Biomedical subjects

R N Beachy

Publications and source records attributed to R N Beachy.

At least 127 records · Page 7Linked to original sources

PubMed1989

Are the PR1 proteins of tobacco involved in genetically engineered resistance to TMV?

Transgenic tobacco plants constitutively expressing the coat protein (CP) of tobacco mosaic virus (TMV) exhibit enhanced resistance ot TMV (P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, Science, 232, 738-743, 1986; R. S. Nelson, P. Powell Abel, and R. N. Beachy, Virology 158, 128-132, 1987). To determine if this enhanced resistance might be mediated through the PR1 family of pathogenesis-related (PR) proteins, their synthesis was examined. In transgenic plants derived from NN genotypic tobacco, a high proportion (approximately 80%) of those producing CP also expressed the PR1 genes at low levels. However, this correlation between TMV CP and PR1 gene expression was not observed in similarly transformed nn genotypic tobacco plants. Therefore, it appears unlikely that PR1 proteins play a critical role in genetically engineered resistance in transgenic plants producing TMV CP.

Gene Expression Regulation↗

PubMed1989

Decreased levels of TMV coat protein in transgenic tobacco plants at elevated temperatures reduce resistance to TMV infection.

Transgenic tobacco plants that accumulate tobacco mosaic virus (TMV) coat protein (CP) are resistant to TMV infection under standard growth conditions. The amount of CP accumulated and the degree of resistance to TMV were found to be temperature dependent. Exposure to continuous high temperatures (30-35 degrees) results in a sharp decrease in the amount of CP within 6 hr with no further change for at least 6 days. Under these conditions the transgenic plants developed typical systemic disease symptoms when inoculated with TMV although disease development was delayed. Transgenic plants which were moved from 35 to 22 degrees accumulated the normal level of CP within several hours. Transgenic tobacco plants inoculated and held at 35/25 degrees day/night cycles retained resistance to TMV infection. The level of CP mRNA was constant at each temperature and was associated with polyribosomes. On the basis of these results we suggest that the low level of CP under elevated temperature is due to instability of the TMV CP. In contrast, TMV CP levels in transgenic tomato plants also dropped under elevated temperatures yet retained high resistance to TMV.

Blotting, Northern↗

PubMed1989

Effect of protein aggregation state on coat protein-mediated protection against tobacco mosaic virus using a transient protoplast assay.

To address the mechanism(s) of protection against tobacco mosaic virus (TMV) infection conferred by expression of the TMV capsid protein (CP) gene in transgenic tobacco plants, a transient protection assay has been developed. Introduction of either purified viral CP or virus inactivated by ultraviolet irradiation into tobacco protoplasts induced a transient protection to challenge virus introduced concomitantly or shortly thereafter. The transient protection was characterized and the effects of different aggregation states of TMV CP were tested in the transient assay system. Tobacco mosaic virus CP preparations composed largely of helical, virus-like, aggregates conferred a less transient protection against TMV and greater protection against a distantly related virus than did preparations composed primarily of smaller aggregates.

Capsid↗

PubMed1989

Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA.

Transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus (TMV) coat protein (CP) coding sequence with or without the tRNA-like structure at the 3' end of the TMV RNA were produced. Progeny of self-pollinated plants were challenged with TMV to determine their resistance to infection. Plants that expressed RNA sequences complementary to the CP coding region and the 3' untranslated region, including the tRNA-like sequences, were protected from infection by TMV at low levels of inoculum. However, plants that expressed RNA complementary to the CP coding sequence alone were not protected from infection. These results indicate that sequences complementary to the terminal 117 nucleotides of TMV, which include a putative replicase binding site, are responsible for the protection. However, the level of protection in these plants was considerably less than in transgenic plants that expressed the TMV CP gene and accumulated CP. Since the mechanisms of protection in the two systems are different, it may be possible to increase protection by introducing both sequences into transgenic plants.

