Marie-Thérèse Esquerré-Tugayé

Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity, such as 13-keto-9(Z),11(E),15(Z)-octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly, this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms.

A Novel Arabidopsis-Colletotrichum Pathosystem for the Molecular Dissection of Plant-Fungal Interactions

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.

Cell wall degrading enzymes, inhibitory proteins, and oligosaccharides participate in the molecular dialogue between plants and pathogens

The wall interface between plants and pathogens plays an important role in the outcome of their interactions. Studying the degradation of plant pectic polysaccharides by microbial pectinases, and of microbial β-glucans by plant glucanases has shown that these polymers are a source of oligosaccharides which elicit defence responses in plants. The extent of degradation appears to be controlled by the presence of inhibitory proteins which counteract enzyme hydrolysis. Thus, plant cell walls participate in the molecular dialogue established between plants and pathogens.

Cellulose Binding Domains of a Phytophthora Cell Wall Protein Are Novel Pathogen-Associated Molecular Patterns

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.

The CBEL glycoprotein of Phytophthora parasitica var-nicotianae is involved in cell wall deposition and adhesion to cellulosic substrates.

The cell wall of the oomycete plant pathogen Phytophthora parasitica var. nicotianae contains a protein called CBEL that shows cellulose-binding (CB), elicitor (E) of defense in plants and lectin-like (L) activities. The biological role of this molecule in Phytophthora was investigated by generating transgenic strains suppressed in CBEL expression. Phenotypic characterization of these strains showed that they were severely impaired in adhesion to a cellophane membrane, differentiation of lobed structures in contact with cellophane, and formation of branched aggregating hyphae on cellophane and on flax cellulose fibres. Infection assays revealed that the strains suppressed in CBEL expression were not greatly affected in pathogenicity and formed branched aggregating hyphae in contact with the roots of the host plant, thereby indicating that CBEL is involved in the perception of cellulose rather than in the morphogenesis of hyphal aggregates. Interestingly, the absence of CBEL was correlated with abnormal formation of papillae-like cell wall thickenings in vitro, suggesting that CBEL is involved in cell wall deposition in Phytophthora. Reverse genetics in oomycetes has long been hampered by their diploid nature and difficulties in transformation and regeneration. The gene inactivation approach reported in this work provides the first direct evidence for intrinsic functions of an elicitor and cell wall protein in oomycetes.

Cloning and Characterization of a cDNA Encoding an Elicitor of Phytophthora parasitica var. nicotianae That Shows Cellulose-Binding and Lectin-Like Activities

Phytophthora parasitica var. nicotianae produces a 34-kDa glycoprotein elicitor (CBEL) that is localized in the cell wall. A cDNA encoding the protein moiety of this elicitor was cloned and characterized. The deduced amino acid sequence consisted of two direct repeats of a cysteine-rich domain, joined by a Thr/Pro-rich region. Although having no general homology with published sequences, the positions of the cysteine residues in the two repeats show a conserved pattern, similar to that of the cellulose-binding domain of fungal glycanases. CBEL did not possess hydrolytic activity on a variety of glycans, but bound to fibrous cellulose and plant cell walls. In addition, it exerted a lectin-like hemagglutinating activity. Infiltration of tobacco leaves (cultivar 46-8) with this molecule elicited necrosis and defense gene expression at 150 nM. Elicitor pretreatment of this tobacco cultivar resulted in protection against subsequent inoculation with an otherwise virulent race of P. parasitica var. nicotianae. All these biological activities were exerted within a low concentration range. This is the first report that a fungal elicitor exhibits cellulose-binding and lectin-like activities. The possible implications of such a multifunctional elicitor in plant-microbe interactions are discussed.

Lipoxygenase Gene Expression in the Tobacco-Phytophthora parasitica nicotianae Interaction

A recently isolated cDNA clone of tobacco (Nicotiana tabacum L.) lipoxygenase (LOX) was used to study LOX gene expression in tobacco cell-suspension cultures and intact plants in response to infection with Phytophthora parasitica nicotianae (Ppn). Southern blot analysis of tobacco DNA indicated that only a small number of LOX genes hybridize to this probe. These genes were not constitutively expressed to a detectable level in control cells and healthy plants. In contrast, a rapid and transient accumulation of transcripts occurred in cells and plants after treatment with elicitor and inoculation with zoospores of Ppn, respectively. In cell cultures LOX gene expression could also be induced by linolenic acid, a LOX substrate, and by methyl jasmonate, one of the products derived from the action of LOX on linolenic acid. In the infection assays, LOX gene expression and enzyme activity were observed earlier when the plants carried a resistance gene against the race of Ppn used for inoculation. The differential expression of LOX during the race-cultivar-specific interaction between tobacco and Ppn, as well as its regulation by elicitors and jasmonate, suggest a role of LOX in plant resistance and establishment of the defense status against this pathogen.

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii.

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.

Endopolygalacturonase genes from Colletotrichum lindemuthianum : Cloning of CLPG2 and comparison of its expression to that of CLPG1 during saprophytic and parasitic growth of the fungus

Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.

Ulvan, a Sulfated Polysaccharide from Green Algae, Activates Plant Immunity through the Jasmonic Acid Signaling Pathway

The industrial use of elicitors as alternative tools for disease control needs the identification of abundant sources of them. We report on an elicitor obtained from the green algae Ulva spp. A fraction containing most exclusively the sulfated polysaccharide known as ulvan-induced expression of a GUS gene placed under the control of a lipoxygenase gene promoter. Gene expression profiling was performed upon ulvan treatments on Medicago truncatula and compared to phytohormone effects. Ulvan induced a gene expression signature similar to that observed upon methyl jasmonate treatment (MeJA). Involvement of jasmonic acid (JA) in ulvan response was confirmed by detecting induction of protease inhibitory activity and by hormonal profiling of JA, salicylic acid (SA) and abscisic acid (ABA). Ulvan activity on the hormonal pathway was further consolidated by using Arabidopsis hormonal mutants. Altogether, our results demonstrate that green algae are a potential reservoir of ulvan elicitor which acts through the JA pathway.