Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post‐symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early‐warning sentinels potentially have tremendous utility as wide‐area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis‐acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time‐course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.     

A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco

When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PR1b protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells.     

转基因烟草的大田抗病性分析

为了尽快地将抗病转基因烟草品种应用于生产,在选育抗病优良株系的同时,进行了转基因株系的大田抗病性鉴定。结果表明：（１）在田间自然发病情况下,转基因烟草的ＮＣ８９各株系的发病率及病情指数显著低于对照ＮＣ８９,对ＣＭＶ的相对防治效果为５５％－７０％,表现较强的抗病性;同时对ＴＭＶ也有一定的抗病力;（２）转基因烟草的产量、产值也明显高于对照。     

Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes

A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the proteincoding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots

The secretion of a functional, full-length monoclonal antibody complex from transgenic Nicotiana tabacum roots has been demonstrated. Initially, seeds were germinated on nitrocellulose membranes and antibody secretion detected from the developing roots. Plants were then established in hydroponic culture and secretion into the growth medium measured over 25 days. Western blotting indicated that full-length antibody was present in the medium along with other fragments. Secreted antibody was shown to be functional by binding to antigen in ELISA studies. In contrast, no antibody could be detected from transgenic Nicotiana in which the same antibody was expressed as a membrane protein in the plasmalemma. These results indicate that antibody accumulation in the growth medium is genuinely caused by rhizosecretion and not cell damage. Addition of gelatin to plant growth medium markedly increased levels of antibody accumulation. The mean antibody yield per plant was calculated to be 11.7 g per gram root dry weight per day. Rhizosecretion may be a viable alternative to agricultural production or cell culture for the generation of monoclonal antibodies in transgenic plants. It may also give rise to novel applications for antibodies expressed in plants such as removal or neutralisation of environmental pollutants and attenuation of pathogens which infect the plant via the rhizosphere.     

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds

Chimeric genes composed of the -phaseolin promoter, an -zein coding sequence and its modified versions containing lysine codons, and a -zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. -Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa -zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an -zein content that was approximately 0.003% of the total seed protein. The amount of -zein in other transgenic plants varied between 1 × 10^–4% and 1 × 10^–5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the -zein protein. Polysomes translating -zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized -zein was degraded in tobacco seeds with a half-life of less than 1 hour.     

Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene

Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Production of a functional single-chain Fv protein in transgenic tobacco root exudates

Nicotiana tabacum was transformed with a gene encoding anti-PreS1 of hepatitis B surface antigen single-chain Fv antibody (scFv) and bearing an N-terminal endoplasmic reticulum protein signal peptide sequence. The scFv antibody protein was continuously secreted from the transgenic tobacco roots into a simple hydroponic medium at 630 to 760 ng g^–1 dry wt root day^–1. The antibody was about 2% of the total secreted protein and still possessed antigen-binding activity.     

ToMV-Resistant transgenic tomato as a material for the first field experiment of genetically engineered plants in Japan

Summary An F₁ hybrid betweenLycopersicon esculentum andL. peruvianum was transformed using a Ti-plasmid binary vector with a coat protein gene cDNA of an attenuated tomato mosaic virus (ToMV) strain L₁₁A which was expressible by the 35S promoter of cauliflower mosaic virus (CaMV). A transgenic plant which expressed the most resistance to ToMV was chosen as a material to be tested in a nonisolated greenhouse and in the field. This transgenic tomato plant was propagated by cutting. In the first test using an isolated greenhouse and in the second test conducted in an nonisolated greenhouse, no major morphologic and physiologic differences were found between the transgenic plants and the nontransgenic control plants. Also, there was no evidence that the transgenic plants produced any new hazardous substances. Both the transgenic and the nontransgenic plants were self-sterile, and crossing of the cultivated species with pollen of these plants produced few seeds. These features of the transgenic plants satisfied the requirements for a small scale field test. The field test of the transgenic plants are in progress. Presented in the Session-in-Depth “Field Test Requirements and Performance of Transgenic Plants” at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Application of transgenic plants in understanding responses to atmospheric change

