Biomedical subjects

D Roby

Publications and source records attributed to D Roby.

At least 19 recordsLinked to original sources

PubMed2001

Developmentally regulated expression of a cyclic nucleotide-gated ion channel from Arabidopsis indicates its involvement in programmed cell death.

Cyclic nucleotide-gated ion channels have been identified in animals and plants. However, the physiological role of these ion channels in plant cells and in non-receptor cells of animals is still unknown. Here, we focused on one member of the large gene family of cyclic nucleotide-gated ion channels from Arabidopsis thaliana (L.) Heynh., AtCNGC2. The analysis of the transcriptional regulation revealed that expression of AtCNGC2 is low in etiolated seedlings but increases substantially during de etiolation. The use of promoter::GUS plants revealed that expression of AtCNGC2 in seedlings is highest in cotyledons after release of the developmental arrest by light. Expression of AtCNGC2 was also observed in later stages of plant development. Investigations using the promoter::GUS plants demonstrated that AtCNGC2 is expressed in flowers during organ senescence and in the dehiscence zone of siliques. Furthermore, expression of AtCNGC2 was transiently induced during leaf and cell culture senescence. These results indicate a potential function for AtCNGC2 in the initiation of developmentally regulated cell death programs.

Animals↗

PubMed2001

Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria.

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyses the conversion of cinnamoyl-CoAs into their corresponding cinnamaldehydes, i.e. the first step of the phenylpropanoid pathway specifically dedicated to the monolignol biosynthetic branch. In previous work, we described the isolation and characterisation of the first cDNA encoding CCR in Eucalyptus (Lacombe, E., Hawkins, S., Van Dorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M., Grima-Pettenati, J., 1997. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant Journal 11, 429--441) and shown the role of this enzyme in controlling the carbon flux into lignins (Piquemal, J., Lapierre, C., Myton, K., O'Connell, A., Schuch, W., Grima-Pettenati, J., Boudet, A.M., 1998. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant Journal 13, 71--83). Here, we report the characterisation of two functionally and structurally distinct cDNA clones, AtCCR1 and AtCCR2 (81.6% protein sequence identity) in Arabidopsis thaliana. The two recombinant proteins expressed in Escherichia coli are able to use the three cinnamoyl-CoAs tested but with different levels of efficiency. AtCCR1 is five times more efficient with feruloyl-CoA and sinapoyl-CoA than AtCCR2. In addition, the two genes are differentially expressed during development and in response to infection. AtCCR1 is preferentially expressed in tissues undergoing lignification. In contrast, AtCCR2, which is poorly expressed during development, is strongly and transiently induced during the incompatible interaction with Xanthomonas campestris pv. campestris leading to a hypersensitive response. Altogether, these data suggest that AtCCR1 is involved in constitutive lignification whereas AtCCR2 is involved in the biosynthesis of phenolics whose accumulation may lead to resistance.

Aldehyde Oxidoreductases↗

PubMed2001

Identification of a novel pathogen-responsive element in the promoter of the tobacco gene HSR203J, a molecular marker of the hypersensitive response.

The tobacco gene, HSR203J, which is specifically activated during the early steps of incompatible plant/pathogen interactions has been shown to be a molecular marker of the hypersensitive response (HR). It constitutes an ideal model for the identification of HR-responsive cis-regulatory elements. As a first step in the promoter dissection, deletion mutants of the 5' flanking sequence of HSR203J fused to the GUS reporter gene were analyzed. Then, the construction and study of chimeric constructs containing HSR203J promoter fragments fused to a minimal promoter enabled us to identify a 28-bp regulatory element located between -106 and -79 upstream of the transcription initiation site. This element has been shown to be necessary and sufficient for transcriptional activation in response to pathogen. It contains a 10-bp palindrome followed by its imperfect repeat. The mutagenesis of these two sequence elements led to the identification of a 12-bp motif termed HSRE (HSR203 responsive element) responsible for the marked induction of the HSR203J gene during the HR. Since this DNA region did not show any homology with known regulatory sequences, this 12 bp motif corresponds to a novel cis-regulatory element.

