Yves Martinez

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.

Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1–. In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1– exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors’ knowledge, this is the first functional characterization of CCR1 in a grass species.

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.

Biochemical and phylogenetic analysis of CEBiP-like LysM domain-containing extracellular proteins in higher plants

The chitin elicitor-binding protein (CEBiP) from rice was the first plant lysin motif (LysM) protein for which the biological and biochemical function had been established. It belongs to a plant-specific family of extracellular LysM proteins (LYMs) for which we analyzed the phylogeny. LYMs are present in vascular plants only, where an early gene duplication event might have resulted in two types which were retained in present day genomes. LYMs consist of a signal peptide, three consecutive LysMs, separated by cysteine pairs, and a C-terminal region without any known signature, whose length allows the distinction between the two types, and which may be followed by a glycosylphosphatidylinositol (GPI) anchor motif. We analyzed a representative of each type, MtLYM1 and MtLYM2, from Medicago truncatula at the biochemical level and with respect to their expression patterns and observed some similarities but also marked differences. MtLYM1 and MtLYM2 proved to be very different with regard to abundance and apparent molecular mass on SDS-PAGE. Both undergo several post-translational modifications, including N-glycosylation and the addition of a GPI anchor, which would position the proteins at the outer face of the plasma membrane. Only MtLYM2, but not MtLYM1, showed specific binding to biotinylated N-acetylchitooctaose in a manner similar to CEBiP, which belongs to the same type. We postulate that LYM2-type proteins likely function in the perception of chitin-related molecules, whereas possible functions of LYM1-type proteins remain to be elucidated.

The sunflower downy mildew pathogen Plasmopara halstedii.

UNLABELLEDnDowny mildew of sunflower is caused by Plasmopara halstedii (Farlow) Berlese & de Toni. Plasmopara halstedii is an obligate biotrophic oomycete pathogen that attacks annual Helianthus species and cultivated sunflower, Helianthus annuus. Depending on the sunflower developmental stage at which infection occurs, the characteristic symptoms range from young seedling death, plant dwarfing, leaf bleaching and sporulation to the production of infertile flowers. Downy mildew attacks can have a great economic impact on sunflower crops, and several Pl resistance genes are present in cultivars to protect them against the disease. Nevertheless, some of these resistances have been overcome by the occurrence of novel isolates of the pathogen showing increased virulence. A better characterization of P.u2009halstedii infection and dissemination mechanisms, and the identification of the molecular basis of the interaction with sunflower, is a prerequisite to efficiently fight this pathogen. This review summarizes what is currently known about P.u2009halstedii, provides new insights into its infection cycle on resistant and susceptible sunflower lines using scanning electron and light microscopy imaging, and sheds light on the pathogenicity factors of P.u2009halstedii obtained from recent molecular data.nnnTAXONOMYnKingdom Stramenopila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Plasmopara; Species Plasmopara halstedii.nnnDISEASE SYMPTOMSnSunflower seedling damping off, dwarfing of the plant, bleaching of leaves, starting from veins, and visible white sporulation, initially on the lower side of cotyledons and leaves. Plasmopara halstedii infection may severely impact sunflower seed yield.nnnINFECTION PROCESSnIn spring, germination of overwintered sexual oospores leads to sunflower root infection. Intercellular hyphae are responsible for systemic plant colonization and the induction of disease symptoms. Under humid and fresh conditions, dissemination structures are produced by the pathogen on all plant organs to release asexual zoosporangia. These zoosporangia play an important role in pathogen dissemination, as they release motile zoospores that are responsible for leaf infections on neighbouring plants.nnnDISEASE CONTROLnDisease control is obtained by both chemical seed treatment (mefenoxam) and the deployment of dominant major resistance genes, denoted Pl. However, the pathogen has developed fungicide resistance and has overcome some plant resistance genes. Research for more sustainable strategies based on the identification of the molecular basis of the interaction are in progress.nnnUSEFUL WEBSITESnhttp://www.heliagene.org/HP, http://lipm-helianthus.toulouse.inra.fr/dokuwiki/doku.php?id=start, https://www.heliagene.org/PlasmoparaSpecies (soon available).

CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA‐binding proteins which trigger host DNA damage response

To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P.xa0capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A.xa0euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR.

Oxidative injury and antioxidant genes regulation in cadmium-exposed radicles of six contrasted Medicago truncatula genotypes.

