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1.
J Vet Med Sci ; 83(11): 1708-1714, 2021 Nov 16.
Article in English | MEDLINE | ID: mdl-34556603

ABSTRACT

Porcine edema disease (ED) is a life-threatening toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) in weaned piglets. We previously reported that the stx2eB-transgenic lettuce 2BH strain shows potential for use as an oral vaccine candidate against ED. However, the 2BH strain expressed a hemagglutinin (HA)-tag together with Stx2eB and contained non-canonical N-glycosylation. Therefore, we developed two Stx2eB-lettuce strains, the 3 (G+) strain in which the HA-tag was removed from 2BH, and the 3 (G-) lettuce strain, in which the 73rd Asn was replaced with Ser to prevent non-canonical N-glycosylation of Stx2eB from the 3 (G+) strain. We examined the protective effect of these newly developed two strains compared with the previous 2BH strain against ED using a colostrum-deprived piglet STEC infection model. We found that the N-glycosylated 2BH and 3 (G+) strains relieved the pathogenic symptoms of ED in STEC-challenged piglets, whereas the non-glycosylated 3 (G-) strain did not. N-Glycosylation of the Stx2eB product in lettuce may be involved in the immune response in piglets.


Subject(s)
Escherichia coli Infections , Shiga-Toxigenic Escherichia coli , Animals , Bacterial Vaccines , Edema/veterinary , Escherichia coli Infections/prevention & control , Escherichia coli Infections/veterinary , Glycosylation , Lactuca , Shiga Toxin , Shiga Toxin 2/genetics , Swine
2.
Plant Biotechnol (Tokyo) ; 38(2): 239-246, 2021 Jun 25.
Article in English | MEDLINE | ID: mdl-34393602

ABSTRACT

Plant-made oral vaccines can be a cost-effective method to control infectious diseases of humans and farm animals. Pig edema is a bacterial disease caused by enterohemorrhagic Escherichia coli producing the toxin Shiga toxin 2e (Stx2e). In our previous report, we chose the non-toxic B subunit of Stx2e (Stx2eB) as a vaccine antigen, and Stx2eB was expressed in lettuce (Lactuca sativa L., cv. Green wave). We found that a double repeated Stx2eB (2×Stx2eB) accumulates to higher levels than a single Stx2eB. In this study, we analyzed progeny plants introduced with 2×Stx2eB in which the gene was expressed under the control of conventional cauliflower mosaic virus 35S RNA (CaMV 35S) promoter, and found that the lettuce underwent transgene silencing and bore few seeds. We resolved these problems by using a transgene cassette which harbored a transcriptional promoter derived from the lettuce ubiquitin gene and a longer version of HSPT. The lettuce harboring this expression construct will be valuable in establishing the seed lot system on the basis that thousands of seeds can be obtained from one plant body and the resulting progeny plants accumulate 2×Stx2eB at high levels without the transgene silencing.

3.
Plant Biotechnol (Tokyo) ; 38(4): 391-400, 2021 Dec 25.
Article in English | MEDLINE | ID: mdl-35087303

ABSTRACT

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:ß-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

4.
iScience ; 23(7): 101332, 2020 Jul 24.
Article in English | MEDLINE | ID: mdl-32668199

ABSTRACT

Somatic plant cells can regenerate shoots and/or roots or adventitious embryonic calluses, which may induce organ formation under certain conditions. Such regenerations occur via dedifferentiation of somatic cells, induction of organs, and their subsequent outgrowth. Despite recent advances in understanding of plant regeneration, many details of shoot induction remain unclear. Here, we artificially induced shoot stem-like green organs (SSOs) in Arabidopsis thaliana roots via simultaneous induction of two transcription factors (TFs), ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25, At5g65410) and the B3 family transcription factor REPRODUCTIVE MERISTEM 7 (REM7, At3g18960). The SSOs exhibited negative gravitropism and differentiated vascular bundle phenotypes. The ATHB25/REM7 induced the expression of genes controlling shoot stem characteristics by ectopic expression in roots. Intriguingly, the restoration of root growth was seen in the consecutive and adjacent parts of the SSOs under gene induction conditions. Our findings thus provide insights into the development and regeneration of plant shoot stems.

5.
Anim Sci J ; 90(11): 1460-1467, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31502390

ABSTRACT

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB-transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB-lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB-lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB-lettuce is a promising first oral vaccine candidate against ED.


