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D C Baulcombe

Publications and source records attributed to D C Baulcombe.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter.

The promoter of the bean PAL2 gene (encoding phenylalanine ammonia-lyase; EC 4.3.1.5) is a model for studies of tissue-restricted gene expression in plants. Petal epidermis is one of the tissues in which this promoter is activated in tobacco. Previous work suggested that a major factor establishing the pattern of PAL2 expression in tobacco petals is the tissue distribution of a protein closely related to Myb305, which is a Myb-like transcriptional activator from snapdragon. In the present work, we show that Myb305 expression in tobacco leaves causes ectopic activation of the PAL2 promoter. To achieve Myb305 expression in planta, a viral expression vector was used. This approach combines the utility of transient assays with the possibility of direct biochemical detection of the introduced factor and may have wider application for studying the function of plant transcription factors.

Base Sequence↗
PubMed1995

Cell-to-cell movement of potato virus X revealed by micro-injection of a viral vector tagged with the beta-glucuronidase gene.

The replication and cell-to-cell movement of potato virus X (PVX) has been studied using a PVX vector construct which expressed the beta-glucuronidase (GUS) reporter gene in infected cells. Nicotiana clevelandii leaf trichome cells were micro-injected with the PVX-GUS vector and histochemical staining was used to locate GUS activity. The distribution of GUS activity revealed that PVX had moved from the injected cell into other trichome cells and into the cells along the leaf margin. GUS activity was always restricted to the cells at the edge of the leaf suggesting that PVX was unable to move out of the marginal cells. At the infection front, scattered along the leaf margin, there were isolated groups of cells staining for GUS activity. The absence of GUS activity in the intervening cells suggests that PVX had moved through several cells without replicating within them. This latter observation is consistent with previously reported observations that viral movement proteins are capable of moving between cells.

Genetic Vectors↗
PubMed1995

Jellyfish green fluorescent protein as a reporter for virus infections.

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.

Animals↗
PubMed1995

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato.

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a nonvirion form of the coat protein.

Base Sequence↗
PubMed1995

Use of a gene expression system based on potato virus X to rapidly identify and characterize a tomato Pto homolog that controls fenthion sensitivity.

A novel transient gene expression system was used to study both the tomato disease resistance gene Pto and a Pto homolog designated Fen. The gene expression system was based on potato virus X (PVX). Tomato plants that were both susceptible to strains of Pseudomonas syringae pv tomato carrying the corresponding avirulence gene avrPto and insensitive to the insecticide fenthion were infected with in vitro-generated transcripts of PVX derivatives containing either Pto or Fen. Expression of the Pto gene from the virus genome failed to elicit P.s. tomato resistance, indicating that the PVX system is not suitable for the study of Pto. However, expression of the Fen gene resulted in sensitivity to fenthion. The utility of the PVX gene expression system was further demonstrated through structure/function studies of the Fen gene. A correlation was shown between Fen protein kinase activity and the ability of this protein to confer fenthion sensitivity to tomato. Furthermore, it was demonstrated that mutation of a putative N-terminal myristoylation site, proposed to be involved in membrane targeting, rendered the Fen protein inactive. Analysis of a Pto-Fen chimeric gene allowed the fenthion sensitivity domain to be localized to the C-terminal part of the Fen protein. Interestingly, expression of the Fen kinase from the PVX genome in Nicotiana spp resulted in a fenthion-independent necrotic response. Our results support the involvement of the Fen gene in a signal transduction pathway(s).

Amino Acid Sequence↗
PubMed1993

Sub-cellular localization of the 25-kDa protein encoded in the triple gene block of potato virus X.

The 25-kDa protein of potato virus X (PVX) is encoded by the 5' open reading frame of the overlapping triple gene block, has a GKS sequence motif characteristic of nucleotide binding proteins, and is thought to play a role in movement between cells. We have produced a polyclonal antibody to the 25-kDa protein to investigate its synthesis and intracellular location. On Western blots of infected tissue extracts this antibody detected a single major protein of the expected mobility, produced earlier than the viral coat protein in infected protoplasts and located predominantly in the 1000 g pellet fraction of infected cells. Immunogold labeling of thin sections of infected tissue revealed that the 25-kDa protein is associated with the cytoplasmic inclusion bodies characteristic of PVX infections, but not with the cell wall or plasmodesmata. Therefore, although mutational studies indicate a role for the 25-kDa protein in the cell to cell movement of viral infection it is unlikely to act in precisely the same manner as the other well-characterized movement proteins of tobacco mosaic and cowpea mosaic viruses.