Molecular Weight↗

PubMed1989

The nucleotide sequence of a soybean mosaic virus coat protein-coding region and its expression in Escherichia coli, Agrobacterium tumefaciens and tobacco callus.

A DNA complementary to the 3'-terminal 1168 nucleotides of the genome of the N strain of soybean mosaic virus (SMV) has been cloned and sequenced. cDNA sequence and coat protein analyses indicate that the SMV coat protein-coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein. The coat protein-coding sequence is predicted to be 795 nucleotides in length, encoding a protein of 265 amino acids with a calculated Mr of 29,857. The 3' untranslated region is 259 nucleotides in length and is followed by a polyadenylate tract. The SMV coat protein-coding region, along with a small amount of upstream sequence, has been expressed in Escherichia coli as a beta-galactosidase fusion protein. The size of the protein was less than predicted for the fusion protein, suggesting processing in E. coli. The coat protein-coding region has also been expressed in Agrobacterium tumefaciens and transgenic tobacco callus as an unfused protein under the control of the cauliflower mosaic virus 35S promoter. The coat protein produced in transgenic tobacco callus had an electrophoretic mobility identical to that of SMV coat protein and constituted approximately 0.05% (w/w) of the total extracted protein.

Amino Acid Sequence↗

PubMed1989

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus.

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.

Journal Article↗

PubMed1989

Nuclear factors interact with a soybean beta-conglycinin enhancer.

Upstream sequences of the gene encoding the alpha' subunit of beta-conglycinin were analyzed for interactions with nuclear proteins from immature soybean seeds. Two factors were identified that interact with specific sequence elements within 257 base pairs 5' of the transcription start site. One factor, SEF 3, binds exclusively to a region composed of two elements located at -183 to -169 base pairs and -153 to -134 base pairs relative to the start of transcription. Each of these sites includes the hexanucleotide sequence AACCCA, which may serve as a primary recognition sequence. During seed development, SEF 3 binding activity was found to increase in soybean embryos during the time of beta-conglycinin synthesis and to decrease as seeds neared maturity. The position of the SEF 3 binding sequence corresponds with a previously reported seed-specific enhancer region, and it seems likely that this factor may act as a positive regulator of transcription of the beta-conglycinin, alpha' subunit gene in developing soybean seeds. The second factor, SEF 4, also binds within the -257 to -77 region but also interacts with sites located further upstream.

Antigens, Plant↗

PubMed1988

Selective recovery of foreign gene transcripts as virus-like particles in TMV-infected transgenic tobaccos.

A short origin-of-assembly sequence (OAS) located in the 30kDa movement protein gene, about 1.0kb from the 3'-end of the common strain of tobacco mosaic virus (TMV) RNA, nucleates encapsidation of the 6395-nucleotide-long genome by TMV coat protein in vitro, and presumably also in vivo. Single-stranded RNAs containing a foreign reporter gene sequence and the TMV OAS at their 5' - and 3' -ends, respectively, can be synthesized in vitro from recombinant SP6-transcription plasmids and will assemble spontaneously in vitro to form TMV-like 'pseudovirus' particles. In this paper, we show that foreign gene transcripts derived from the nuclear DNA of plants transformed by Agrobacterium tumefaciens, and which contain the TMV OAS, can be assembled into stable 'pseudovirus' particles in vivo during a systemic infection by TMV (helper). This is the first report of structural complementation between a heritable function bestowed on a transgenic plant and an infecting virus. As a route to protect, accumulate and recover a specific mRNA in vivo, in transgenic plant cells, this novel approach may find wider applications in developmental plant molecular biology.

Cloning, Molecular↗

PubMed1988

A DNA sequence element that confers seed-specific enhancement to a constitutive promoter.