Base Sequence↗

PubMed2001

HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death.

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.

Cell Division↗

PubMed2000

hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris.

A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.

Arabidopsis↗

PubMed1999

Identification of new early markers of the hypersensitive response in Arabidopsis thaliana(1).

New molecular markers of the hypersensitive response (HR) of Arabidopsis thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris (X.c.c.) have been identified by differential screening of a cDNA library constructed from suspension cells inoculated by an HR-inducing strain in the presence of cycloheximide. Seven families of genes (called Athsr) have been isolated, show similarities to voltage-dependent anion channels (VDAC) and alternative oxidases, or are novel proteins. Athsr genes have shown to be specifically or preferentially expressed during the HR. These data suggest that Athsr genes might be involved in early events conditioning the establishment of the HR.

Amino Acid Sequence↗

PubMed1999

Markers for hypersensitive response and senescence show distinct patterns of expression.

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.

Apoptosis↗

PubMed1999

A novel myb oncogene homologue in Arabidopsis thaliana related to hypersensitive cell death.

A novel myb oncogene homologue (AtMYB30) has been isolated by differential screening of a cDNA library prepared from Xanthomonas campestris pv. campestris (X. campestris)-inoculated Arabidopsis thaliana cells cultured in the presence of cycloheximide. AtMYB30 is a single-copy gene, and the encoded protein contains a MYB domain highly homologous to other plant and animal MYB proteins. Analyses of transcript levels in A. thaliana plants, or in cultured A. thaliana cells infected with either virulent or avirulent strains of the pathogens X. campestris and Pseudomonas syringae pv. tomato, showed that maximal levels of transcription of this gene occurred during the hypersensitive response. Furthermore, in A. thaliana mutants affected in the control of cell death initiation (lsd3, lsd4 and lsd5), constitutive expression or expression in lesion-positive plants was observed, while in suppressors of the mutations lsd5 and lsd4, AtMYB30 transcripts did not accumulate. However, AtMYB30 expression could not be detected in the lsd1 mutant, which was hyper-responsive to cell death initiators and unable to limit the extent of cell death, whatever the environmental conditions. The results presented here suggest a strong correlation between AtMYB30 and genetically controlled cell death, with a role in the initiation of cell death rather than in the limitation of its extent. Our results further indicate that the lsd mutants constitute an appropriate genetic model for studying the role of this gene in hypersensitive cell death, and their relation to different steps of the pathway(s) leading to cell death.

Amino Acid Sequence↗

PubMed1999

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.

Algal Proteins↗

PubMed1998

The hypersensitive response. A programmed cell death associated with plant resistance.

In plants, the hypersensitive response (HR) is defined as a rapid cell death occurring in response to pathogen attack, and is closely related to active resistance. Initiation of the HR process begins with the recognition of the pathogen by the plant, which is mediated mainly by the pathogen avirulence genes and the plant resistance genes. Then, complex signal transduction pathways intervene, involving changes in protein phosphorylation, production of reactive oxygen species and modification of ion fluxes. Components required for the regulation of the HR cell death are now being identified genetically by the isolation of mutants, in contrast to those involved in the execution of the cell death programme, which are still largely unknown. Further genetic and biochemical analyses will undoubtedly answer the question as to whether this form of programmed cell death (PCD) can be compared with other forms of PCD in plants and with apoptosis in animals.

