Arnaud Bottin

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.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinklers, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

NFP, a LysM protein controlling Nod factor perception, also intervenes in Medicago truncatula resistance to pathogens

Plant LysM proteins control the perception of microbial-derived N-acetylglucosamine compounds for the establishment of symbiosis or activation of plant immunity. This raises questions about how plants, and notably legumes, can differentiate friends and foes using similar molecular actors and whether any receptors can intervene in both symbiosis and resistance. To study this question, nfp and lyk3 LysM-receptor like kinase mutants of Medicago truncatula that are affected in the early steps of nodulation, were analysed following inoculation with Aphanomyces euteiches, a root oomycete. The role of NFP in this interaction was further analysed by overexpression of NFP and by transcriptome analyses. nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A. euteiches, whereas NFP overexpression increased resistance. Transcriptome analyses on A. euteiches inoculation showed that mutation in the NFP gene led to significant changes in the expression of c. 500 genes, notably involved in cell dynamic processes previously associated with resistance to pathogen penetration. nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M. truncatula immunity, suggesting an unsuspected role for NFP in the perception of pathogenic signals.

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Abstract. Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions.

Partial resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes.

A pathosystem between Aphanomyces euteiches, the causal agent of pea root rot disease, and the model legume Medicago truncatula was developed to gain insights into mechanisms involved in resistance to this oomycete. The F83005.5 French accession and the A17-Jemalong reference line, susceptible and partially resistant, respectively, to A. euteiches, were selected for further cytological and genetic analyses. Microscopy analyses of thin root sections revealed that a major difference between the two inoculated lines occurred in the root stele, which remained pathogen free in A17. Striking features were observed in A17 roots only, including i) frequent pericycle cell divisions, ii) lignin deposition around the pericycle, and iii) accumulation of soluble phenolic compounds. Genetic analysis of resistance was performed on an F7 population of 139 recombinant inbred lines and identified a major quantitative trait locus (QTL) near the top of chromosome 3. A second study, with near-isogenic line responses to A. euteiches confirmed the role of this QTL in expression of resistance. Fine-mapping allowed the identification of a 135-kb sequenced genomic DNA region rich in proteasome-related genes. Most of these genes were shown to be induced only in inoculated A17. Novel mechanisms possibly involved in the observed partial resistance are proposed.

Cell wall chitosaccharides are essential components and exposed patterns of the phytopathogenic oomycete Aphanomyces euteiches.

ABSTRACT Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-d-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants.

Sterol biosynthesis in oomycete pathogens

Oomycetes are a diverse group of filamentous eukaryotic microbes comprising devastating animal and plant pathogens. They share many characteristics with fungi, including polarized hyphal extension and production of spores but phylogenetics studies have clearly placed oomycetes outside the fungal kingdom, in the kingdom Stramenopila which also includes marine organisms such diatoms and brown algae. Oomycetes display various specific biochemical features, including sterol metabolism. Sterols are essential isoprenoid compounds involved in membrane function and hormone signaling. Oomycetes belonging to Peronosporales, such as Phytophthora sp., are unable to synthesize their own sterols and must acquire them from their plant or animal hosts. In contrast, a combination of biochemical and molecular approaches allowed us to decipher a nearly complete sterol biosynthetic pathway leading to fucosterol in the legume pathogen Aphanomyces euteiches, an oomycete belonging to Sparolegniales. Importantly, sterol demethylase, a key enzyme from this pathway, is susceptible to chemicals widely used in agriculture and medicine as antifungal drugs suggesting that similar products could be used against plant and animal diseases caused by Saprolegniales.

Aphanomyces euteiches cell wall fractions containing novel glucan-chitosaccharides induce defense genes and nuclear calcium oscillations in the plant host Medicago truncatula.

N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.

The unique architecture and function of cellulose-interacting proteins in oomycetes revealed by genomic and structural analyses

BackgroundOomycetes are fungal-like microorganisms evolutionary distinct from true fungi, belonging to the Stramenopile lineage and comprising major plant pathogens. Both oomycetes and fungi express proteins able to interact with cellulose, a major component of plant and oomycete cell walls, through the presence of carbohydrate-binding module belonging to the family 1 (CBM1). Fungal CBM1-containing proteins were implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding Elicitor Lectin (CBEL), a well-characterized CBM1-protein from Phytophthora parasitica, was implicated in cell wall integrity, adhesion to cellulosic substrates and induction of plant immunity.ResultsTo extend our knowledge on CBM1-containing proteins in oomycetes, we have conducted a comprehensive analysis on 60 fungi and 7 oomycetes genomes leading to the identification of 518 CBM1-containing proteins. In plant-interacting microorganisms, the larger number of CBM1-protein coding genes is expressed by necrotroph and hemibiotrophic pathogens, whereas a strong reduction of these genes is observed in symbionts and biotrophs. In fungi, more than 70% of CBM1-containing proteins correspond to enzymatic proteins in which CBM1 is associated with a catalytic unit involved in cellulose degradation. In oomycetes more than 90% of proteins are similar to CBEL in which CBM1 is associated with a non-catalytic PAN/Apple domain, known to interact with specific carbohydrates or proteins. Distinct Stramenopile genomes like diatoms and brown algae are devoid of CBM1 coding genes. A CBM1-PAN/Apple association 3D structural modeling was built allowing the identification of amino acid residues interacting with cellulose and suggesting the putative interaction of the PAN/Apple domain with another type of glucan. By Surface Plasmon Resonance experiments, we showed that CBEL binds to glycoproteins through galactose or N-acetyl-galactosamine motifs.ConclusionsThis study provides insight into the evolution and biological roles of CBM1-containing proteins from oomycetes. We show that while CBM1s from fungi and oomycetes are similar, they team up with different protein domains, either in proteins implicated in the degradation of plant cell wall components in the case of fungi or in proteins involved in adhesion to polysaccharidic substrates in the case of oomycetes. This work highlighted the unique role and evolution of CBM1 proteins in oomycete among the Stramenopile lineage.

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors

Key messageA fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity byM. truncatulain a newly designed versatile root elicitation assay.AbstractThe root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora β-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.