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1.
FEBS Lett ; 508(2): 191-5, 2001 Nov 16.
Article in English | MEDLINE | ID: mdl-11718714

ABSTRACT

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Glycine max/metabolism , Hydrogen Peroxide/metabolism , Soybean Proteins/metabolism , Aequorin/metabolism , Benzopyrans/analysis , Blotting, Northern , Calcium Signaling/drug effects , Cells, Cultured , Glucans/pharmacology , Inhibitory Concentration 50 , Ion Channel Gating/drug effects , Nitrobenzoates/pharmacology , Plant Extracts/analysis , Pterocarpans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Reactive Oxygen Species/pharmacology , Sesquiterpenes , Glycine max/cytology , Glycine max/drug effects , Glycine max/physiology , Terpenes , Phytoalexins
2.
Plant Physiol ; 126(1): 87-96, 2001 May.
Article in English | MEDLINE | ID: mdl-11351073

ABSTRACT

Transgenic soybean (Glycine max) culture cells expressing apoaequorin, a Ca2+ indicator, were exposed to glucan fragments derived from Phytophthora sojae or to chitin oligomers. The effects of these elicitors on cytosolic Ca2+ concentrations and on mRNA levels of two beta-tubulin isoforms, tubB1 and tubB2, were investigated. The glucan elicitors, to which the cells are known to react with a biphasic cytosolic Ca2+ increase, induced a down-regulation of the tubB1 mRNA levels while the tubB2 mRNA level remained constant. The decrease of tubB1 mRNA level was observed after 1 hour of glucan treatment. In contrast, chitin oligomers, known to provoke a monophasic Ca2+ increase of short duration, did not affect the tubB1 mRNA level. Pre-incubation with 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an extracellular Ca2+ chelator, blocked the cytosolic Ca2+ increase as well as the decrease of tubB1 mRNA levels induced by glucan elicitors. Likewise, pre-incubation with 1 mM neomycin, which reduced only the second glucan-induced Ca2+ peak, blocked the decrease of tubB1 mRNA level. Experiments with cordycepin, a transcription inhibitor, indicated that glucan fragments induced the degradation of tubB1 mRNA. In conclusion, the glucan-induced cytosolic Ca2+ changes are correlated with a strong increase in tubB1 mRNA degradation.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Glucans/metabolism , Glycine max/metabolism , Protein Isoforms/genetics , RNA, Messenger/metabolism , Tubulin/genetics , Base Sequence , DNA Primers , Hydrolysis , Plants, Genetically Modified/metabolism , Glycine max/cytology
3.
Plant Physiol ; 124(2): 733-40, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11027722

ABSTRACT

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.


Subject(s)
Aequorin/metabolism , Calcium/metabolism , Glycine max/metabolism , Lipopolysaccharides/pharmacology , Oligosaccharides/pharmacology , Aequorin/genetics , Carbohydrate Sequence , Cells, Cultured , Cytosol/metabolism , Lipopolysaccharides/chemistry , Molecular Sequence Data , Oligosaccharides/chemistry , Plants, Genetically Modified , Glycine max/drug effects , Glycine max/genetics , Glycine max/microbiology
4.
Biol Chem ; 381(8): 705-13, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11030428

ABSTRACT

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.


Subject(s)
Carrier Proteins/genetics , Fabaceae/chemistry , Plants, Medicinal , Receptors, Drug/metabolism , Base Sequence , Binding Sites , Binding, Competitive , Blotting, Northern , Blotting, Southern , Carrier Proteins/chemistry , Carrier Proteins/metabolism , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Glucans , Lectins , Ligands , Solanum lycopersicum/chemistry , Solanum lycopersicum/genetics , Membrane Proteins , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/chemistry , Plants, Genetically Modified/genetics , Receptors, Drug/genetics , Sequence Alignment , Soybean Proteins/chemistry , Soybean Proteins/genetics , Soybean Proteins/metabolism
6.
Plant Cell ; 6(6): 827-834, 1994 Jun.
Article in English | MEDLINE | ID: mdl-12244260