Apoptosis↗

PubMed1998

Activation of hsr203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death.

hsr203J is a tobacco gene whose activation is rapid, highly localized, and specific for incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. The effect of other hypersensitive response (HR)-inducing pathogens and elicitors has been tested with transgenic plants containing the hsr203J promoter-GUS reporter gene fusion, and confirms the generality of the preferential inducibility of the hsr203J gene promoter during incompatible interactions: bacterial and viral pathogens inducing an HR in tobacco were able to induce the promoter fusion, as were inducers of HR-like responses such as harpin, elicitins, and PopA1 proteins. A tomato hsr203 homologous cDNA was isolated (Lehsr203) and used to examine the effect of avr gene products on the expression of such genes. Lehsr203 was shown to be rapidly and transiently induced in leaves of the tomato Cf-9 line, following Avr9 product infiltration, but not in those of the Cf-0 line. Among potential effectors of HR or resistance such as H2O2, salicylic acid, methyl jasmonate, and 2,6-dichloro-isonicotinic acid (INA), none is able to induce a significant increase in promoter activation. In contrast, heavy metals that cause leaf necrosis can trigger such an activation. In addition, hsr203-GUS fusion expression is detected in transgenic tobacco lines expressing the bO gene and exhibiting spontaneous HR-like lesions. Taken together, these results demonstrate a strong correlation between hsr203 and genetically controlled cell death in tobacco and tomato. The expression of this gene should be a useful marker for programmed cell death occurring in response not only to diverse pathogens, but also to diverse death-triggering extracellular agents.

Amino Acid Sequence↗

PubMed1997

Functional expression of a tobacco gene related to the serine hydrolase family -- esterase activity towards short-chain dinitrophenyl acylesters.

We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.

Butyrates↗

PubMed1997

Differential activation of two ACC oxidase gene promoters from melon during plant development and in response to pathogen attack.

ACC (1-aminocyclopropane-1-carboxylate) oxidase genes are differentially expressed in melon during development and in response to various stresses. We investigated the molecular basis of their transcription by analyzing the 5' untranslated regions of the ACC oxidase genes CM-ACO1 and CM-ACO3. In order to determine how their temporal and spatial expression patterns were established, we fused the promoter regions of CM-ACO1 (726 bp) and CM-ACO3 (2260 bp) to the beta-glucuronidase (GUS) reporter gene and examined their regulation in transgenic tobacco plants. The CM-ACO1 promoter was able to drive GUS expression in response to wounding, and to treatment with ethylene or copper sulfate. It was also rapidly induced (8-12 h postinoculation) in tobacco leaves inoculated with the hypersensitive response (HR)-inducing bacterium Ralstonia solanacearum. Expression was also observed during compatible interactions but was delayed. In contrast, the CM-ACO3 promoter was not expressed in response to infection, but was up-regulated during flower development. Both promoters were regulated during leaf senescence but in different patterns. The CM-ACO1-driven GUS activity increased sharply concomitantly with the onset of chlorophyll breakdown, while the CM-ACO3 promoter drove strong GUS expression in green, fully expanded leaves and this declined at the onset of senescence. This result is consistent with the expression patterns of these two genes in senescent melon leaves. These data suggest that the regulation of expression of CM-ACO1 is related preferentially to stress responses, whereas CM-ACO3 seems to be associated with developmental processes. The possible role of ethylene is discussed, particularly in the regulation of the CM-ACO1 gene in response to stress and during senescence.

Amino Acid Oxidoreductases↗

PubMed1997

Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.

A receptor-like kinase, SRK, has been implicated in the autoincompatible response that leads to the rejection of self-pollen in Brassica plants. SRK is encoded by one member of a multigene family, which includes several receptor-like kinase genes with patterns of expression very different from that of SRK but of unknown function. Here, we report the characterization of a novel member of the Brassica S gene family, SFR2. RNA gel blot analysis demonstrated that SFR2 mRNA accumulated rapidly in response both to wounding and to infiltration with either of two bacteria: Xanthomonas campestris, a pathogen, and Escherichia coli, a saprophyte. SFR2 mRNA also accumulated rapidly after treatment with salicylic acid, a molecule that has been implicated in plant defense response signaling pathways. A SFR2 promoter and reporter gene fusion was introduced into tobacco and was shown to be induced by bacteria of another genus, Ralstonia (Pseudomonas) solanacearum. The accumulation of SFR2 mRNA in response to wounding and pathogen invasion is typical of a gene involved in the defense responses of the plant. The rapidity of SFR2 mRNA accumulation is consistent with SFR2 playing a role in the signal transduction pathway that leads to induction of plant defense proteins, such as pathogenesis-related proteins or enzymes of phenylpropanoid metabolism.

