The agroinfiltration of plant tissue is a robust method that allows the rapid and transient expression of recombinant proteins. Using wild-type plants as biomass, agroinfiltration exploits the ability of plants to synthesize even complex multimeric proteins that require oxidative folding and/or post-translational modifications, while avoiding the expensive and time-consuming creation of stably transformed plant lines. Here we describe a generic method for the transient expression of recombinant proteins in Nicotiana benthamiana at the small to medium laboratory scale, including appropriate binary vectors, the design and cloning of expression constructs, the transformation, selection, and cultivation of recombinant Agrobacterium tumefaciens, the infiltration of plants using a syringe or vacuum device, and finally the extraction of recombinant proteins from plant tissues.
Ornithine decarboxylase (ODC) is a cytosolic enzyme that catalyses the direct decarboxylation of L-ornithine to putrescine, one of the rate-limiting steps of polyamine biosynthesis in plants. We targeted recombinant human ODC to the cytosol and apoplast of transformed tobacco ( Nicotiana tabacum ) plants, and evaluated the impact of subcellular compartmentalization on the accumulation of the enzyme and its corresponding metabolic product. Immunoblot analysis showed that human ODC accumulated to high levels in both the cytosol and apoplast of transiently transformed tobacco leaves. In stably transformed tobacco plants with ODC targeted to the apoplast, enzyme activity increased by up to 32- fold (P < 0.001) and putrescine levels increased by up to 8.5-fold (P < 0.05) compared to wild type plants. These results demonstrate that the subcellular targeting of polyamine pathway enzymes may provide a useful strategy to enhance the accumulation and activity of enzymes involved in polyamine biosynthesis and may increase metabolic flux toward desired end products.
Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low-alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low-alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics-based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT-RNAi) or PR50-protein (PR50-RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT-RNAi and TN90 PR50-RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low-alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild-type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT-RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50-RNAi plants was intermediate between PMT-RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT-RNAi plants. For PMT-RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50-RNAi transgenic plants looked more similar to the WT than PMT-RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT-RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening.
Aspergillus fumigatus and A. flavus are fungal pathogens linked to most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum using specific antibodies allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. The use of known mAbs in the IA diagnosis is often associated with false positive results due to cross-reactivity with some bacterial polysaccharides , that challenge the search of more specific mAbs. Here we describe the characterization of the Aspergillus-specific monoclonal antibody (mAb) AP3 (IgG1κ), including the identification of its specific target antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind specifically to Aspergillus spp. Immunofluorescence microscopy revealed the presence of the target antigen in the cell wall. However, the analysis of culture media by immunoprecipitation and ELISA showed that the antigen was also secreted into the medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf)-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicate that AP3 recognizes Galf residues linked to O-glycans on Aspergillus proteins. A glycoarray demonstrated that mAb AP3 could effectively recognize oligo-[β-D-Galf-1,5]-sequences starting from a tetramer while longer chains are recognized more efficiently. Moreover, AP3 captured GM in the serum, suggesting that this antibody may be a useful tool for the diagnosis of IA.
ABSTRACT Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 μg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus , with detection limits of 2 to 60 ng/ml.
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