Acclimation of plants to an increase in atmospheric carbon dioxide concentration is a well described phenomenon. It is characterized by an increase in leaf carbohydrates and a degradation of ribulose 1, 5-bisphosphate carboxylase protein (Rubisco) leading in the long term to a lower rate of CO₂ assimilation than expected from the kinetic constants of Rubisco. This article summarizes studies with transgenic plants grown in elevated pCO₂ which are modified in their capacity of CO₂ fixation, of sucrose and starch synthesis, of triosephosphate and sucrose transport and of sink metabolism of sucrose. These studies show that a feedback accumulation of carbohydrates in leaves play only a minor role in acclimation, because leaf starch synthesis functions as an efficient buffer for photoassimilates. There is some evidence that in elevated pCO₂, plants grow faster and senescence is induced earlier.     

利用转基因植物生产口服疫苗的研究现状

本文概述了利用转基因植物生产口服疫苗的研究现状。分别对转基因植物生产口服疫苗的优点、作用原理、研究方法、已研究的口服疫苗及问题和前景进行了介绍。     

Long-term resistance to tomato spotted wilt virus in transgenic tobacco cultivars expressing the viral nucleoprotein gene: greenhouse and field tests

We examined the resistance phenotype of a large number of transgenic tobacco plants originating from 12 commercial (Nicotiana tabacum) cultivars expressing the sense form of the nucleoprotein (N) gene of L3, a Bulgarian isolate of tomato spotted wilt virus (TSWV). The analysis revealed that transgenic plants are completely protected against the homologous L3 isolate of TSWV irrespective of whether or not they contain detectable levels of translational product. The effectiveness of protection against the virus was investigated upon mechanical inoculation under greenhouse conditions and in field trials. Non-segregating resistant lines were selected and the inheritance of the resistance to TSWV was analysed in successive generations (R₃–R₆). Extensive tests under controlled conditions and two-year field trials proved that the resistance to TSWV is stable in different environments and is a stably inherited trait.     

Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase

Cysteine synthase [O-acetyl-L-serine(thiol)lyase] catalyzes the final step for L-cysteine biosynthesis in plants. The tolerance of transgenic tobacco plants over-expressing cysteine synthase cDNA in cytosol (3F), chloroplasts (4F) and in both organelles (F1) was investigated towards heavy metals such as Cd, Se, Ni, Pb and Cu. The transgenic plants were significantly more tolerant than wild-type plants in agar medium containing Cd, Se and Ni. The F1 transgenic plants had a higher resistance than other transgenic lines towards these metals and could enhance accumulation of Cd in shoot. These results suggest that the transgenic plants over-expressing cysteine synthase both in cytosol and chloroplasts can be applicable to phyto-remediation of Cd from contaminated soils.     

Expression and production of bioactive human interleukin-18 in transgenic tobacco plants

The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively. The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein. Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN- on IL-18-responsive J6-1 cells by the extracts obtained from the transformants. The expression level of hIL-18 (351 ng g^–1 tobacco tissue) obtained in the present study may be sufficient to induce responses/effects in vivo.     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

Field performance of derived generations of transgenic tobacco

Two inbred cultivars of Nicotiana tabacum (tobacco), Samsun and Xanthi, were transformed with the plasmid pBI 121 using Bin 19 in Agrobacterium tumefaciens. The plasmid carries the nptII gene conferring kanamycin resistance and the uidA gene encoding -glucuronidase (GUS). Progeny carrying the genes in the homozygous condition were identified and selfed over several generations. One line homozygous for the introduced genes and one untransformed control from each cultivar were then selected and crossed reciprocally to give four families per cultivar. Seeds from each family were grown in a replicated field trial and all plants scored for a range of morphological and agronomic characters. In addition, leaf samples were taken and GUS activity measured. In the Samsun material, which contained one copy of the introduced gene at a single locus and showed high levels of GUS expression, the transformed homozygote showed twice the level of GUS activity as the hemizygotes, wheareas in the Xanthi line, which had a lower level of GUS, the hemizygotes showed the same level of GUS activity as the transformed homozygote. The agronomic data showed differences between the families, but the source of such differences could not be ascribed unambiguously. The results are discussed in the light of related information on gene expression and field performance from other transgenic material.