Amino Acid Sequence↗
PubMed1993

A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness.

The coat protein of PVX determines whether isolates of PVX are affected by Rx-mediated resistance in potato. Isolates with the coat protein of PVXHB are not affected by the resistance; those with the coat protein of PVXUK3 elicit an extreme resistance in the Rx potato that prevents virus accumulation, even on the inoculated leaf. In this paper we describe the analysis of a series of hybrid and mutant isolates of PVXHB and PVXCP4 which were inoculated to plants and protoplasts of Rx and rx cultivars of potato. From the virulence phenotypes of these isolates we conclude that elicitation of the resistance is affected by amino acids 121 and 127 of the viral coat protein, with codon 121 being the major determinant. PVXHB and hybrid or mutant isolates with lysine and arginine at positions 121 and 127 were able to overcome the resistance of Rx, whereas those with threonine and arginine were resistance sensitive both on plants and in protoplasts. The viral isolates with single mutations at either codon 121 or 127 were less infectious than the wild-type or double mutant isolates although, in protoplasts of the susceptible cultivar of potato, they accumulated as well as the wild-type virus. Taken together these data suggest that amino acids 121 and 127 affect a feature of the viral coat protein which may interact with cellular components involved in the spread of PVX and with the product of the Rx resistance gene.

Amino Acid Sequence↗
PubMed1993

Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X.

A British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.

Amino Acid Sequence↗
PubMed1992

Infectious in vitro transcripts from amplified cDNAs of the Y and Kin strains of cucumber mosaic virus.

Using a method based on the polymerase chain reaction (PCR) with primers that include the phi 10 promoter of bacteriophage T7, we obtained cDNA clones of the three RNA genomes of two different strains of cucumber mosaic virus (CMV; Kin and Y strains) from which infectious in vitro transcripts were generated, and demonstrated that the same primers could be used for amplification of at least two other strains of CMV (O and Py). This method is rapid and requires only limited nucleotide (nt) sequence data (16-18 nt) from the termini of the RNA species. Either viral RNA or unpurified RNA samples from infected plants can be used as template for first-strand cDNA synthesis. For cDNAs of RNA1 and RNA2 of the Y strain, the transcription efficiency was substantially lower than with the Kin strain, unless the primer sequence included transcribed G residues on the 5' side of the viral cDNA, so that the promoter for T7 RNA polymerase resembled more closely the canonical sequence from the bacteriophage T7 phi 10 promoter. The lower specific infectivity of transcripts of the modified cDNAs was more than compensated for by increased transcription efficiency. The possibility that the PCR process may introduce deleterious mutations into the viral cDNA was investigated by re-amplification of a functional cloned cDNA of RNA2: all six cDNA clones of the re-amplified cDNA produced transcripts as infectious as those from the progenitor cDNA.

Base Sequence↗
PubMed1992

Analysis of the gibberellin-responsive promoter of a cathepsin B-like gene from wheat.

A wheat gene (A121) encoding a protein with sequence similarity to mammalian cathepsin B is regulated by gibberellic acid (GA) in aleurone layers of germinating grains. To analyse the mechanism of A121 regulation, its promoter was fused to the beta-glucuronidase reporter gene (GUS) and introduced by micro-projectile bombardment into aleurone layers of oat. With 2.3 kb of promoter sequence, the GUS expression was enhanced by GA treatment. This effect was reversed by abscisic acid (ABA). This result showed for A121, like the alpha-amylase genes, that the regulation by GA and ABA was at the level of transcription. The GA responsiveness of the promoter was retained with as little as 276 bp of promoter sequence. Sequence comparison with a GA responsive promoter of an alpha-amylase gene identified the conserved element GCAACGGCAACGATGG which is required intact for full expression of both promoters. However, there was no identifiable similarity in the cathepsin-like promoter with the GA-responsive element of alpha-amylase promoters with the consensus sequence TAACAAA, suggesting that GA affects more than one mechanism of transcriptional control.

