggregated. However, in T.T.8, organelle structure remained largely intact Selective autophagy predominated in T.T.8, whereas non-selective autophagy dominated in NC82, resembling cellular disorder. Proteomic analysis of T.T.8 revealed differentially expressed proteins (DEPs) mostly associated with photosynthesis, respiration, reactive oxygen species (ROS) scavenging, and cellular autophagy. Biochemical analyses revealed that ROS-related catalase, autophagy-related disulfide isomerase, and jasmonic acid and antioxidant secondary metabolite synthesis-related 4-coumarate:CoA ligase (Nt4CL) exhibited different trends and significant differences in expression in the two cultivars after CMV inoculation. Furthermore, mutant phenotyping verified that reduced Nt4CL expression impaired resistance in T.T.8. The identified DEPs are crucial for maintaining intracellular homeostatic balance and likely contribute to the mechanism of CMV resistance in tobacco. These findings increase our understanding of plant cytological mechanisms conferring resistance to CMV infection.

Physiological effects of Acmella ciliata essential oil on root development of Nicotiana tabacum

In this study, the essential oil of Acmella ciliata, characterized by the presence of significant amounts of spilanthol, α-phellandrene epoxide, and carvotanacetone, was assessed for its influence on the growth of Nicotiana tabacum cv. Xanthi, with the focus primarily on root hair density and primary root length. Following its extraction through microwave-assisted hydrodistillation, the oil was stored at 4°C in amber vials, distinguishable by its unique yellowish-reddish hue, with a refractive index of 1.3478 and a density of 0.847 g cm-3. Among the various dilutions evaluated, the undiluted oil (T3) and the dilution 1.5:0.5 (Oil:EtOH) (T6) demonstrated the most prominent effects. The T3 and T6 treatments markedly enhanced root hair numbers, with T6 additionally promoving root length compared to other treatments. Considering the presence of bioactive alkamides such as spilanthol in the oil, these compounds may have contributed to the observed root growth modulation. When compared against the positive control, affinin, Acmella ciliata essential oil displayed a more pronounced effect on root hair proliferation, while affinin predominantly boosted primary root elongation. The findings highlight the differential effects of the essential oil on specific plant growth parameters.

Simulation and optimization of nutrient uptake and biomass formation using a multi-parameter Monod-type model of tobacco BY-2 cell suspension cultures in a stirred-tank bioreactor.

Tobacco (Nicotiana tabacum) cv Bright Yellow-2 (BY-2) cell suspension cultures enable the rapid production of complex protein-based biopharmaceuticals but currently achieve low volumetric productivity due to slow biomass formation. The biomass yield can be improved with tailored media, which can be designed either by laborious trial-and-error experiments or systematic, rational design using mechanistic models, linking nutrient consumption and biomass formation. Here we developed an iterative experiment-modeling-optimization workflow to gradually refine such a model and its predictions, based on collected data concerning BY-2 cell macronutrient consumption (sucrose, ammonium, nitrate and phosphate) and biomass formation. The biomass formation was well predicted by an unstructured segregated mechanistic Monod-type model as long as the nutrient concentrations did not approach zero (we omitted phosphate, which was completely depleted). Multi-criteria optimization for sucrose and biomass formation indicated the best tradeoff (in a Paretian sense) between maximum biomass yield and minimum process time by reducing the initial sucrose concentration, whereas the inoculation biomass could be increased to maximize the biomass yield or minimize the process time, which we confirmed in calibration experiments. The model became inaccurate at biomass densities > 8 g L-1 dry mass when sucrose was almost depleted. We compensated for this limitation by including glucose and fructose as sucrose hydrolysis products in the model. The remaining offset between the simulation and experimental data might be resolved by including intracellular pools of sucrose, ammonium, nitrate and phosphate. Overall, we demonstrated that iterative models can be used to systematically optimize conditions for bioreactor-based processes.

Antioxidant and drought-acclimation responses in UV-B-exposed transgenic Nicotiana tabacum displaying constitutive overproduction of H2O2.

