Biomedical subjects

C M Rommens

Publications and source records attributed to C M Rommens.

14 recordsLinked to original sources

PubMed2000

Exploiting the full potential of disease-resistance genes for agricultural use.

Effective and sustained control of fungal pathogens and nematodes is an important issue for all agricultural systems. Global losses caused by pathogens are estimated to be 12% of the potential crop production [1], despite the continued release of new resistant cultivars and pesticides. Furthermore, fungi are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons. In addition to reducing crop yield, fungal diseases often lower crop quality by producing toxins that affect humans and human health. Additional methods of disease control are therefore highly desirable. Breeding programs based on plant disease-resistance genes are being optimized by incorporating molecular marker techniques and biotechnology. These efforts can be expected to result in the first launches of new disease-resistant crops within the next five years.

Agriculture↗

PubMed2000

Fungal pathogen protection in potato by expression of a plant defensin peptide.

Defensins are small cysteine-rich peptides with antimicrobial activity. We demonstrate that the alfalfa antifungal peptide (alfAFP) defensin isolated from seeds of Medicago sativa displays strong activity against the agronomically important fungal pathogen Verticillium dahliae. Expression of the alfAFP peptide in transgenic potato plants provides robust resistance in the greenhouse. Importantly, this resistance is maintained under field conditions. There have been no previous demonstrations of a single transgene imparting a disease resistance phenotype that is at least equivalent to those achieved through current practices using fumigants.

Amino Acid Sequence↗

PubMed1997

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Bacteriorhodopsins↗

PubMed1996

Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster.

In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.

Alleles↗

PubMed1995

Intergeneric transfer and functional expression of the tomato disease resistance gene Pto.

Plant disease resistance loci have been used successfully in breeding programs to transfer traits from resistant germplasm to susceptible plant cultivars. The molecular cloning of plant disease resistance genes now permits the transfer of such traits across species boundaries by genetic transformation of recipient hosts. The tomato disease resistance gene Pto confers resistance to strains of the bacterial pathogen Pseudomonas syringae pv tomato expressing the avirulence gene avrPto. Transformation of Nicotiana benthamiana with Pto results in specific resistance to P. s. pv tabaci strains carrying avrPto. The resistant phenotype is manifested by a strong inhibition of bacterial growth and the ability to exhibit a hypersensitive response. Resistance cosegregates with the Pto gene in transgene selfings and testcrosses. Our results demonstrate the conservation of disease resistance functions across genus boundaries and suggest that the utility of host-specific resistance genes can be extended by intergeneric transfer.

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

Differential repair of excision gaps generated by transposable elements of the 'Ac family'.

Studies on transposable elements of the Ac family have led to different models for excision gap repair in either plants or Drosophila. Excision products generated by the plant transposable elements Ac and Tam3 imply a more or less straightforward ligation of broken ends; excision products of the Drosophila P element indicate the involvement of 'double-strand break' (DSB) repair. Recent findings that excision products of Ac and Tam3 can also contain traces of the element ends indicate, however, that DSB repair might be an alternative repair mechanism in plants. A functional DSB repair mechanism in plants can also be deduced from the observed rapid increases of Ac copy number during plant development and from the involvement of Ac in the generation of internal Ac deletions. On the other hand, alternative repair mechanisms may also be functional in Drosophila, because some of the 'footprints' generated upon P excision can be explained by a mechanism that has been postulated for excision gap repair in plants. It is concluded that plants and Drosophila can use similar repair mechanisms, but that the predominance of a certain repair mechanism is determined by the host.

Animals↗

PubMed1993

Supported PCR: an efficient procedure to amplify sequences flanking a known DNA segment.

We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.

Base Sequence↗

PubMed1993

Transposition pattern of a modified Ds element in tomato.

Several aspects of transposition of an in vitro modified Ds element are described. This Ds element, designated Ds-r, is equipped with bacterial plasmid sequences and can, therefore, be rescued from the plant genome. Our results indicate that the Ds-r element has a 'late' timing of transposition from T-DNAs. This feature of the element might be advantageous for tagging experiments because it leads to independently transposed germinally transmitted elements. Furthermore, it is shown that Ds-r transposition generates clusters of insertions, indicating that 'genes to be tagged' should be located in genomic regions covered by insertions.

Base Sequence↗

PubMed1992

Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue.

We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within the genome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active) Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flanking Ac. In another case, a 1878 bp internal Ac sequence is deleted.

Base Sequence↗

PubMed1992

Transactivation of Ds by Ac-transposase gene fusions in tobacco.

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Acyltransferases↗

PubMed1991

The use of transgenic plants to understand transposition mechanisms and to develop transposon tagging strategies.

This review compares the activity of the plant transposable elements Ac, Tam3, En/Spm and Mu in heterologous plant species and in their original host. Mutational analysis of the autonomous transposable elements and two-element systems have supplied data that revealed some fundamental properties of the transposition mechanism. Functional parts of Ac and En/Spm were detected by in vitro binding studies of purified transposase protein and have been tested for their importance in the function of these transposable elements in heterologous plant species. Experiments that have been carried out to regulate the activity of the Ac transposable element are in progress and preliminary results have been compiled. Perspectives for manipulated transposable elements in transposon tagging strategies within heterologous plant species are discussed.

DNA Mutational Analysis↗

PubMed1991

Ac-induced disruption of the double Ds structure in tomato.

The maize doubleDs element is stably maintained in the tomato genome. Upon the subsequent introduction of Ac into a plant containing doubleDs, disruption of the doubleDs structure and DNA rearrangements at the site of the doubleDs element were observed. No indications were obtained for excision of the complete doubleDs structure. The consequences of transactivation of doubleDs in these experiments are different from those described for transactivation of single Ds elements in tomato. The mechanisms by which such rearrangements could have occurred in tomato are discussed in relation to complex insertions containing doubleDs in maize.

Base Sequence↗

PubMed1989

A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia plants.

Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the beta-glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.

Agrobacterium tumefaciens↗