Transgenic Plant Extracts Research Articles

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.

Secretion of Bacterial Xenobiotic-Degrading Enzymes from Transgenic Plants by an Apoplastic Expressional System: An Applicability for Phytoremediation

In search of an effective method for phytoremediation of wastewater contaminated with organic compounds, we investigated the application of an apoplastic expressional system that secretes useful bacterial enzymes from transgenic plants into hydroponic media through the addition of a targeting signal. We constructed transgenic Arabidopsis expressing the aromatic-cleaving extradiol dioxygenase (DbfB), which degrades 2,3-dihydroxybiphenyl (2,3-DHB), and transgenic tobacco expressing haloalkane dehalogenase (DhaA), which catalyzes hydrolytic dechlorination of 1-chlorobutane (1-CB). Although crude leaf extracts of transgenic plants expressing cytoplasm-targeted degradative enzymes showed higher activity than did those from transgenic plants expressing apoplast-targeted enzymes, the hydroponic media of the latter showed 23.2 times (DbfB) and 76.4 times (DhaA) higher activity than plants containing the cytoplasm-targeted enzymes. Addition of crystalline 2,3-DHB to 100 mL of the hydroponic medium of transgenic or wild-type seedlings revealed that only medium from the transgenic Arabidopsis expressing apoplast-targeted DbfB showed rapid ring cleavage of 2,3-DHB. Transgenic tobacco expressing apoplast-targeted DhaA also resulted in the accumulation of the dehalogenation product 1-butanol in the hydroponic medium and showed a higher tolerance to 1-CB than wild-type or transgenic plants expressing cytoplasm-targeted DhaA. These results demonstrate the usefulness of the apoplastic expression of bacterial recombinant proteins in phytoremediation.

Mucosal and systemic immunization elicited by Newcastle disease virus (NDV) transgenic plants as antigens

As a step towards developing a safe and effective edible vaccine against Newcastle disease virus (NDV), we have explored the use of plants genetically engineered to express viral proteins. We report the construction of transgenic potato plants expressing the genes coding for immunogenic proteins of NDV under the regulation of CaMV 35S promoter and its immunogenicity in mice. All mice receiving transgenic plant extracts in icomplete Freund adjuvant produced specific anti-NDV antibodies. Animals fed with transgenic leaves showed a specific response aganist NDV. Detection of IgA released from in vitro-cultured intestinal tissue fragments indicated the presence of IgA-secreting cells in the gut.

Aggregates formed as a result of the expression of yeast Met2 gene in transgenic tobacco plants, stimulate the production of stress-protective metabolites and increased the plants tolerance to heat stress

Methionine biosynthesis has taken different evolutionary pathways in bacteria, fungi and plants. To gain insight into these differences and to search for new ways of manipulating methionine biosynthesis in plants, the yeast (Saccharomyces cerevisiae) Met2 gene and the bacteria (Leptospira meyeri) MetX gene, both encoding homoserine O-acetyltransferase, were expressed in tobacco plants. We found protein aggregates in extracts of these transgenic plants, whose levels were much higher in plants grown at 35 °C than at 25 °C. It appears that the yeast and the bacterial proteins are heat labile and tend to change their intracellular conformation. These conformational changes of the transgenic proteins were more prominent at high temperature and most probably triggered aggregation of the yeast and the bacterial proteins. Moreover, plants expressing the yeast gene that grew at 35 °C over-accumulated stress-associated metabolites, such as phenolic compounds, including tannins, as well as the amino acid arginine. In addition, the transgenic plants expressing high levels of the foreign genes show growth retardation, which further suggests that, these plants suffer from internal stress. The changes in protein conformation and the consequent triggering of stress response may account for the ability of these transgenic plants to tolerate more extreme heat stress (60 °C) than the wild-type plants.

Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp.

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.

Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f 9 coding for spidroin 1 analogs were 3"-fused in-frame with the reporter lichenase gene. The Tr2" weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.

High-yield expression of a viral peptide vaccine in transgenic plants

A high-yield production of a peptide vaccine in transgenic plants is described here. A 21-mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino-terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low-cost production of peptide vaccines.

PCR rescue and analysis of transgene sequences directly from crude extracts of transgenic embryos and plants

We report the application of a PCR-based method in conjunction with automated sequencing for the reliable detection and verification of transgenes in crude extracts of leaf and callus tissue from different plant species. Transformed tissue can be identified easily at any stage of the regeneration process, whether it is via embryogenesis or organogenesis. This allows researchers to focus their attention and resources on truly transformed tissues and avoid unwittingly culturing untransformed tissues. This protocol can also be used to rescue relatively large PCR products as well as duplexing the detection of transgenes. Direct sequencing of the PCR products allows confirmation of the integrity of the transgenein planta.

An enantiomeric assay for the flavonoids medicarpin and vestitone using capillary electrophoresis.