Hydrogen peroxide (H2O2) is an important molecule that regulates antioxidant responses that are crucial for plant stress resistance. Exposure to low levels of ultraviolet-B radiation (UV-B, 280-315nm) can also activate antioxidant defenses and acclimation responses. However, how H2O2 and UV-B interact to promote stress acclimation remains poorly understood. In this work, a transgenic model of Nicotiana tabacum cv Xanthi nc, with elevated Mn-superoxide dismutase (Mn-SOD) activity, was used to study the interaction between the constitutive overproduction of H2O2 and a 14-day UV-B treatment (1.75kJm-2 d-1 biologically effective UV-B). Subsequently, these plants were subjected to a 7-day moderate drought treatment to evaluate the impact on drought resistance of H2O2- and UV-dependent stimulation of the plants' antioxidant system. The UV-B treatment enhanced H2O2 levels and altered the antioxidant status by increasing the epidermal flavonol index, Trolox Equivalent Antioxidant Capacity, and catalase, peroxidase and phenylalanine ammonia lyase activities in the leaves. UV-B also retarded growth and suppressed acclimation responses in highly H2O2-overproducing transgenic plants. Plants not exposed to UV-B had a higher drought resistance in the form of higher relative water content of leaves. Our data associate the interaction between Mn-SOD transgene overexpression and the UV-B treatment with a stress response. Finally, we propose a hormetic biphasic drought resistance response curve as a function of leaf H2O2 content in N. tabacum cv Xanthi.

Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

Plastid dsRNA transgenes trigger phased small RNA-based gene silencing of nuclear-encoded genes.

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis.

Analysis of the Spatial Dispersion of Tomato Brown Rugose Fruit Virus on Surfaces in a Commercial Tomato Production Site

The tomato brown rugose fruit virus (ToBRFV) causes severe damage to tomato cultivars and has international economic importance. The harmful tobamovirus is easily mechanically transmissible and highly stable. An ongoing cultivation of infected tomato plants may lead to the spread of ToBRFV in and around the production area of the infested tomato farm. We conducted a study in which we collected a representative number of swab samples from various inanimate surfaces in greenhouses, packaging halls, and shared and private accommodations. In addition, numerous fabrics, such as outer clothing, bed linen, and items used by greenhouse workers, were tested. The infectivity of ToBRFV-contaminated surfaces was tested in bioassays using Nicotiana tabacum cv. Xanthi NN and confirmed using DAS-ELISA. The proportion of ToBRFV-contaminated surfaces varied among locations, from 48.7% in greenhouses to 0% in offices with limited access to staff. Samples from shared accommodation and private accommodation were 18.4% and 3.6% ToBRFV positive, respectively. Clothing and protective items were found to be highly contaminated with ToBRFV, and even around the sleeping area, infective ToBRFV was detected in a few apartments. This study provides evidence for the first time on how and where infectious ToBRFV can be spread by humans beyond the production area. To avoid further dissemination, strict hygiene protocols are required to interrupt transmission routes.

A novel transcription factor PsMYBM enhances the biosynthesis of anthocyanins in response to light in tree peony

Light is a vital environmental factor that affects the biosynthesis of anthocyanin, and the ornamental and economic value of ornamental plants can be improved by properly regulating the conditions of light for their growth. Tree peony (Paeonia suffruticosa Andrews), “the king of flowers” in China, is popular in the whole country and contributes to cultivar breeding throughout the world. In the tree peony industry, light affects not only flower color and quality when the cultivation of flowers is forced but also its ornamental value in specific gardens and fields. However, the molecular mechanism on how light regulates the biosynthesis of anthocyanin is unclear. In this study, darkness was found to decrease the pigmentation of flower petals in the tree peony cultivar ‘Higurashi’. The differentially expressed genes responsible for anthocyanin biosynthesis were obtained through a comparative transcriptome analysis of flower petals under light/dark conditions. This led to the identification of an R2R3-MYB transcription factor designated PsMYBM. The transient silencing of PsMYBM reduced the biosynthesis of anthocyanins in the petals, while its overexpression promoted the formation of various colors in organs in transgenic tobacco (Nicotiana tabacum cv. Nc89) plants. In addition, it has been demonstrated that PsMYBM can directly activate the promotors of PsCHS and PsF3H. Moreover, light-responsive elements were also identified in the PsMYBM promoter, which could be directly bound by the light signal factor PsHY5. This study illuminates the mechanism of anthocyanin biosynthesis in response to light in tree peony, which could be applied to other woody ornamental plants. It also provides key gene resources to improve the flower color and germplasm innovation.

