[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding a protein, NtrbohD, located on the plasma membrane and homologue to the flavocytochrome of the neutrophil NADPH oxidase, was cloned in tobacco. The corresponding mRNA was accumulated when tobacco leaves and cells were treated with the fungal elicitor cryptogein. After elicitation with cryptogein, tobacco cells transformed with antisense constructs of NtrbohD showed the same extracellular alkalinization as the control, but no longer produced active oxygen species (AOS). This work represents the first demonstration of the function of a homologue of gp91-phox in AOS production in elicited tobacco cells.
The Plant Journal 08/2002; 31(2):137-47. DOI:10.1046/j.1365-313X.2002.01342.x · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY‐2 tobacco cells exposed to H 2 O 2 . It was shown that H 2 O 2 is able to induce various morphological cell death features in cultured tobacco BY‐2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H 2 O 2 added to the cell culture. The appearance of nuclear fragmentation similar to ‘apoptotic bodies’ associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H 2 O 2 . The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H 2 O 2 induces high necrotic cell death. These data suggest that H 2 O 2 ‐induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.
[Show abstract][Hide abstract] ABSTRACT: To examine whether molecular similarities exist between the animal and plant Rho GTPase signaling pathways, we have developed a heterologous two-hybrid screening method. By this technique, we have cloned a cDNA encoding a tobacco Rac-like protein able to interact with a mammalian Rho-GDI. In a second screen this tobacco Rac was used as a bait and a tobacco homologue of Rho-GDI was identified. These results show that some components of the animal and plant Rac signaling pathways are similar enough to allow their interaction in an heterologous approach. Moreover these data suggest a similar regulation of Rho GTPases in animals and plants.
[Show abstract][Hide abstract] ABSTRACT: We previously isolated, by differential display and 5′ RACE (rapid amplification of cDNA ends), cDNAs corresponding to genes activated following cryptogein treatment of tobacco cell suspensions, among them tcI 7 (tcI for tobacco cryptogein Induced), a gene encoding a β-subunit of proteasome. Here, we report that tcI 7 was up-regulated in tobacco plants treated with elicitins (cryptogein and parasiticein) that have been shown to induce a systemic acquired resistance (SAR). Moreover, subsequent inoculation of tobacco with the pathogen Phytophthora parasitica var. nicotianae (Ppn) was shown to induce an additional activation of tcI 7 in tobacco plants pretreated with cryptogein. We also showed an up-regulation of tcI 7 by salicylic acid (SA). Moreover, accumulation of tcI 7 transcripts after treatment with cryptogein or with SA only occurred in NahG 9− tobacco plants that do not express the salicylate hydroxylase and thus are able to accumulate SA and develop a SAR. Suppressed accumulation of tcI 7 transcripts in NahG 8+ tobacco plants after cryptogein or SA treatment correlated with the loss of SAR. H2O2 was also shown to up-regulate tcI 7 in tobacco plants. Using gene walking by PCR we cloned and sequenced the 5′ flanking region of tcI 7 containing hypothetical regulatory sequences, especially myb and NF-κB boxes, that could be responsible for the regulation of tcI 7 by salicylic acid and H2O2 respectively.
[Show abstract][Hide abstract] ABSTRACT: The major yellow compounds extracted from the mycelium of
Cercospora beticola, the beticolins, can be divided into two
subgroups, the o- and the p-beticolins. It has been
found that crystallogenesis conditions which allowed the X-ray analyses
of the first subgroup are unsuccessful for the second. New conditions
affording microcrystals for three p-beticolins, B1, B3 and B13
are described. Diffraction experiments have been performed on a
synchrotron beam line; the results confirm the proposed structures for
B1 and B3 and allow that of B13 to be elucidated.
Journal of the Chemical Society Perkin Transactions 2 09/1997; DOI:10.1039/a608511c
[Show abstract][Hide abstract] ABSTRACT: We report the successful combination of mRNA differential-display reverse-transcription PCR (DDRT-PCR) and 5-rapid amplification of cDNA ends (5-RACE) in order to isolate full-length cDNAs corresponding to genes activated in tobacco cells treated with cryptogein within 60 min. Cloning and sequencing of two cDNAs, called tcI 7 and tcI 14 (for tobacco cryptogein-induced), allowed the identification of open reading frames. Deduced amino-acid sequences of tcI 7 and tcI 14 showed significant homologies with a -type proteasome subunit and a transformer-2-like serine/arginine-rich (SR) ribonucleoprotein, respectively. The accumulation of mRNAs corresponding to tcI 7 started 30 min after the addition of cryptogein to tobacco cell suspensions and continued up to 180 min, whereas the accumulation of tcI 14 corresponding mRNAs was transitory between 30 and 60 min. These results indicated a transcriptional activation of the corresponding genes early after elicitation of tobacco cells by cryptogein. The biological significance of this activation remains to be elucidated.
[Show abstract][Hide abstract] ABSTRACT: The fluorescent dye 3,3′-diethylthiadicarbocyanine iodide [diS-C2-(5)] was used to observe plasmalemma transmembrane potential variations of tobacco cells treated with uncoupler (FCCP), respiratory inhibitors (azide and cyanide), and H+-ATPase inhibitors (DCCD and a carbanilate derivative). These chemicals induced an increase in fluorescence, indicating a dissipation of the transmembrane potential. The [diS-C2-(5)] was also used to study the effects of two Cercospora beticola toxins on tobacco cells. Changes in fluorescence of [diS-C2-(5)] suggested that these two toxins caused a dissipation of the transmembrane potential with a different magnitude whereas kinetics of their association with membranes were comparable.
