J. Fournier

Isolation and sequence analysis of Clpgl, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race beta and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed on ORF encoding a 363-amino-acid (aa) polypeptide beginning with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5 non-coding region. This genomic clone was thereafter designated Clpg1. Southern analysis, performed with a Clpg1-specific probe, showed that this gene is present as a single copy in the Cl genome.

Differential regulation in tobacco cell suspensions of genes involved in plant-bacteria interactions by pathogen-related signals

Six cDNA clones whose corresponding mRNAs accumulate early during the hypersensitive reaction in tobacco leaves have been classified into 2 groups according to their maximum levels of accumulation in an incompatible versus a compatible interaction withPseudomonas solanacearum. We present evidence that, at least in the first stages of the interaction, tobacco cell suspensions retain the ability to respond differentially to compatible and incompatible isolates ofP. solanacearum.In addition, studies on the effect of a fungal elicitor on the accumulation of the mRNAs corresponding to the cDNA clones in cell suspensions indicate that only one group of genes responds to this treatment.

Coordinated transcriptional regulation of the divinyl ether biosynthetic genes in tobacco by signal molecules related to defense.

In tobacco, 9-divinyl ethers (DVEs) produced by the lipoxygenase NtLOX1 and DVE synthase NtDES1 are important for full resistance to pathogens. In this work, the regulation of NtLOX1 and NtDES1 expression by signal molecules was investigated in LOX1 promoter-reporter transgenic plants and by RT-qPCR. Methyl jasmonate, ACC and elicitor were shown to coordinately trigger the DVE pathway. Induction was strongly attenuated in the presence of salicylic acid, which seems to act as a negative regulator of the 9-DVE biosynthetic enzymes. Our data suggest that, in tobacco, DVE biosynthesis is cross-regulated by jasmonates, and by other hormonal and signal molecules such as ethylene and SA.

Hydroxyproline-containing fragments in the cell wall of Phytophthora parasitica

Abstract The composition of a cell wall derived elicitor preparation from Phytophthora parasitica var. nicotianae which induced ethylene and proteinase inhibitor production in tobacco plants, was analysed. The peptide part which accounted for 77% of the dry mass was characterized by high levels of Glx, Ala, Thr, Asx, Ser and Hyp; the carbohydrate moiety was mainly mannose and glucose. Unlike most plants, in Phytophthora cell walls and elicitor, most of the Hyp residues were non-glycosylated. The glycosylated Hyp (7%) carried oligoarabinose side chains composed of three or four arabinose residues.

Lipoxygenases in Plant Signalling

Lipoxygenases have been reported since a long time in Animals as well as in Plants. They catalyze the oxidation of polyunsaturated fatty acids released from membranes by the prior action of phospholipases. In Animals, the hydroxyperoxides that are formed from arachidonic acid are converted into leukotrienes and lipoxins which play important roles during inflammation. In Plants, they have been mainly described in seeds and storage organs. At the DNA level, a comparison of nucleotide sequences indicates that the extent of homology is quite variable between species. In the recent years, it was found that lipoxygenase activity is enhanced in response to pathogens and their elicitors. In race-cultivar specific interactions, early increases accompany the development of incompatibility. The data obtained on the Tobacco-Phytophthora parasitica nicotianae system strongly support the hypothesis that lipoxygenase plays a key role in the transduction cascade leading from recognition to defence induction in the host.

Ethylene in Early Signalling Phenomena at the Plant-Microorganism Interface

An early increase in ethylene production was recorded in melon, tobacco and alfalfa in response to infection by Colletotrichum, Phytophthora and Rhizobium respectively. This effect was mimicked by elicitor treatment of plant tissues and cells. Measurements of ACC synthase and ACC showed that the methionine pathway of ethylene biosynthesis was activated. The effect of exogenous and of endogenous ethylene on defence induction was investigated.

Cloning of an Elicitor-Induced Lipoxygenase cDNA from Tobacco

Plant lipoxygenases (Iinoleate:oxygen oxidoreductase, E.C. 1.13.11.12, LOX) are dioxygenases which catalyze the oxidation of polyunsaturated fatty acids (PUFAs) such as linoleic acid or linolenic acid by molecular oxygen. The hydroperoxides formed by LOX action can be converted into a series of compounds, some of them showing biological activity (e.g. traumatic acid, jasmonic acid). We previously reported an increase of LOX activity in tobacco (Nicotiana tabacum) cell suspensions treated with an elicitor preparation from the cell walls of Phytophthora parasitica var. nicotianae (Ppn), the causal agent of black shank disease in this plant [1, 2]. The induced LOX is supposed to be involved in a signaling pathway leading to the production of defense proteins via membrane lipid metabolisation, and biosynthesis of PUFAderived compounds such as jasmonic acid. The latter is able to induce defense gene expression in plants [3]. In order to obtain a suitable probe for studying LOX gene expression in tobacco, we prepared and screened a cDNA library of elicitor-treated tobacco cells. Here, we report the isolation and characterization of a LOX cDNA clone. It corresponds to a gene which is not expressed in tobacco cells under standard culture conditions and responds to elicitor treatment.

Cellular and Molecular Approaches of Defense in Plants

The signals exchanged during plant-microorganism interactions account for a three-step phenomenon leading from signal generation to cell responses, via transduction pathways. Recent reviews on signalling molecules and on their effects have been published (Darvill and Albersheim, 1984; Ryan, 1988). Attempts to integrate general features and more specific data from our work are presented.

Signals and Defense Responses Associated with the Race-Cultivar Specific Interaction Between Tobacco and Phytophthora Parasitica Nicotianae

Cell surface interactions between plants and pathogens induce defense responses in plants. In the past twenty years, it has been established that cell wall fragments of either pathogen or plant origin have the ability to induce the same defense reactions as pathogen attack. Such fragments, also called elicitors, are supposed to be among the earliest signals of the interaction (Darvill and Albersheim 1984). Distinct classes of elicitors (oligosaccharides, glycans, glycopeptides, peptides, lipids) have thus been identified. Similarly, a wide array of defense responses has been characterized, and several relevant genes and products isolated (Collinge and Slusarenko 1987). Despite such advances, our knowledge of transduction events leading from elicitor or pathogen recognition to defense gene expression remains scanty. Understanding signal transduction pathways requires the availability of model systems on which cellular and molecular approaches can be performed. The tobacco-Phytophthora parasitica nicotianae system is presented in this paper. A biochemical study of host responses, possibly related to signal processing in this system, is then reported.

Molecular characterization and expression of Colletotrichum lindemuthianum genes encoding endopolygalacturonase

Abstract Colletotrichum lindemuthianum , a fungal pathogen causing anthracnose on bean seedlings, secretes an endopolygalacturonase (endoPG) when grown on liquid medium containing pectin. The endolytic nature of the enzyme was demonstrated both by chemical analysis of the products it releases from the substrate, and through its specific interaction with the Polygalacturonase Inhibitory Protein (PGIP) isolated from the host-plant. Oligodeoxyribonucleotide primers designed from the N-terminal amino acid sequence of the endoPG of C. lindemuthianum race s and from an internal sequence conserved among different fungal endoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A fragment of 542-bp was obtained and used as a probe to screen a partial genomic library of C. lindemuthianum . Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363 amino-acid polypeptide showing a high degree of homology to ten fungal endoPG sequences. This genomic clone was thereafter designated Clpgl . Southern analysis, performed with a Clpgl -specific probe, showed that this gene is present as a single copy in the C. lindemuthianum genome.