Genes encoding beta-conglycinin, a soybean seed storage protein, are expressed only in seeds during mid-to-late stages in embryogeny. It was previously determined that a DNA sequence 200 nucleotides upstream of the transcriptional start site of the gene encoding the alpha'-subunit of beta-conglycinin is essential for regulated gene expression in transgenic plants. The regulatory effect of this DNA element was tested by inserting the element in different positions and different orientations within a chimeric constitutively expressed reporter gene. The reporter gene was comprised of the 35S promoter from cauliflower mosaic virus (CaMV), a gene encoding chloramphenicol acetyltransferase (CAT) and the polyadenylation signal from the alpha'-subunit gene. The element had no significant effect on the expression of the CAT gene in roots, stems, or leaves, regardless of the position of its insertion (i.e. 5 or 3 of the gene). However, there was 25- to 40-fold enhancement of CAT gene expression in seeds during mid-to-late stages of embryo development when the element was placed in either orientation within the 35S promoter. There was 2- to 4-fold enhancement of CAT activity when the element was placed 3' of the CAT coding sequence. No enhancement was detected when the element was placed downstream of the 3' non-coding region. This is, to our knowledge, the first identification of a cis-acting element that enhances gene expression in a tissue-specific and temporally regulated manner during embryo development in plants.

Journal Article↗

PubMed1988

Resistance to TMV in transgenic plants results from interference with an early event in infection.

Constitutive expression of the tobacco mosaic virus (TMV) coat protein (CP) gene in transgenic tobacco plants results in inhibition of disease symptom development following inoculation with TMV. Evidence is presented here that this protection is also observed in leaf mesophyll protoplasts isolated from these plants. Protoplasts were resistant to infection by TMV at concentrations of 10 microgram/ml to 1 mg/ml when introduced by either electroporation or polyethylene glycol-mediated inoculation. There was little protection against infection by TMV RNA and the protection was lost as the concentration of TMV RNA in the inoculum increased. When virus was incubated briefly at pH 8.0 prior to inoculation, protection broke down in a manner similar to that observed following RNA inoculation. Analogous results were obtained in experiments with whole plants. Because virus treated in this manner has presumably lost little or no CP, these results suggest that expression of the TMV CP gene in transgenic plant cells prevents TMV from uncoating. A model is presented for the mechanism of this blockage which relates these results to early events in TMV infection.

Capsid↗

PubMed1988

Expression of a maize storage protein gene in petunia plants is not restricted to seeds.

Genes encoding maize seed storage proteins, zeins, are expressed in developing endosperm tissue. To determine whether the DNA sequences controlling the developmental expression of these genes are recognized in dicots, we introduced a gene encoding a M(r) 19,000 zein protein into petunia by Agrobacterium tumefaciens mediated transformation. Southern blot analysis of DNA from regenerated transgenic plants showed that between 1 and 12 copies of the zein gene were integrated at various locations in the petunia genome. S1 nuclease mapping with 5' and 3' probes for zein mRNA showed that transcription of the gene was correctly initiated and terminated in seeds of the transgenic plants. The mRNA was first detected in petunia seeds 10 days after pollination and disappeared 17 days after pollination. However, only small amounts of zein transcripts were synthesized and protein could not be detected at any stage of development. We also found low levels of zein mRNA in leaves, stems, and flowers of the transgenic plants, suggesting that DNA sequences responsible for developmental regulation are not readily recognized in petunia plants.

Journal Article↗

PubMed1987

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants.

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.

Journal Article↗

PubMed1987

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons.

Analysis of the expression of genes encoding the beta-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The alpha, alpha', and beta subunits of beta-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the alpha, alpha', and beta subunits of beta-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of alpha' and alpha subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The beta subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in beta-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.

Journal Article↗

PubMed1987

Protein trafficking in plant cells.

The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co-translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation.

Journal Article↗

PubMed1986

The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris. Structural homologies of genes and proteins.