Oxidative disorders were triggered in the presence of Cd toxicity in early seedling growth of six Medicago truncatula genotypes. Interactions between root growth inhibition, cadmium uptake, as well as the occurrence of oxidative injury suggest differential responses of the genotypes, with susceptible or tolerant accessions. ROS enhancement was observed in situ and H2O2 content was measured, that did not seem related to tolerance or susceptibility. Oxidative burst impact on cell membrane integrity was analyzed in agreement with MDA content and glucose exudation, which suggest an active role of this burst in susceptible lines. Transcriptional changes in response to cadmium treatment were analyzed on target genes involved in (1) ROS-scavenging enzymes (superoxide dismutase (SOD; EC1.15.1.1) and peroxidase (PRX; EC 1.11.1.7)), (2) reduced glutathione (γ-Glu-Cys-Gly, GSH) metabolism (glutathione-S-transferase (GST; EC: 2.5.1.18) and glutathione reductase (GR; EC 1.8.1.7)), and (3) metal-chelating metabolism (PCS). The susceptible line shows no response or non-timely gene expression patterns. This research work gave an overview of the deleterious effects and oxidative injury of cadmium stress in Medicago truncatula. Oxidative defense efficiency and gene upregulation should explain relative tolerance in tested genotypes.

The Arabidopsis Lipid Transfer Protein 2 (AtLTP2) Is Involved in Cuticle-Cell Wall Interface Integrity and in Etiolated Hypocotyl Permeability

Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family implicated in diverse physiological processes. However, their function and mode of action remain unclear probably because of functional redundancy. Among the different roles proposed for nsLTPs, it has long been suggested that they could transport cuticular precursor across the cell wall during the formation of the cuticle, which constitutes the first physical barrier for plant interactions with their aerial environment. Here, we took advantage of the Arabidopsis thaliana etiolated hypocotyl model in which AtLTP2 was previously identified as the unique and abundant nsLTP member in the cell wall proteome, to investigate its function. AtLTP2 expression was restricted to epidermal cells of aerial organs, in agreement with the place of cuticle deposition. Furthermore, transient AtLTP2-TagRFP over-expression in Nicotiana benthamiana leaf epidermal cells resulted in its localization to the cell wall, as expected, but surprisingly also to the plastids, indicating an original dual trafficking for a nsLTP. Remarkably, in etiolated hypocotyls, the atltp2-1 mutant displayed modifications in cuticle permeability together with a disorganized ultra-structure at the cuticle-cell wall interface completely recovered in complemented lines, whereas only slight differences in cuticular composition were observed. Thus, AtLTP2 may not play the historical purported nsLTP shuttling role across the cell wall, but we rather hypothesize that AtLTP2 could play a major structural role by maintaining the integrity of the adhesion between the mainly hydrophobic cuticle and the hydrophilic underlying cell wall. Altogether, these results gave new insights into nsLTP functions.

Assessment of Ustilago maydis as a fungal model for root infection studies

Ustilago maydis is a fungus infecting aerial parts of maize to form smutted galls. Due to its interest as a genetic tool in plant pathology, we evaluated its ability to penetrate into plant roots. The fungus can penetrate between epidermic root cells, forming inter and intracellular pseudohyphae. Root infection didnt provoke gall formation on the maize lines tested, and targeted PCR detection showed that U. maydis, unlike the other maize smut fungus Sporisorium reilianum, has a weak aptitude to grow from the roots up to the aerial part of maize. We also observed that U. maydis can infect Medicago truncatula hairy roots as an alternative host. This plant species is a model host to study root symbiosis, and this pathosystem can provide new insights on root-microbe interactions. Considering that U. maydis could be a soil fungus, we tested its responsiveness to GR24, a strigolactone analogue. Strigolactones are root exuded molecules which activate mitochondrial metabolism of arbuscular mycorrhizal (AM) fungi. Physiologic and molecular analysis revealed that GR24 also increases cell respiration of U. maydis. This result points out that strigolactones could have an incidence on several rhizospheric microbes. These data provide evidences that the biotrophic pathogen U. maydis has to be considered for studying root infection.

The life cycle of the smut fungus Moesziomyces penicillariae is adapted to the short-cycle of the host, Pennisetum glaucum

Moesziomyces penicillariae (Brefield) Vànky is a basidiomycete fungus responsible for smut disease on pearl millet, an important staple food in the sub-Sahelian zone. We revisited the life cycle of this fungus. Unlike other Ustilaginales, mating of sporidia was never observed and monoclonal cultures of monokaryotic sporidia were infectious in the absence of mating with compatible partner. These data argued for an atypical monokaryotic diploid cell cycle of M. penicillariae, where teliospores only form solopathogenic sporidia. After inoculation of monoclonal solopathogenic strains on spikelets, the fungus infects the ovaries and induces the folding of the micropilar lips, as observed during early pollination steps. The infected embryo then becomes disorganized and the fungus invades peripheral ovary tissues before sporulating. We evaluated the systemic growth abilities of the fungus. After root inoculation, mycelium was observed around and inside the roots. As argued by transmission electron microscopy (TEM) observations and polymerase chain reaction (PCR) detection using specific primers for M. penicillariae, the fungus can grow from roots to the caulinar meristems. In spite of this systemic growth, no sori were formed on the varieties of pearl millet tested after root inoculation. All together, these data suggest that the reduced life cycle of M. penicillariae--i.e. dispersal of ready to infect solopathogenic sporidia, floral infection--is an adaptation to the aetiology of this disease to short-cycle pearl millet varieties from the sub-Sahel.