Subject(s)
Animals, Newborn , Bacterial Vaccines/administration & dosage , Edema Disease of Swine/etiology , Edema Disease of Swine/prevention & control , Escherichia coli Infections/complications , Escherichia coli Infections/veterinary , Lactuca , Shiga Toxin 2/immunology , Shiga-Toxigenic Escherichia coli , Swine , Weaning , Administration, Oral , Animals , Shiga-Toxigenic Escherichia coli/pathogenicity , Virulence
6.
Anim Sci J ; 88(5): 826-831, 2017 May.
Article in English | MEDLINE | ID: mdl-28145027

ABSTRACT

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 1011  colony-forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum-deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.


Subject(s)
Colostrum/physiology , Disease Models, Animal , Disease Susceptibility , Escherichia coli Infections , Shiga-Toxigenic Escherichia coli , Animals , Edema Disease of Swine/microbiology , Escherichia coli Infections/microbiology , Shiga Toxin 2/biosynthesis , Shiga-Toxigenic Escherichia coli/metabolism , Swine
7.
Plant J ; 83(6): 1114-22, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26227242

ABSTRACT

We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.


Subject(s)
Arabidopsis/genetics , Chromosomes, Artificial , Physical Chromosome Mapping/methods , Chloroplasts/genetics , Genome, Mitochondrial , Genome, Plant , Genomic Library , Molecular Sequence Data , Software
8.
DNA Res ; 20(6): 583-92, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23897972

ABSTRACT

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.


Subject(s)
Cloning, Molecular/methods , DNA/chemistry , Arabidopsis/genetics , DNA/genetics , DNA, Plant/chemistry , DNA, Plant/genetics , Genetic Vectors , Plasmids
9.
J Vet Med Sci ; 75(10): 1309-15, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23728257

ABSTRACT

Porcine edema disease (ED) is a communicable disease of shoats caused by infection with Shiga toxin (Stx)-producing Escherichia coli. Stx2e is classified as a 1A5B-type toxin and is a decisive virulence determinant of ED. The single A subunit of Stx2e possesses enzymatic activity and is accompanied by a pentamer of B subunits, which binds to the host receptor and delivers the A subunit into the cell. In the present study, we used a mouse model to evaluate the immunogenicity of 3 ED vaccine candidates: a non-toxic mutant holotoxin mStx2e and 2 Stx2eB-based fusion proteins, Stx2eA2B-His and Stx2eB-His. Systemic inoculation of mice with mStx2e- and the Stx2eB-derived antigens induced anti-Stx2e IgG responses that were fully and partially capable of neutralizing Stx2e cellular cytotoxicity, respectively. Intranasal immunization with mStx2e protected the mice from subsequent intraperitoneal challenge with a lethal dose of Stx2e, whereas immunization with Stx2eA2B-His and Stx2eB-His afforded partial protection. Analysis of serum cytokines revealed that mStx2e, but not the Stx2eB-based antigens, was capable of inducing a Th2-type immune response. These results suggest that although the Stx2eB-based antigens elicited an immune response to Stx2e, they did so through a different mechanism to the Th2-type response induced by mStx2e.


Subject(s)
Edema/veterinary , Escherichia coli Infections/veterinary , Recombinant Proteins/immunology , Shiga Toxin 2/immunology , Shiga-Toxigenic Escherichia coli/immunology , Swine Diseases/microbiology , Administration, Intranasal/veterinary , Animals , Antibodies, Bacterial/blood , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Edema/immunology , Edema/microbiology , Edema/prevention & control , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli Infections/prevention & control , Female , Immunization/methods , Immunization/veterinary , Injections, Intraperitoneal/veterinary , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Shiga Toxin 2/genetics , Shiga-Toxigenic Escherichia coli/genetics , Specific Pathogen-Free Organisms , Swine , Swine Diseases/immunology , Swine Diseases/prevention & control
10.
Transgenic Res ; 20(4): 735-48, 2011 Aug.
Article in English | MEDLINE | ID: mdl-20972886

ABSTRACT

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.


