Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa)

Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.     

Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants

The pentameric B subunit of Escherichia coli heat-labile enterotoxin (LTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic tobacco plants to express a fusion protein consisting of the synthetic LTB and a synthetic neutralizing epitope of porcine epidemic diarrhea virus (PEDV), causing an enteric disease that is especially severe in piglets. Both components of the fusion proteins were detected in Western blot analysis, and binding assay confirmed that plant-synthesized pentameric LTB-PEDV fusion bound to the intestinal membrane GM1-ganglioside receptor. This suggested that the fusion protein retained both its native antigenicity and the ability to form pentamers.     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

lyc1, a transgenic mutant of Lactuca sativa (lettuce) tightly linked to chloroplast biogenesis.

A T-DNA tagging mutant of lettuce was studied. The transgenic lettuce mutant, designated light yellowish cotyledon1 (lyc1), exhibited a chloroplast mutant phenotype in which the chloroplast development was arrested at the cotyledon stage and the cotyledon became a light yellowish due to the reduced level of chlorophyll content. A microscopic observation of the mutant showed that the chloroplasts failed to position along the cell wall, but were scattered in the cytoplasm. The number of chloroplasts in the mutant was reduced to 66% compared to that in the wild-type plant. These results suggest that the lyc1 mutation is due to a defect in the biogenesis of chloroplasts. Also, the homozygous lyc1 mutant became lethal in a T2 and F1 progeny analysis, indicating that the mutation is recessive.     

Efficacy of heat-labile enterotoxin B subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets

Applied Microbiology and Biotechnology - Devastating outbreaks of porcine epidemic diarrhea (PED) started in China in late 2010 and rapidly spread to North America and Asia causing severe diarrhea...     

Transactivation of Ds elements in plants of lettuce (Lactuca sativa)

The maize transposable element, Activator (Ac), is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. In this paper, we describe somatic and germinal transactivation of Ds by chimeric transposase genes in whole plants. Constructs containing either the Ds element or the Ac transposase open reading frame (ORF) were introduced into lettue. The Ds element was located between either the 35S or the Nos promoter and a chimeric spectinomycin resistance gene (which included a transit peptide), preventing expression of spectinomycin resistance. The genomic coding region of the Ac transposase was expressed from the 35S promoter. Crosses were made between 104 independent R₁ plants containing Ds and three independent R₁ plants expressing transposase. The excision of Ds in F₁ progenies was monitored using a phenotypic assay on spectinomycin-containing medium. Green sectors in one-third of the F₁ families indicated transactivation of Ds by the transposase at different developmental stages and at different frequencies in lettuce plants. Excision was confirmed using PCR and by Southern analysis. The lack of green sectors in the majority of F₁ families suggests that the majority of T-DNA insertion sites are not conducive to excision. In subsequent experiments, the F₁ plants containing both Ds and the transposase were grown to maturity and the F₂ seeds screened on medium containing spectinomycin. Somatic excision was again observed in several F₂ progeny; however, evidence for germinal excision was observed in only one F₂ family.     

Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.     

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R₂ seedlings on media containing G418.Abbreviations IAA indole acetic acid - KIN kinetin - BA benzyladenine - NOS nopaline synthase - NPTII neomycin phosphotransferase II - RMNO tobacco nutrient medium (Marton and Maliga, 1975) - SH Shenk & Hildebrandt nutrient medium (Shenk & Hildebrandt, 1972; Michelmore and Eash, 1985) Present address: Agriculture Canada, P.O. Box 457, St. Jean-sur-Richelieu, Quebec, Canada, J3B 6Z8     

Propagation of lettuce (Lactuca sativa) breeding material by tissue culture

A tissue culture method using Murashige and Skoog's (MS) medium was devised to propagate healthy plants from field grown lettuce plants selected for seed production. Explants (2–3 mm long) from axillary buds were successfully grown on MS + 1.0 or 2.0 mg litre^-1 kinetin and 6.4 mg litre^-1 IAA to promote shoot growth. Concentrations of 0.5 and 4.0 mg litre^-1 kinetin gave poor shoot growth. The cultures were successfully rooted after 3–4 wk on MS + 6.4 mg litres^-1 IAA after transfer from MS + 1.0 mg litre^-1 kinetin and on MS + 4.8 mg litre^-1 IAA after transfer from MS + 2.0 mg litre^-1 kinetin. Concentrations of 3.2 and 8.0 mg litre^-1 IAA gave poor root initiation. Root initiation was more successful when cultures were grown at 40 Wm^-2 than in cultures grown at 5 Wm^-2. Rooted cultures were established in compost with a 90–95% success rate and the regenerated plants flowered c. 18 wk after the cultures were initiated.     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.     

Identification of a phenylalanine ammonia-Iyase inactivating factor in harvested head lettuce (Lactuca sativa)

Exposing head lettuce ( Lactuca sativa L., crisphead or Iceberg type) leaf tissue to hormonal levels of ethylene (10 μl l⁻¹) at 5°C promotes the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and an increase in its activity. It also promotes the appearance of the postharvest physiological disorder called russet spotting (RS). Discontinuing ethylene exposure after 4 days resulted in a rapid decline in PAL activity which was delayed by treating excised midrib leaf tissue with actinornycin D or cycloheximide at 5°C. Only cycloheximide delayed the loss of PAL activity in tissue that was transferred from 5 to 15°C. Activity of PAL from Rhodolorula glutinis was. slowly lost during incubation in buffer alone, but there was a logarithmic decline in its activity over time when it was incubated with aliquots of the resuspended 10000 g pellet from homogenized, lettuce tissue affected with RS. The in vitro loss in PAL activity was 9–fold higher in extracts from lettuce showing RS symptoms than from control lettuce, boiled samples or the buffer control. The PAL-inactivating factor isolated from lettuce affected with RS had a pH optimum around 8.0. It appears that the rapid loss in PAL activity after the discontinuation of exposure to ethylene is dependent on the de novo synthesis of a PAL-inactivating factor.     

Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains

Lettuce mosaic potyvirus (LMV) can be very destructive on lettuce crops worldwide. The LMV strain 0 (LMV-0) coat protein (CP) gene was engineered for expression in plants. It was introduced into three susceptible cultivars of Lactuca sativa using an improved procedure for transformation and regeneration of lettuce, by co-cultivation of leaf explants with Agrobacterium tumefaciens. Several transformants accumulated detectable levels of LMV CP. The R1 progeny of twelve R0 transformants (four plants per cultivar) with T-DNA integration at one single locus, was studied for protection against LMV. The progeny from five R0 transformants showed resistance to LMV-0, with the effectiveness of resistance depending on the development stage of the plants at the time of inoculation. The R1 and R2 progeny from one of these R0 transformants, Cocarde-9a, were more extensively analysed. The homozygous but not the hemizygous R1 plants displayed protection to LMV-0. The R2 progeny from one homozygous R1 plant were shown to be resistant to infection by LMV-0 and other LMV strains. As previously observed in other cases of potyvirus sequence-mediated protection, a phenomenon of recovery was observed in some plants, as well as complete resistance. However, this recovery phenotype was not always maintained, as opposed to the previous described cases, leading to a late progression of viral infection.     

Germination and growth of lettuce (Lactuca sativa) at low atmospheric pressure

The response of lettuce ( Lactuca sativa L. cv. Waldmann's Green) to low atmospheric pressure was examined during the initial 5 days of germination and emergence, and also during subsequent growth to vegetative maturity at 30 days. Growth took place inside a 66-l-volume low pressure chamber maintained at 70 kPa, and plant response was compared to that of plants in a second, matching chamber that was at ambient pressure (approximately 101 kPa) as a control. In other experiments, to determine short-term effects of low pressure transients, plants were grown at ambient pressure until maturity and then subjected to alternating periods of 24 h of low and ambient atmospheric pressures. In all treatments the partial pressure of O₂ was maintained at 21 kPa (approximately the partial pressure in air at normal pressure), and the partial pressure of CO₂ was in the range 66.5–73.5 Pa (about twice that in normal air) in both chambers, with the addition of CO₂ during the light phase. With continuous exposure to low pressure, shoot and root growth was at least as rapid as at ambient pressure, with an overall trend towards slightly greater performance at the lower pressure. Dark respiration rates were greater at low pressure. Transient periods at low pressure decreased transpiration and increased dark respiration but only during the period of exposure to low pressure. We conclude that long-term or short-term exposure to subambient pressure (70 kPa) was without detectable detriment to vegetative growth and development.     

Vesicular-arbuscular mycorrhizal infection in lettuce (Lactuca sativa) in relation to calcium supply

Summary Infection of lettuce roots (Lactuca sativa L.) by the vesicular-arbuscular mycorrhizal fungiGlomus caledonium andGlomus mosseae was dependent on the amount of calcium (supplied as CaCl₂·2H₂O or CaSO₄·2H₂O) in the nutrient solution; those plants growing at low calcium concentrations being poorly infected.     

Intracellular localization of sucrose-Phosphate Phosphate in photosynthetic cells of lettuce (Lactuca sativa)

The intracellular localization of sucrose-phosphate phosphatase (SPP) in photosynthetic cells of lettuce ( Lactuca sativa ) was investigated by using protoplasts isolated directly from leaf tissue. No SPP activity was detected in either vacuolar, chloroplast or mitochondrial preparations. The recovered activity of SPP was found in the cytosolic fractions at proportions parallel with those of the cytoplasmic marker alcohol dehydrogenase. Maximal activity was measured between pH 6. 0 and 7. 0. The data demonstrate a cytosolic location for SPP. Neither sucrose nor any other saccharide tested at 100 m M was a strong inhibitor of the enzyme, which was inactive in the presence of 35 m M ethylenediammetetraacetic acid.     

Temperature sensitive phases during the germination of lettuce (Lactuca sativa) seeds

Lettuce seeds cvs Hilde, Feltham King and Avoncrisp were subjected, at different phases during imbibition at 22°C, to a high temperature (33°C) inhibitory for germination, for periods ranging from 4 to 144 h, before returning them to 22°C. The results showed, that the first 4h of imbibition and also the phase between the commencement of mitosis and the onset of radicle emergence were more sensitive to the effects of high temperature than other phases in the germination process. Short exposures (8–24 h) to 33°C commencing at the latter phase delayed germination by up to 4 days, and at the earlier by up to 8 days. Percentage germination was unaffectd except after prolonged exposures (> 48 h) from the beginning of imbibition, which reduced it. Seedling emergence from moist sieved soil was both delayed and reduced when imbibing seeds were exposed for a short period from the beginning of imbibition to 33°C compared with seeds imbibing continuously at 19°C. Germination was delayed and not reduced when seed was exposed to 33°C at the phase between commencement of mitosis and the onset of radicle emergence.     

Infrequent transposition of Ac in lettuce,Lactuca sativa

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R₁ plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2 promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.