Abscisic Acid↗
PubMed1992

A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells.

A previous report described several cDNAs corresponding to mRNAs which accumulated in wheat aleurone layers treated with gibberellic acid (GA) (Baulcombe and Buffard, 1983). The protein sequence deduced from one of these clones (2529) has extensive similarity to the thiol protease, cathepsin B from mammalian cells. Southern analysis of wheat DNA has shown that the 2529 mRNA is encoded by a small family of genes carried on the group 4 chromosome. The nucleotide sequence of a member of the gene family expressed at a low level in aleurone layers and the use of a primer extension assay to identify a clone of a member of the gene family producing an abundant mRNA are reported. The 2529 mRNA accumulates in the scutellum and the aleurone layer of germinating grains where its expression is regulated by GA. In the scutellum the expression was restricted to the parenchyma, suggesting that the 2529 product may have a role other than for mobilization of the endosperm.

Amino Acid Sequence↗
PubMed1991

Mutation and replacement of the 16-kDa protein gene in RNA-1 of tobacco rattle virus.

The function of the 16-kDa protein encoded by tobacco rattle virus (TRV) RNA-1 was investigated by a mutational analysis of the 16-kDa protein gene. Transcripts of TRV RNA-1 produced from a full-length cDNA clone of TRV RNA-1 (SYM strain) remained infectious when the 16-kDa protein gene was disrupted by premature termination codons and a deletion which removed 73% of the coding region. A deletion which included the intergenic region between the 29-kDa protein gene and the 16-kDa protein gene, the entire 16-kDa protein coding region, and 57% of the 3' noncoding region was not infectious. Transcripts in which the 16-kDa protein coding region was replaced by the tobacco mosaic virus (TMV) (L strain) coat protein gene were also infectious and expressed TMV coat protein in infected tissue. Inclusion of the TMV origin of assembly sequence in the chimaeric constructs resulted in the accumulation of TMV-like virus particles in infected tissue.

Blotting, Northern↗
PubMed1991

Tobacco rattle virus RNA-1 29K gene product potentiates viral movement and also affects symptom induction in tobacco.

In order to investigate the function of the 29K protein of tobacco rattle virus (TRV), we introduced different mutations in the 29K protein gene and analyzed the biological properties of the subsequent transcripts in tobacco plants. Although none of the mutant RNAs was able to accumulate to a detectable level, the defects in the 29K protein could be complemented by coinoculation with wild-type TRV or tobacco mosaic virus (TMV). Complementation was also achieved in transgenic plants expressing the homologous TMV 30K protein which is involved in cell-to-cell movement, but without inducing distinctive symptoms. Transcripts of chimeric TRV clones containing duplicate genes for the 29K protein initiated infections with formation of necrotic lesions and the progeny retained only one copy of the gene. These experiments demonstrate that the 29K protein is not required for viral RNA replication and, because the TRV transcripts do not encode the coat protein, that the 29K and 30K proteins act on nonencapsidated RNA. In addition to potentiating viral movement, the TRV 29K protein may also play a role in symptom induction on tobacco.

Blotting, Northern↗
PubMed1990

Structure and tissue-specific regulation of genes encoding barley (1----3, 1----4)-beta-glucan endohydrolases.

Two genes encode (1----3, 1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzymes in barley. A gene for isoenzyme EI has been isolated from a barley genomic library and the nucleotide sequence of a 4643 bp fragment determined. The gene is located on barley chromosome 5 while the gene for (1----3, 1----4)-beta-glucanase isoenzyme EII is carried on chromosome 1. The isoenzyme EI gene contains a single 2514 bp intron that is inserted in codon 25 of a sequence encoding a signal peptide of 28 amino acids. The coding region of the mature enzyme is characterized by a high G+C content, which results from an extreme bias towards the use of these nucleotides in the wobble base position of codons. Determination of the nucleotide sequence of the gene has enabled the complete primary structure of the enzyme to be deduced: isoenzyme EI shows 92% positional identity with the primary sequence of (1----3, 1----4)-beta-glucanase isoenzyme EII at both the nucleotide and amino acid level. However, the nucleotide sequences of the two genes diverge markedly in their 3' untranslated regions. Expression sites of the two genes were defined by Northern analysis using oligonucleotide probes specific for these 3' untranslated regions and by amplifying specific cDNAs through the polymerase chain reaction. In the tissues examined, transcription of the isoenzyme EII gene is restricted to the aleurone layer of germinated grain. In contrast, the gene for isoenzyme EI is transcribed at relatively high levels in young leaves, but also in the scutellum and aleurone of germinated grain.