Base Sequence↗

PubMed1996

Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria.

A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotransferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hypersensitive reaction, as compared to virulent pathogens, which lead to disease.

Amino Acid Sequence↗

PubMed1996

Clinicopathologic study of paraneoplastic brainstem encephalitis and ophthalmoparesis.

We report three patients who exhibited ophthalmoparesis as an early manifestation of progressive paraneoplastic brainstem encephalitis. In two patients, anti-Hu antibodies were detected, whereas in a third, found at postmortem to have thyroid cancer, no antibodies were identified. Postmortem examination of two patients disclosed extensive gliosis, perivascular inflammation, and cell loss in the midbrain and pontine tegmentum. In one of these patients, there was selective neuronal loss within the third, fourth, and sixth nerve nuclei. We conclude that supranuclear or nuclear ophthalmoparesis may be the initial manifestation of paraneoplastic brainstem encephalitis. Our pathologic data suggest that the ophthalmoparesis may result from selective neuronal death within the brainstem tegmentum and ocular motor nuclei.

Brain Stem↗

PubMed1995

Developmental and pathogen-induced activation of an msr gene, str 246C, from tobacco involves multiple regulatory elements.

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5' flanking DNA sequence from the str246C gene fused to the beta-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5' deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 bp was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.

Base Sequence↗

PubMed1994

Phase I and pharmacokinetic study of ormaplatin (tetraplatin, NSC 363812) administered on a day 1 and day 8 schedule.

Ormaplatin (tetraplatin, NSC 363812) is a platinum(IV) analogue that is active against cisplatin-resistant cell lines in preclinical models. A schedule previously shown to be active and well tolerated for cisplatin was evaluated in 26 patients. Ormaplatin was administered over a dose range of 4.4-60.8 mg/m2 i.v. given over 30 min on a day 1 and day 8 schedule every 28 days. Twenty-three patients had received prior chemotherapy, and the median performance status was 1. Nausea/vomiting (> or = grade 2) occurred in 40% of patients but was well controlled with standard antiemetic therapy. One patient had grade 2 renal toxicity and 1 patient had grade 3 hepatotoxicity (grade 2 pretreatment). No toxicity limited the dose given during the first course. With repeated drug administration delayed severe neurotoxicity developed in 4 patients, manifested as a sensory polyneuropathy in 3 patients and a possible autonomic neuropathy in one. Prospective nerve conduction studies did not detect subclinical neuropathy prior to the onset of symptoms. Patients who received cumulative doses above 200 mg/m2 were at increased risk for developing neurotoxicity. Plasma elimination of ultrafilterable platinum (measured by atomic absorption spectrometry) was biphasic with a harmonic mean terminal half-life of 15.8 h. The mean total body clearance and renal clearance of ultrafilterable platinum were 173 and 29.8 ml/min/m2, respectively. Thus, renal clearance accounted for 16% of total clearance suggesting that extensive protein/tissue binding was responsible for the majority of platinum clearance. Approximately 60% of the platinum is protein bound (one-half irreversibly) at the end of the infusion. Pharmacokinetic parameters were not dose dependent. No pharmacokinetic parameters were more predictive of neurotoxicity than the cumulative ormaplatin dose. A phase II dose cannot be recommended on this schedule because severe and unpredictable neurotoxicity precludes the administration of more than three cycles at the three highest doses levels tested.

Adult↗