Long Term Monocropping Effects Tobacco Yield by Regulating Rhizosphere Allelochemicals and Microbial Community

Long term monocropping is considered to change soil chemical properties and affect the yield. It is significant to study the relationship among soil character, rhizosphere microorganism and crop yield under long term monocropping, to clarify the mechanism of monocropping obstacle. In this work, a continuous cropping area of tobacco in Southwest China was selected, in which Nicotiana tabacum cv. Yunyan87 was planted. The disease incidence, yield, soil properties, rhizosphere allelochemicals and microbial community were determined in the mature phase of tobacco with treatments for 2 (MCC2), 3 (MCC3), 4 (MCC4), 5 (MCC5), and 15 years (MCC15) of continuous cropping. In the results, the continuous cropping barrier was serious in the first 5 years, however, after 15-year continuous cropping, the yield was gradually recovered, and the soil-borne diseases were significantly inhibited. In terms of allelochemicals, the concentration of myristic acid increased with the time of monocropping, except for MCC15. Additionally, 2,2′-methylene bis-(4-methyl-6-tert-butylphenol) and dioctyl phthalate were lower in MCC15 than other treatments. After different monocropping years, the structure of bacterial community was distinct, with no significant differences in diversity index. For bacteria, the dominant phyla in all the treatments were Proteobacteria, followed by Acidobacteria, and Actinobacteria. In addition, in MCC15, showed relative abundances in beneficial bacterial genera like Sphingomonas and Gemmatimonas, that are involved in disease suppression and plant growth, were increased. For fungi, the dominant phyla in all the treatments were Ascomycota, followed by Mortierellomycota. Moreover, compared with MCC5 treatment, the relative abundance of pathotroph in MCC15 treatment decreased by 31.52%, the relative abundance of saprotroph increased by 31.90%. The relative abundance of potential pathogen fusarium in rhizosphere soil was the highest at the mature stage of tobacco crop rotation (MCR). Hence, different from the short-term monocropping, the 15-year long-term tobacco monocropping showed a gradual restoration of microbial community structure and specific genera, while reducing the accumulation of specific allelochemicals, thus relatively recovering the yield. It is expected to provide a reference for further understanding the mechanism of continuous cropping obstacle.

Identification of Stigma Specific Expression Fragment in the Promoter of a Soybean Chitinase Class I Gene

The expression level of heterologous genes in transgenic plants serves as an important indicator of gene efficiency. The small number of currently known effective promoters, limits the possibilities in fine-tuning the expression of transgenes. We cloned and characterized a tissue-specific promoter fragment of the soybean chitinase class I gene (GmChi1). The GmChi1 promoter (GmChi1P) was cloned from Jungery soybean. The promoter sequence contains a number of putative cis-acting elements, including tissue-specific and stress-regulated motifs. By histochemical analysis, the GmChi1P-controlled β-glucuronidase (GUS) reporter enzyme activity was shown to be highest in the roots of transgenic Nicotiana tabacum cv. NC89 at the four-leaf sprout formation stage. Interestingly, the high GUS activity in transgenic tobacco roots was effectively suppressed by salicylic acid (SA) treatment. Deletion analysis of GmChi1P revealed that the sequences located between positions -719 and -382 contain key cis-elements responsible for the reporter uidA gene expression (encoding GUS) in leaves, roots, and wounds of Nicotiana tabacum. In addition, fluorometric analysis showed that the activity of the shortened ChiP(-1292) to ChiP(-719) promoters in the roots of transgenic tobacco was significantly suppressed by abscisic acid and completely suppressed by SA. The ChiP(-382) promoter was also found to be expressed exclusively in the stigma of transgenic tobacco flowers. Using the GUS reporter enzyme, no staining was detected in other flower organs in transgenic Nicotiana tabacum, including sepals, petals, anthers, filaments, and ovaries, or in any vegetative tissues. The results indicate that the promoter fragment ChiP(-382) can be used in tissue-specific regulation of gene expression and plant genetic engineering.