[Show abstract][Hide abstract] ABSTRACT: The O2− · scavenging properties of beticolin-1, a fungal toxin, have been studied using peroxidase-mediated luminol chemiluminescence. A comparison between beticolin-1, vitamin E and tiron is reported: beticolin-1 displays an anti-radical effect without inhibiting peroxidase activity in a larger range of concentrations (1 × 10−8−1 × 10−5 M) than vitamin E (3 × 10−7−1 × 10−5 M) or tiron (5 × 10−7−4 × 10−5 M). Maximal scavenging efficacy was higher for beticolin-1 and vitamin E than for tiron (88, 80 and 52%, respectively).
[Show abstract][Hide abstract] ABSTRACT: Cercospora beticola toxins were extracted from the mycelium and purified by flash chromatography and crystallization. TLC was used to monitor the purification. The pure compounds were used to determine the optimum conditions for HPLC analysis. The HPLC method was used to quantify these metabolites in crude extracts.
Journal of Chromatography A 05/1995; 699(1):277-283. DOI:10.1016/0021-9673(95)00079-3 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment of excised tobacco leaves with the fungal elicitor cryptogein progressively induced lipid peroxidation. In a first step, evidence was provided by the accumulation of thiobarbituric acid reactive substances (TBARS) and in a second step, the process was monitored for a 26 h period by high-temperature thermoluminescence (TL) emission, showing a close correlationship with the TBARS data. Differences in the temperature-associated F0 rise (constant fluorescence) and in fluorescence emission spectra point to a progressive destabilization of the thylakoid membrane, especially affecting Photosystem II (PS II). In parallel, the PS II quantum efficiency ΔF / Fm and the Fv / Fm ratio of chlorophyll fluorescence induction decreased significantly over the 24 h period.
[Show abstract][Hide abstract] ABSTRACT: With the exception of Phytophthora parasitica var. nicotianae (Ppn), the tobacco black-shank causing agent, Phytophthoras give rise to non-host interactions with tobacco. The resulting
local hypersensitive response (HR) is accompanied by necrotic spots on the leaves at distance from the infection site . Low molecular weight proteins are excreted by these Phytophthoras, both in planta and in vitro. They form a family of highly homologous holoproteins, called elicitins . Tobacco plants treated with purified elicitins develop necrotic symptoms similar to those induced by the live fungus, and
become resistant to further inoculation with Ppn . Elicitin-treated tobacco represent an attractive model for the analysis of HR and its relation to acquired resistance.
[Show abstract][Hide abstract] ABSTRACT: Changes in lipid composition occurred when tobacco cells (Nicotiana tabacum var. Xanthi) were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. The most striking change was an increase in acylated steryl glycosides and steryl esters levels, certainly resulting from the glycosylation and/or esterification of free sterols. Moreover, in vivo pulse-labelling experiments with [14C]acetate also showed that a progressive decline in the incorporation rate of [14C]acetate into free sterols started with the induction of sesquiterpenoid synthesis and lasted when sesquiterpenoid synthesis stops. This phenomenon was accompanied by a significant increase in the synthesis rate of phosphatidylethanolamine occuring after a period of 12 h (80% of [14C]incorporated into lipids was found in PE). These pulse-labelling experiments also indicated a transient neosynthesis of high levels of acylated steryl glycosides and steryl esters in elicited cells. These results demonstrated that glycosylation and/or esterification of sterols (preexistent or neosynthetised) is an initial event among plant cell responses associated with the hypersensitive reaction.
[Show abstract][Hide abstract] ABSTRACT: Among the secondary metabolites produced by Cercospora beticola, two new compounds have been isolated and their structures elucidated by NMR and MS analysis : they have the same basic skeleton as beticolins 1 and 2 and were named beticolins 3 and 4.
[Show abstract][Hide abstract] ABSTRACT: Tobacco cells treated with cryptogein, an extracellular protein produced by Phytophthora cryptogea, accumulated ethylene and sesquiterpenoid compounds, mainly capsidiol. This protein, which causes hypersensitive-like necroses and ultimately makes tobacco resistant to the pathogen P. parasitica var. nicotianae is, therefore, an elicitor of defence mechanisms.
[Show abstract][Hide abstract] ABSTRACT: The activity of 23 substituted ureas was assayed on nonphotosynthetic Acer pseudoplatanus cells, in batch suspension culture. They inhibited growth, stimulated oxygen consumption of cells, and caused a change in the pH of the culture medium. Some substituted ureas could modify the membrane permeability to protons, whereas some others could change the membrane permeabilities to ions and neutral solutes.
[Show abstract][Hide abstract] ABSTRACT: Twelve a and b 20S proteasome subunits cDNAs showing 70-82% identity with the corresponding genes in Arabidopsis or rice, and features of eukaryotic proteasome subunits were cloned in tobacco. Only b1-tcI 7, a3 and a6, 20S proteasome subunits encoding genes were up-regulated by cryptogein, a proteinaceous elicitor of plant defence reactions. These results led to the hypothesis that the activation of b1-tcI 7, a3 and a6 could induce a specific proteolysis involved in the hypersensitive response and systemic acquired resistance monitored by cryptogein. In eukaryotes, the 26S proteasome is the central multicatalytic proteinase complex comprising two subcomplexes: the 20S core particle that performs proteolysis and the 19S regulatory particle that recognizes the protein targeted for degradation. The 20S proteasome is a stack of four seven-membered rings, the two outer rings being formed by seven a sub- units and the two inner rings by seven b subunits. The central b rings enclose a cavity that houses the active sites allowing the progressive degradation of proteins. In mammals, proteasomes are shown to be involved in the degradation of misfolded proteins and also in biological functions such as cell cycle progression or cell death, and specific proteo- lysis such as the cleavage of propeptides in order to activate peptides