Amino Acid Sequence↗
PubMed1989

A wheat alpha-Amy2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts.

Gibberellin (GA(3))-responsive aleurone protoplasts isolated from Avena sativa have been successfully used as a transient expression system to analyse promoter fusions between the wheat alpha-amylase gene alpha-Amy2/54 and the reporter gene GUS. Following PEG-mediated uptake of plasmid DNA, transient expression directed by the alpha-Amy2/54 promoter was found to be regulated in the same way as the endogenous oat alpha-amylase genes. Expression was thus dependent on the inclusion of GA(3) in the protoplast incubation media, could not be detected before a lag phase of 2 days following transformation and was inhibited by simultaneous addition of abscisic acid (ABA) with GA(3) to the media. In contrast, expression from the CaMV 35S promoter in the same system was not affected by GA(3) or ABA and could be detected 1 day after transformation. Introduction of a further three different promoters into the aleurone protoplasts confirmed that GA(3) specifically controlled transient expression from the alpha-Amy2/54 promoter only. Promoter deletions of the alpha-Amy2/54: GUS fusion demonstrated that sequences within 300 bp of the start of transcription of the gene were sufficient to direct high-level expression that was regulated by GA(3) and ABA.

Journal Article↗
PubMed1989

An unusual wheat insertion sequence (WIS1) lies upstream of an alpha-amylase gene in hexaploid wheat, and carries a "minisatellite" array.

Comparison of the 5' flanking regions of three alpha-amylase genes from chromosome 6B of hexaploid wheat by heteroduplex and sequence analysis revealed the presence of a 1.6 kb stem-loop insertion sequence (WIS1) in one of them. Polymorphism among hexaploid wheat varieties suggests the relatively recent insertion/excision of this sequence from its present position. The complete sequence of the stem-loop insertion shows that it has many of the features found in transposable elements, including target site duplication and terminal inverted repeats. One unusual feature is a tandem array of direct repeats comprising a wheat "minisatellite" sequence. Both the insertion sequence and the minisatellite are found at multiple locations in the wheat genome, but the functional significance of their association in WIS1 is unknown. The minisatellite arrays share a common core structure, and long arrays are polymorphic between different hexaploid varieties.

Base Sequence↗
PubMed1988

Sequence heterogeneity and differential expression of the alpha-Amy2 gene family in wheat.

The alpha-Amy2 genes of wheat are a multigene family which is expressed in the aleurone cells of germinating grain under control of the plant hormone gibberellin. A subset of the genes are also expressed in developing grain. Comparison of five genomic clones containing alpha-Amy2 genes, using DNA sequence analysis and Southern hybridisation, showed that the extent of similarity between genes differed. Two of the most heterogeneous genes compared were located to the same group 7 chromosome while the most similar genes alpha-Amy2/54 and alpha-Amy2/8 were located to different ones; hence sequence variation could not be correlated to the ancestry of the alpha-Amy2 genes during the separate existence of the constituent genomes of hexaploid wheat. Expression of the cloned genes was measured using an S1 nuclease protection assay and this identified alpha-Amy2/54 and alpha-Amy2/8 as part of the subset of alpha-Amy2 genes expressed in both the developing grain and in aleurone cells. Comparison of the 5' upstream regions of all five genes showed high similarity, with the exception of one gene, up to -280 nucleotides from the transcriptional start, while similarity between alpha-Amy2/54 and alpha-Amy2/8 extended a further 90 bp upstream of this point. It is suggested that regulatory elements responsible for tissue specificity and gibberellin regulation may be located within these regions of similarity.

Amylases↗