Considerable information is now available concerning the 7 S seed storage proteins of legumes and the genes that encode them. Our study compares the gene encoding a beta-type subunit of phaseolin (Pvu beta), the 7 S protein of common bean (Phaseolus vulgaris), with the gene encoding an alpha'-subunit of beta-conglycinin (Gma alpha'), the 7 S protein of soybean (Glycine max). The comparison involves 2880 base pairs of Pvu beta and 3636 base pairs of Gma alpha' and includes approximately 1 kilobase pair of 5'-flanking sequences, and 5' and 3' untranslated sequences, as well as the six exons and five introns that are found to occur in similar positions in both genes. Conserved sequences in the 5'-flanking regions of these genes are discussed in light of their potential regulatory role. Published sequences for 7 S genes of pea (Pisum sativum) permit the inference of the nature and direction of evolutionary change and, in particular, show that the major size difference between the large Gma alpha' polypeptide and the smaller polypeptides of pea and common bean is due to a large insertion in the first exon of Gma alpha'. Comparisons of protein primary structure, potential glycosylation sites, and predicted protein hydropathy show that strongly conserved features of 7 S proteins cut across exon boundaries and that nonconserved regions exist that may have potential for protein modification.

Amino Acid Sequence↗

PubMed1986

Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene.

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

DNA↗

PubMed1986

In vitro transcription and translation of cloned cDNAs encoding the 30-kDa protein gene of TMV.

A cDNA clone encoding the nonstructural, 30-kDa protein of the common (U1) strain of tobacco mosaic virus (TMV) was isolated and characterized. cDNA clones representing the intact gene as well as deletions from the 5' end of the gene were subcloned into SP6 vectors. Capped RNAs produced by in vitro transcription reactions were translated in a wheat germ cell-free system. The resultant proteins were compared to proteins obtained from the in vitro translation of intermediate length (I2) rods of TMV. Transcripts of the cDNA clones encoded polypeptides of 30, 28, or 18 kDa that were immunoprecipitated by antibody prepared against a synthetic peptide representing the carboxy terminus of the 30-kDa protein. cDNA clones containing the intact 30-kDa sequence coded for 30-kDa polypeptides while clones lacking the 30-kDa initiation codon produced 28-kDa polypeptides. Surprisingly, translation of a transcript from a cDNA clone containing the 30-kDa gene plus 390 nucleotides 5' of the initiator AUG yielded a polypeptide with an approximate molecular mass of 18 kDa. The results indicate that an intact and functional 30-kDa protein gene has been cloned. The significance of these results, with respect to determining the function of the 30-kDa protein, is discussed.

Cell-Free System↗

PubMed1986

Functional analysis of regulatory elements in a plant embryo-specific gene.

Previously we demonstrated the expression of a plant embryo-specific gene encoding the alpha' subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), in transgenic petunia plants. To examine the regulatory elements that control the expression of this embryo-specific gene (Gmg17.1), a series of deletion mutants was made that contain the alpha'-subunit gene flanked in the 5' direction from +14 nucleotides to -8.5 kilobases (kb) relative to the site of transcription initiation. Each of these deletion mutants was introduced into the genome of petunia cells with the help of Ti-plasmid-derived vectors. Petunia plants were regenerated from transformed cells and expression of the introduced soybean gene was examined. When the alpha'-subunit gene was flanked by 159 nucleotides upstream (Gmg17.1 delta-159), the gene was expressed at a low level in immature embryos. When the gene was flanked by 257 nucleotides upstream of the site of transcription initiation (Gmg17.1 delta-257), a high level of expression was obtained. An additional 8 kb of DNA sequence (which includes the sequence GTGGATAG at -560, which is identical to the core enhancer sequence of simian virus 40 and some animal genes) did not significantly increase the level of expression. The increase in expression level between the delta-159 and delta-257 mutants was at least 20-fold. Analysis of the nucleotides between delta-159 and delta-257 reveals four repeats of a 6-base-pair (G + C)-rich sequence (see formula in text). The deletion Gmg17.1 delta-159 contains a single AACCCA sequence. We suggest that the (G + C)-rich repeats play a critical role in determining the level of expression of the transgenic plants.

Base Sequence↗