PubMed Health⌕ Search

Biomedical subjects

D C Baulcombe

Publications and source records attributed to D C Baulcombe.

At least 37 records · Page 2Linked to original sources
PubMed1999

A species of small antisense RNA in posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.

Amino Acid Oxidoreductases↗
PubMed1999

Gene silencing: RNA makes RNA makes no protein.

A mutation that disrupts post-transcriptional gene silencing in Neurospora crassa has been found to affect the homologue of a plant-encoded RNA-dependent RNA polymerase. This enzyme may produce a specificity determinant of gene silencing and mediate an epigenetic conversion at the RNA level.

Fungal Proteins↗
PubMed1999

Fast forward genetics based on virus-induced gene silencing.

Gene expression in plants can be suppressed in a sequence-specific manner by infection with virus vectors carrying fragments of host genes. Recent developments have revealed that the mechanism of this gene silencing is based on an RNA-mediated defence against viruses. It has also emerged that a related mechanism is involved in the post-transcriptional silencing that accounts for between line variation in transgene expression and cosuppresion of transgenes and endogenous genes. The technology of virus-induced gene silencing is being refined and adapted as a high throughput procedure for functional genomics in plants.

Gene Expression Regulation, Plant↗
PubMed1999

Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes.

The disease caused by rice yellow mottle virus (RYMV) is a serious problem for African rice growers in large-scale irrigated programs. As there are very few suitable natural sources of RYMV resistance, we have investigated a transgenic approach using widely grown, RYMV-susceptible cultivars of rice and a transgene encoding the RNA-dependent RNA polymerase of RYMV. Transformed lines were resistant to RYMV strains from different African locations. In the most extreme examples there was complete suppression of virus multiplication. Resistance was stable over at least three generations. Subject to satisfactory field testing, these transgenic lines may be suitable for introduction into RYMV-affected rice-growing areas. In the most resistant line, transcription analysis indicated that the resistance derives from an RNA-based mechanism associated with posttranscriptional gene silencing.

Genome, Viral↗
PubMed1999

Technical advance: potato virus X amplicon-mediated silencing of nuclear genes.

Transgenic expression of a replicating potato virus X (PVX) construct (termed an 'amplicon') reproducibly and consistently activates post-transcriptional gene silencing (PTGS) in every plant. The amplicon-mediated PTGS mechanism can target transiently expressed RNAs that share homology with the amplicon transgene. We show that amplicons can also be used to silence stably integrated transgenes and endogenous genes. Plants expressing both a transgene and an amplicon targeting part of the transgene show low accumulation of the transgene RNA. Similarly, plants expressing an amplicon targeting an endogenous gene show low accumulation of the endogenous RNA and display a mutant phenotype matching a previously characterised mutation. These data demonstrate that PVX amplicons present a novel approach for the consistent activation of PTGS that can be used to specifically target and suppress gene expression in plants.

Cell Nucleus↗
PubMed1999

RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing.

Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP-transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X-rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA-DNA interaction that ensures that PTGS is maintained.

DNA Methylation↗
PubMed1999

The Rx gene from potato controls separate virus resistance and cell death responses.

Rx-mediated extreme resistance against potato virus X in potato does not involve a necrotic hypersensitive response at the site of initial infection and thereby differs from the more usual type of disease resistance in plants. However, the Rx protein is structurally similar to products of disease resistance genes conferring the hypersensitive response. We show in both Nicotiana spp and potato that Rx has the potential to initiate a cell death response but that extreme resistance is separate and epistatic to necrosis. These data indicate that cell death and pathogen arrest are separate disease resistance responses in plants.

Amino Acid Sequence↗
PubMed1998

Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.

Post-transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV-encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

Cucumovirus↗
PubMed1998

Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA.

Systemic, posttranscriptional silencing of transgenes in Nicotiana benthamiana was initiated in localized regions of the plant by introduction of transgene-homologous DNA fragments, including those without a promoter. Following this initiation step, a sequence-specific signal of gene silencing spread from cells that had received the ectopic DNA via a relay mechanism that employs plasmodesmatal and phloem channels. The nature of this mechanism, the mode of its initiation, and the ability of the signal to move long distances indicate the existence of a sequence-specific signaling mechanism in plants that may have roles in developmental control as well as in protection against transposons and viruses.

Genes, Plant↗
PubMed1998

In vivo translation of the triple gene block of potato virus X requires two subgenomic mRNAs.

The 25-kilodalton (25K), 12K, and 8K movement proteins of potato virus X are derived from overlapping open reading frames (ORFs). Using an in vivo complementation assay, we have shown that the 25K protein is expressed from a functionally monocistronic mRNA, whereas the 12K and 8K proteins are from a bicistronic mRNA. Translation of the 8K ORF is by leaky ribosome scanning through the 12K ORF.

Base Sequence↗
PubMed1997

Concurrent suppression of virus replication and rescue of movement-defective virus in transgenic plants expressing the coat protein of potato virus X.