Subject(s)
Bacterial Vaccines/genetics , Edema Disease of Swine/therapy , Enterohemorrhagic Escherichia coli , Shiga Toxin 2/biosynthesis , Shiga Toxin 2/therapeutic use , Animals , Bacterial Vaccines/therapeutic use , Genetic Vectors , Lactuca/genetics , Lactuca/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Protein Subunits/genetics , Shiga Toxin 2/genetics , Swine , Vaccines, Edible/genetics , Vaccines, Edible/therapeutic use
11.
Glycobiology ; 21(8): 994-9, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21123369

ABSTRACT

The vesicular transport pathway in plant cells is often used for higher accumulation of recombinant proteins. In the endoplasmic reticulum, which acts as a gateway to the vesicular transport pathway, N-glycosylation occurs on specific Asn residues. This N-glycosylation in recombinant proteins must be carefully regulated as it can impact their enzymatic activity, half lives in serum when injected, structural stability, etc. In eukaryotic cells, including plant cells, N-glycans were found to be attached to Asn residues in Asn-X-Ser/Thr (X ≠ Pro) sequences. However, recently, N-glycosylations at noncanonical Asn-X-Cys sequences have been found in mammals and yeast. Our laboratory has discovered that N-glycans are attached to Asn residues at Asn-Thr-Cys sequences of double-repeated B subunit of Shiga toxin 2e produced in plant cells, the first reported case of N-glycosylation at a noncanonical Asn-X-Cys sequence in plant cells.


Subject(s)
Arabidopsis/cytology , Asparagine/metabolism , Cysteine/metabolism , Lactuca/cytology , Plant Cells/chemistry , Plant Cells/metabolism , Asparagine/chemistry , Cysteine/chemistry , Glycosylation , Polysaccharides/chemistry , Shiga Toxin 2/biosynthesis , Shiga Toxin 2/chemistry
12.
J Exp Bot ; 60(4): 1319-32, 2009.
Article in English | MEDLINE | ID: mdl-19204032

ABSTRACT

Brassica napus (canola) plants were genetically manipulated to increase the amount and composition of carotenoids in seeds by using seven key enzyme genes involved in ketocarotenoid formation, which originated from a soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3), and marine bacteria Brevundimonas sp. strain SD212 and Paracoccus sp. strain N81106 (formerly called Agrobacterium aurantiacum). The seven key gene cassettes, in which each gene was surrounded by an appropriate promoter and terminator, were connected in a tandem manner, and the resulting constructs (17 kb) were inserted into a binary vector and used for transformation of B. napus. Surprisingly, 73-85% of the regenerated plants retained all seven genes, and formed orange- or pinkish orange-coloured seeds (embryos), while untransformed controls had light yellow-coloured seeds with predominant accumulation of lutein. Three of the transgenic lines were analysed further. The total amount of carotenoids in these seeds was 412-657 microg g(-1) fresh weight, which was a 19- to 30-fold increase compared with that of untransformed controls. The total amount of ketocarotenoids was 60-190 microg g(-1) fresh weight. beta-Carotene was the predominant carotenoid, with significant amounts of alpha-carotene, echinenone, phytoene, lutein, and canthaxanthin also detected in the transgenic seeds. The ratio of hydroxylated carotenoids to overall carotenoids was quite small relative to the ratio of ketocarotenoids to overall carotenoids. Interestingly, expression of many endogenous carotenogenic genes was also altered in the transgenic seeds, suggesting that their expression was affected by an increase in carotenoid biosynthesis.


Subject(s)
Brassica napus/enzymology , Brassica napus/genetics , Carotenoids/biosynthesis , Genetic Engineering , Metabolic Networks and Pathways/genetics , Seeds/enzymology , Seeds/genetics , Chromatography, High Pressure Liquid , DNA, Bacterial/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Genetic Vectors/genetics , Herbicide Resistance , Plant Leaves/metabolism , Plants, Genetically Modified , Plasmids/genetics , Transgenes
13.
Biosci Biotechnol Biochem ; 70(7): 1780-3, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16861815

ABSTRACT

Transgenic Arabidopsis thaliana plants carrying a single copy of integrated DNA can be identified by single-step genomic polymerase chain reaction. The reaction employs two sets of primer pairs with the same melting temperature that amplify the amplicons derived from the integrated T-DNA together with those from an endogenous single-copy gene as a reference. When the band intensity ratio is one, this means that the transgenic plants are carrying a single copy of the integrated gene per haploid.


Subject(s)
Arabidopsis/genetics , DNA, Plant/genetics , Gene Dosage , Plants, Genetically Modified , Genome, Plant , Heterozygote , Homozygote , Polymerase Chain Reaction
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