An enantiomeric assay for the flavonoids vestitone and medicarpin from transgenic plant extracts was developed using capillary electrophoresis. It was found that no single cyclodextrin proved capable of resolving the enantiomers of both medicarpin and vestitone. Instead, hydroxypropyl-beta-cyclodextrin provided the best selectivity for the vestitones while hydroxypropyl-gamma-cyclodextrin was best for the medicarpins. The addition of organic modifiers improved the resolution of both enantiomers. Acetonitrile proved best for the vestitones and only methanol improved the resolution of the medicarpins. An optimization study of mixed hydroxypropyl-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin containing electrolytes revealed that the separation of the medicarpin enantiomers was intolerant to the presence of hydroxypropyl-beta-cyclodextrin. Our optimized running electrolyte was composed of 2 mM hydroxypropyl-beta-cyclodextrin, 20 mM hydroxypropyl-gamma-cyclodextrin, and 25 mM borate at pH 10.0 with 10% v/v methanol. This system provided a resolution of 1.47 and 1.80 for the medicarpin and vestitone enantiomers, respectively. This analysis was completed in 12 min. This separation provided a rapid screen to determine the enantiomeric purity of key flavonoids biosynthesized by transgenic legumes.

068 Development of the Bacterial Disease Resistant Potatoes by Introduction of Shiva Gene

Antimicrobial peptide gene (shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) was transformed into potato (Solanum tuberosum L.) plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified its nucleotide seqnence to increase antimicrobial activity. Phenylalanine ammonia lyase 5 (PAL5) gene was known to express highly by wounding, irradiation, and infection by pathogens. It also expresses specifically on vessel tissues of young roots, stems, and leaves. The vector with shiva and CaMV35S promoter was also introduced into potatoes. The efficiency of regeneration was maximized at the medium containing Zeatin 2 mg/L, NAA 0.01 mg/L, GA3 0.1 mg/L. Putative transgenic potato plants were cultured on the media containing kanamycin 50 mg/L. From the tissue extracts of putative transgenic plants, GUS activity was assayed using 4-methylumbellyferyl glucuronide (MUG) as a substrate for GUS enzyme. In several transformant, GUS activity was 20- to 40-fold higher than non-transformants. Especially, one clone with CaMV35S promoter expressed ≈400-fold higher GUS activity than nontransformants. For histochemical in situ localization of GUS activity, chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide(X-gluc) was used for staining. GUS was highly expressed in the whole tissue of the transformants under CaMV35S promoter, but the other side GUS was expressed especially in the vascular tissues of stems and leaves of transformants with tPAL5 promoter. PCR was carried out at 94 °C for 20 s, 52 °C for 20 s, and 72 °C for 60 s with 45 cycles, using NPTII gene-specific primer set. PCR amplification by NPTII-specific primers confirmed 0.5-kb band in most transformants.

A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluorTM Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 μg/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 μg and 2.11 μg sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 μg mGFP5-ER per mg extractable protein.

Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance.

The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehaloseaccumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance.

Expression of Mammalian Antiviral Enzymes from the 2–5A System in Transgenic Plants

The 2–5A system is an interferon-regulated antiviral RNA decay pathway present in cells of higher vertebrates. Two enzymes are essential, a 2–5A synthetase which produces 5′-phosphorylated, 2′,5′-linked oligoadenylates (2–5A) in response to doublestranded RNA, and the 2–5A-dependent RNase L. To determine if these human proteins would be functional in plants, we expressed the human cDNAs for a 2–5A synthetase and RNase L in separate tobacco plants. Both proteins were enzymatically active in extracts of transgenic plants while such activities were not detected in the control plants. Furthermore, activation by 2–5A of RNase L in the transgenic plant leaves was shown to cause degradation of ribosomal RNA. The requirement for both the synthetase and RNase L for antiviral activity was underscored by the observations that expression of human RNase L alone or 2–5A-synthetase alone was insufficient to protect plants against either tobacco etch virus or tobacco mosaic virus.

Expression of cDNA for a bark lectin of Robinia in transgenic tobacco plants

A cDNA encoding a bark lectin of Robinia pseudoacacia was introduced into tobacco plants. The expression of the lectin cDNA under control of the 35S promoter was confirmed by Western blot analysis and a hemagglutination assay of extracts of transgenic plants. Western blot analysis revealed that the subunit of the lectin from tobacco had a molecular mass of 29 kDa. The sequence of nine amino acids from the N-terminus of the lectin from transgenic tobacco plants was identical to that of the bark lectin from Robinia, indicating that the lectin had been processed correctly at its N-terminus in tobacco. The molecular mass of the purified native lectin produced by tobacco plants was estimated to be 112 kDa by gel filtration on a column of Superdex 200. It is suggested that the lectin subunits assembled to form tetramers in transgenic tobacco plants.

Expression of human ?-interferon cDNA in transgenic rice plants

The plasmid pIG3031 containing human α-interferon cDNA and the neomycin phosphotransferase II coding sequence was successfully transferred into rice protoplasts (Indica type rice) by Lipofectinmediated transformation. Assays for NPT II enzyme activity indicated that the transformation frequency was 10%. Transgenic plants were regenerated from transformed calli. Southern blots showed that the human α-interferon cDNA sequence was present in rice DNA. RNA slot blots indicated apparent transcription of human α-interferon cDNA under the control of the plant-active promoter PI'. Extracts of transgenic cell cultures and plants contained apparent interferon activity as measured by resistance of a human amniotic cell line to viral infection in the presence of plant extracts. These studies demonstrate that human α-interferon cDNA may be correctly expressed in rice cells.

Expression of hepatitis B surface antigen in transgenic plants.

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.