A line of transgenic tobacco expressing the coat protein (CP) of potato virus X (PVX) was resistant against a broad spectrum of PVX strains. Inoculation of leaves and protoplasts with PVX expressing the jellyfish green fluorescent protein reporter gene revealed that this resistance mechanism suppressed PVX replication in the initially infected cell and systemic spread of the virus. Cell-to-cell movement was also slower in the resistant plants. The resistance at the level of replication was effective against wild-type PVX and also against movement-defective isolates with a frameshift mutation or deletion in the CP ORF. However, the cell-to-cell movement defect of the mutant viruses was rescued on the resistant plants. Based on these results it is proposed that the primary resistance mechanism is at the level of replication.

Animals↗
PubMed1997

Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA.

Tobacco plants were transformed with constructs in which the transgene was a cDNA of replicating potato virus X (PVX) RNA. The constructs, referred to here as amplicons, were the intact genome of PVX and PVX constructs modified to carry the beta-glucuronidase (GUS) reporter gene either as an additional gene or as a replacement for the coat protein gene (PVX/GUS/CP and PVX/GUS respectively). Transformed plants carrying these constructs displayed several phenotypes that we attribute to post-transcriptional gene silencing. These phenotypes include the absence of viral symptoms, low accumulation of transgene-derived RNA, extreme strain-specific resistance against PVX, low and non-uniform GUS expression (in the PVX/GUS and PVX/GUS/CP plants) and suppression of transiently expressed RNA sharing homology with the transgene. Importantly, the amplicon-mediated gene silencing was exhibited in all lines tested. There was no evidence of gene silencing in seven lines expressing a PVX RNA that was unable to replicate. From these data we conclude that the replicating viral RNA is a potent trigger of gene silencing. Moreover, amplicon-mediated gene silencing provides an important new strategy for the consistent activation of gene silencing in transgenic plants.

Cloning, Molecular↗
PubMed1997

Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris.

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.

Enzyme Inhibitors↗
PubMed1997

Mutation of the GKS motif of the RNA-dependent RNA polymerase from potato virus X disables or eliminates virus replication.

The RNA-dependent RNA polymerase (RdRp) of potato virus X (PVX) contains a glycine-lysine-serine (GKS) motif. This motif is present in the replication enzyme of many RNA viruses and is thought to be required for nucleoside triphosphate-binding. Three single amino acid changes, glycine to alanine (AKS), lysine to asparagine (GNS) and lysine to glutamate (GES) within the GKS motif of the PVX RdRp were tested for their effect on PVX accumulation. The GNS and GES mutations rendered the virus unable to accumulate in either tobacco plants or protoplasts, whereas substitution of glycine with alanine had only a minor effect on accumulation of PVX. The glycine to alanine mutation reverted to wild-type after passage on Nicotiana clevelandii plants. These findings suggest that the GKS motif is required for PVX replication and that strong selection pressures are active to maintain necessary sequences of the viral RdRp.

Amino Acid Sequence↗
PubMed1997

Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes.

Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.

Brassica↗
PubMed1996

Cell-to-cell movement of potato virus X is associated with a change in the size-exclusion limit of plasmodesmata in trichome cells of Nicotiana clevelandii.

To study the effect of cell-to-cell movement of potato virus X (PVX) on the size-exclusion limit of plasmodesmata we have microinjected virus particles and fluorescently labelled dextrans (F-dextrans) with average molecular mass of 4.4, 10, and 20 kDa into Nicotiana clevelandii trichome cells. A vector construct of PVX expressing GUS (PVX.GUS) moved from the injected cell and modified the plasmodesmata to allow passage of the 4.4- and 10-kDa F-dextrans but not the 20-kDa F-dextran. A mutant PVX with a deletion in the gene for the 25-kDa protein accumulated in inoculated protoplasts to the same level as the wild-type PVX. This mutant virus did not move from the injected cell and did not modify the plasmodesmata to allow passage of the 10-kDa F-dextran. However, the 4.4-kDa F-dextran moved from the cells infected with the mutant virus. These data therefore illustrate that movement of PVX is associated with modification of plasmodesmata. The full plasmodesmatal modification requires a functional 25-kDa protein but partial modification occurs when this protein is present in a mutant form.

Base Sequence↗
PubMed1996

RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants.

Post-transcriptional gene silencing in transgenic plants is the manifestation of a mechanism that suppresses RNA accumulation in a sequence-specific manner. The target RNA species may be the products of transgenes, endogenous plant genes or viral RNAs. For an RNA to be a target it is necessary only that it has sequence homology to the sense RNA product of the transgene. There are three current hypotheses to account for the mechanism of post transcriptional gene silencing. These models all require production of an antisense RNA of the RNA targets to account for the specificity of the mechanism. There could be either direct transcription of the antisense RNA from the transgene, antisense RNA produced in response to over expression of the transgene or antisense RNA produced in response to the production of an aberrant sense RNA product of the transgene. To determine which of these models is correct it will be necessary to find out whether transgene methylation, which is frequently associated with the potential of transgenes to confer post-transcriptional gene silencing, is a cause or a consequence of the process.

Plants, Genetically Modified↗