ABSTRACT

When integrated as a transgene in one or a few copies, the -90 35S promoter of cauliflower mosaic virus confers expression in roots with little or no expression in cotyledons and leaves. The responsible cis element, activation sequence-1 (as-1), can bind to the nuclear factor ASF-1 as well as to the transcription factor TGA1a. Here, we show that microinjection of 104 molecules of TGA1a per cotyledon cell activated transgenes containing as-1-linked promoters. Transgenes with promoters linked to the octopine synthase (ocs) element, which also binds TGA1a, responded similarly. The acidic, N-terminal segment of TGA1a is important for transcription activation in vivo because a deletion mutant without the first 80 amino acids was inactive. Finally, we show that the -90 35S-[beta]-glucuronidase (GUS) fusion gene conferred GUS expression in cotyledon cells when injected at 50,000 copies per cell. Collectively, these results provide support for the hypothesis that the undetectable expression of the as-1-linked transgene in cotyledon cells is most likely a result of its inability to compete for a limiting amount of its cognate transcription factor(s), presumably TGA1a or related proteins.

7.
Plant J ; 5(4): 571-82, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8012407

ABSTRACT

To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20-30 days after injection was proven by PCR analysis.


Subject(s)
Genetic Vectors , Plant Proteins/genetics , Transcription Factors/genetics , Transcriptional Activation , Zea mays/cytology , Base Sequence , Cell Differentiation , Cells, Cultured , DNA Primers , Genetic Engineering , Genetic Markers , Microinjections , Molecular Sequence Data , Protoplasts , Seeds/cytology , Zea mays/embryology , Zea mays/genetics
8.
Plant Cell Rep ; 13(10): 564-9, 1994 Jul.
Article in English | MEDLINE | ID: mdl-24196222

ABSTRACT

The regulatory Lc gene is a member of the R gene family of maize which amongst other transcriptional activators controls anthocyanin biosynthetic genes. The availability of R locus mutants which lack anthocyanin production in all tissues offers the possibility of studying cell lineage by introducing a chimeric Lc gene into defined cells and following cell autonomous anthocyanin production. For this purpose integrative and self-replicating Lc vectors were constructed. Integrative expression vectors contained the 2.4 kbp Lc cDNA with the entire 5' leader fused to the constitutive cauliflower mosaic virus 35S promoter with and without the maize alcohol dehydrogenase 1 intron 1 or to the mesophyll specific phosphoenolpyruvate carboxylase promoter of maize. To enhance expression and to circumvent the necessity of stable integration, extrachromosomally replicating and expressing wheat dwarf virus-Lc constructions were also designed. Both categories of expression vectors were tested in embryogenic callus-derived protoplasts of maize and were found to transactivate anthocyanin biosynthesis. Southern blot analysis indicated that the wheat dwarf virus-Lc constructions were replicating.

9.
Transgenic Res ; 1(1): 23-30, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1668908

ABSTRACT

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basis layer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l-1 kanamycin or 5 mg l-1 paromomycin. Single resistant cells can be recovered from about 10,000 sensitive cells in one alginate layer. Injection of the neo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.


Subject(s)
Kanamycin Resistance/genetics , Nicotiana/genetics , Phosphotransferases/genetics , Plants, Genetically Modified , Plants, Toxic , Transformation, Genetic , Base Sequence , Blotting, Southern , Cells, Cultured , Crosses, Genetic , DNA/genetics , DNA/isolation & purification , Genetic Techniques , Kanamycin Kinase , Microinjections , Molecular Sequence Data , Oligodeoxyribonucleotides , Plasmids , Protoplasts/physiology , Transfection
10.
Biotechnology (N Y) ; 9(11): 1080-5, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1369343

ABSTRACT

We have constructed a novel micro-projectile accelerating system for efficient gene transfer into cells in situ that avoids binding DNA to micro-projectiles and keeps the DNA in solution. Further, instead of a macro-projectile (or the equivalent), it accelerates the particles in a Bernoulli air stream. The micro-targeting approach directs highly dispersed particles to sites with diameters as little as 0.15 mm, allowing precise aiming to restricted tissues. The system is physically flexible and should therefore be adaptable to different tissues and species. Transient expression of the Escherichia coli beta-glucuronidase gene in immature wheat embryo scutella was obtained at a frequency of up to 3% of the treated cells in the surface layer. In tobacco SR1, we achieved many transgenic plants, and the efficiency of stable transformation with the neomycin phosphotransferase (NPTII) gene was approximately 10(-3) per exposed cell.