Epidemiology of ChiVMV and loss assessment in capsicum (Capsicum annum var. grossum Sendt)

The survey was conducted during rabi season (2021) to determine the incidence of mosaic disease of capsicum in major capsicum growing districts namely, Chikkaballapura, Kolar, Bengaluru rural and Ramanagar. The per cent incidence of mosaic disease based on symptoms in field was recorded, highest in Ramanagar (54.85%) and the least incidence of mosaic disease was observed in Chikkaballapura (26.85%). Transmission and host range studies under glasshouse conditions revealed that ChiVMV is transmitted mechanically. Among 16 host plants tested, 7 plant species (Nicotiana tabacum cv. Samsun, N. glutinosa, N. occidentalis, Datura metel, Physalis floridana, S. nigrum, Capsicum annum) were infected with the Chilli veinal mottle virus disease and the symptom could be seen in 20-25 days. The per cent transmission of ChiVMV by aphid Aphis gossypii was studied. The results showed that ChiVMV can be transmitted by A. gossypii. However, five aphids per plant showed highest per cent transmission (100%). The effect of different dates of inoculation on different plant growth parameters was also studied, the highest per cent disease transmission was observed in T1: Inoculation 15 days after sowing (100.00%).

Decontamination of Tomato Brown Rugose Fruit Virus-Contaminated Shoe Soles under Practical Conditions

Due to its global spread, easy mechanical transmissibility inside greenhouses, and detrimental effects on marketability, Tomato brown rugose fruit virus (ToBRFV) is considered to be one of the biggest threats to tomato production. Regarding such crop epidemics, it is essential to identify all conceivable transmission routes and to interrupt them with effective decontamination strategies. We analyzed the potential efficacy of reliable shoe sole cleaning in combination with a disinfection measure. For this purpose, first, a suspension test was undertaken that involved applying different disinfectants to an infected plant homogenate. This was followed by a simulated carrier test. Finally, shoe-sole decontamination was tested under practical conditions. The extent of decontamination was determined by bioassays of the infectivity of the initial load remaining after treatment. Thereby, necrotic local lesions on the susceptible indicator plant Nicotiana tabacum cv. Xanthi NN were counted. Recommendations for practical applications, based on suspension or simulated carrier tests, are limited in their applicability, since very short contact times between ToBRFV and disinfectants reduce efficacy. Under practical conditions, the approved disinfectant MENNO Florades was able to achieve complete inactivation of the virus in the disinfection mat following mechanical depletion from the shoe soles.

PM 7/153 (1) Mechanical inoculation of test plants

PM 7/153 (1) Mechanical inoculation of test plants

Cucumber mosaic virus Is Unable to Self-Assemble in Tobacco Plants When Transmitted by Seed

Cucumber mosaic virus (CMV), which has great impact on agronomic production worldwide, is both aphid and seed transmitted. Although the mechanisms of aphid transmission have been widely studied, those underlying the ability of CMV to survive and remain infectious during the passage from one generation to the next through the seeds are still to be clarified. Moreover, the viral determinants of seed transmission rate are poorly understood. Three viral genotypes produced from same RNA 1 and 2 components of CMV-Fny but differing in RNA 3 (the wild type CMV-Fny, a pseudorecombinant CMV-Fny/CMV-S and a chimeric CMV previously obtained by our group, named F, FS and CS, respectively) were propagated in Nicotiana tabacum cv Xanthi plants in order to assess differences in tobacco seed transmission rate and persistence through plant generations in the absence of aphid transmission. Seed-growth tests revealed CMV infection in the embryos, but not in the integuments. Seedlings from seed-growth tests showed the presence of all considered viruses but at different rates: from 4% (F, FS) to 16% (CS). Electron microscopy revealed absence (CS) of viral particles or virions without the typical central hole (F and FS). In agreement, structural characteristics of purified CMV particles, assessed by circular dichroism spectroscopy, showed anomalous spectra of nucleic acids rather than the expected nucleoproteins. These alterations resulted in no seed transmission beyond the first plant generation. Altogether, the results show for the first time that correct virion assembly is needed for seed infection from the mother plant but not to seedling invasion from the seed. We propose that incorrect virion formation, self-assembly and architecture stability might be explained if during the first stages of germination and seedling development some tobacco seed factors target viral regions responsible for protein-RNA interactions.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.