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David Baulcombe

Publications and source records attributed to David Baulcombe.

11 recordsLinked to original sources
PubMed2026

PotatoRTD and TomatoRTD: Comprehensive Reference Transcript Datasets for Accurate Transcriptome Analysis and Isoform Discovery.

Transcriptome annotations provide essential information on transcript locations, sequences and structures, including transcription start, end sites and splice junctions. They underpin key biological analyses such as gene and transcript quantification, and the study of transcriptional and post-transcriptional regulation, including alternative transcription initiation, polyadenylation and splicing. Accurate characterisation of transcript isoforms is critical for understanding how gene expression relates to functional protein products. However, for many species-including Solanaceae crops such as potato and tomato-current annotations suffer from limited isoform coverage, with tens or hundreds of thousands of splice junctions and transcript isoforms missing. This undermines the completeness and accuracy of transcript-level analyses. Here, by generating Iso-seq and RNA-seq on a range of tissues and samples, we have produced transcriptome annotations for both potato and tomato with improved coverage, diversity, accurate splice junctions, and transcript start and end sites. We have also made these high-quality resources accessible through genome browsers. These enhanced annotations will enable more accurate transcriptome analyses, supporting higher-resolution and novel biological discoveries.

Solanum tuberosum↗
PubMed2006

Sequence analysis of a "true" chalcone synthase (chs_H1) oligofamily from hop (Humulus lupulus L.) and PAP1 activation of chs_H1 in heterologous systems.

Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for "true" chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the "native" chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome.

Acyltransferases↗
PubMed2005

RNA silencing.

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Animals↗
PubMed2004

RNA silencing in plants.

There are at least three RNA silencing pathways for silencing specific genes in plants. In these pathways, silencing signals can be amplified and transmitted between cells, and may even be self-regulated by feedback mechanisms. Diverse biological roles of these pathways have been established, including defence against viruses, regulation of gene expression and the condensation of chromatin into heterochromatin. We are now in a good position to investigate the full extent of this functional diversity in genetic and epigenetic mechanisms of genome control.

Arabidopsis Proteins↗
PubMed2004

The tomato resistance protein Bs4 is a predicted non-nuclear TIR-NB-LRR protein that mediates defense responses to severely truncated derivatives of AvrBs4 and overexpressed AvrBs3.

The Lycopersicon esculentum Bs4 resistance (R) gene specifies recognition of Xanthomonas campestris pv. vesicatoria (Xcv) strains that express the cognate AvrBs4 avirulence protein. Bs4 was isolated by positional cloning and is predicted to encode a nucleotide-binding leucine-rich repeat (NB-LRR) protein that is homologous to tobacco N and potato Y-1 resistance proteins. Xcv infection tests demonstrate that Bs4 confers perception of AvrBs4 but not the 97% identical AvrBs3 protein. However, when delivered via Agrobacterium T-DNA transfer, both, avrBs4 and avrBs3 trigger a Bs4-dependent hypersensitive response, indicating that naturally occurring AvrBs3-homologues provide a unique experimental platform for molecular dissection of recognition specificity. Transcript studies revealed intron retention in Bs4 transcripts. Yet, an intron-deprived Bs4 derivative still mediates AvrBs4 detection, suggesting that the identified splice variants are not crucial to resistance. The L. pennellii bs4 allele, which is >98% identical to L. esculentum Bs4, has a Bs4-like exon-intron structure with exception of a splice polymorphism in intron 2 that causes truncation of the predicted bs4 protein. To test if the receptor-ligand model is a valid molecular description of Bs4-mediated AvrBs4 perception, we conducted yeast two-hybrid studies. However, a direct interaction was not observed. Defense signaling of the Bs4-governed reaction was studied in Nicotiana benthamiana by virus-induced gene silencing and showed that Bs4-mediated resistance is EDS1- and SGT1-dependent.

Agrobacterium tumefaciens↗
PubMed2003

An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.

Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time-consuming regeneration of stably transformed plants.

Gene Expression↗
PubMed2002

Two classes of short interfering RNA in RNA silencing.

RNA silencing is a eukaryotic genome defence system that involves processing of double-stranded RNA (dsRNA) into 21-26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA-directed methylation/transcriptional suppression of homologous genomic DNA. We show here that siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short (21-22 nt) and long (24-26 nt) size classes, whereas those from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these classes of siRNA have different roles. The long siRNA is dispensable for sequence-specific mRNA degradation, but correlates with systemic silencing and methylation of homologous DNA. Conversely, the short siRNA class correlates with mRNA degradation but not with systemic signalling or methylation. These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals.

Adaptation, Physiological↗
PubMed2002

RNA silencing.

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Animals↗
PubMed2002

Viral suppression of systemic silencing.

RNA silencing in plants is a form of antiviral defense that was originally discovered from the anomalous effects of transgenes. The process is associated with a systemic signal, presumed to be RNA, and is suppressed by plant virus-encoded proteins. One of these proteins, the 2b protein of cucumber mosaic virus, prevents systemic spread of the signal molecule but, curiously, is located in the nucleus of infected cells. The antiviral role of silencing might also apply in animals because a suppressor of silencing encoded by an insect virus was identified recently.

Animals↗
PubMed2002

The amplicon-plus system for high-level expression of transgenes in plants.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.

Cysteine Endopeptidases↗