Subject(s)
DNA/genetics , Phosphotransferases/genetics , Transfection , Cloning, Molecular , DNA/administration & dosage , Escherichia coli/genetics , Glucuronidase/genetics , Kanamycin Kinase , Microinjections/instrumentation , Microinjections/methods , Phosphotransferases/metabolism , Plants, Toxic , Plasmids , Restriction Mapping , Nicotiana/genetics , Triticum/genetics
11.
EMBO J ; 5(7): 1437-44, 1986 Jul.
Article in English | MEDLINE | ID: mdl-16453688

ABSTRACT

SV40 DNA and pSV2neo were microinjected into isolated nuclei of Acetabularia mediterranea. The injected nuclei were implanted into anucleate cell fragments of the same species. Such combinations not only survived but also formed progeny. The F(1), F(2) and F(3) generations of these combinations were analyzed. In the case of SV40-treated cells T-antigen was expressed and accumulated in the nuclei of all three generations studied as shown by indirect immunofluorescence. Nuclear exchange experiments revealed expression of the T-antigen only if a transformed nucleus but not if only a transformed cytoplasm was involved. Transformation by pSV2neo, a chimeric gene with a selectable marker was demonstrated by the induction of G-418 resistance as well as immunofluorescence. Genomic DNA was isolated from gametes, originating in cysts from the F(1), F(2) and F(3) generations of injected cells, and subjected to Southern analysis. These experiments demonstrated that both types of DNA are integrated into the host genome.

12.
EMBO J ; 3(9): 2169-72, 1984 Sep.
Article in English | MEDLINE | ID: mdl-6092062

ABSTRACT

Nuclei from Acetabularia mediterranea were isolated, microinjected with simian virus 40 (SV40) DNA and fused with cytoplasts from the same species. Various times after fusion of the injected nuclei the fusion products were screened for expression of the T-antigen by indirect immunofluorescence. One and two days after injection a bright fluorescence could be observed in the nuclei of Acetabularia. On the basis of this immunofluorescence we conclude that in Acetabularia cells the T-antigen is expressed and accumulated in the nucleus. Moreover, evidence is presented that the Acetabularia cell recognizes the SV40 enhancer sequence. The expression product of the SV40 DNA appears significantly earlier than the expression products of other foreign genes in Acetabularia. The results suggest that the well characterized SV40 can be used as a vector system for the introduction and expression of foreign genes in Acetabularia.


Subject(s)
Cell Nucleus/metabolism , Chlorophyta/genetics , Enhancer Elements, Genetic , Genes, Regulator , Genes, Viral , Genes , Simian virus 40/genetics , Viral Proteins/genetics , Fluorescent Antibody Technique , Microinjections , Viral Proteins/analysis
13.
Eur J Cell Biol ; 27(2): 206-12, 1982 Jun.
Article in English | MEDLINE | ID: mdl-6889505

ABSTRACT

As an extension of the observation and interpretation regarding the different microtubule systems of Micrasterias denticulata [12, 19], the existence of intertubular structures, such as microfilaments, which are strongly marked in osmotically treated cells, is especially interesting. The complex of microtubules and microfilaments occurs during post-telophase nuclear migration, probably engaged in the mechanism of movement. The arrangement of microtubules either parallel or perpendicular to the nuclear membrane is characteristic for the stage of nuclear migration. Another microtubule system, the microtubule band in the cortical protoplasm of the isthmus region [12], is described during morphogenesis of the new half cell. Osmotically treated cells in the stage of septum formation demonstrate the presence of cross-linked microtubules near the plasmalemma and microtubule bundles, situated in the protoplasm between the secondary wall and the chloroplast, probably representing the microtubule system in the cortical protoplasm of the old half cell described by Kiermayer [12, 16]. The frequent appearance of microtubules and intertubular structures in differentiating cells of Micrasterias denticulata after osmotic treatment is discussed along with implication for stabilization of microtubules, cross bridges, and microfilaments.


Subject(s)
Chlorophyta/ultrastructure , Cytoskeleton/ultrastructure , Microtubules/ultrastructure , Cell Membrane/ultrastructure , Cell Nucleus/ultrastructure , Microscopy, Electron , Nuclear Envelope/ultrastructure , Osmolar Concentration , Telophase
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