The Phytophthora parasitica cellulose-binding elicitor lectin, (CBEL), is a cell wall-localized protein playing a key role in cell wall organization and adhesion of the mycelium to cellulosic substrates. CBEL is a potent elicitor of plant immune responses and this activity is linked to its ability to bind plant cell wall components. In order to scale up the production of active CBEL, we reported here the cloning and expression of a His-tagged version of CBEL in the yeast Pichia pastoris. Selection of a high-producing P. pastoris clone and optimization of the purification procedure allowed a yield of about 2mg of pure protein per liter of culture filtrate. The identity of the recombinant protein was confirmed by western-blot analysis, N-terminal protein sequencing, and by peptide mass fingerprinting. The cellulose-binding affinity and the lectin activity of the recombinant protein were identical to the native CBEL. Its elicitor activity, tested on Arabidopsis thaliana leaves, was similar to the native CBEL protein as it displays a similar biological activity on plant immune responses inducing defense gene expression and localized necroses of the infiltrated leaf tissues. The present work suggests that P. pastoris can be a suitable host for the production of compounds active on plants or for the development of new agricultural products able to stimulate plant immunity.

The soil-borne oomycete pathogen Aphanomyces euteiches causes devastating root rot diseases in legumes such as pea and alfalfa. The different pathotypes of A. euteiches have been shown to exhibit differential quantitative virulence, but the molecular basis of host adaptation has not yet been clarified. Here, we re-sequenced a pea field reference strain of A. euteiches ATCC201684 with PacBio long-reads and took advantage of the technology to generate the mitochondrial genome. We identified that the secretome of A. euteiches is characterized by a large portfolio of secreted proteases and carbohydrate-active enzymes (CAZymes). We performed Illumina sequencing of four strains of A. euteiches with contrasted specificity to pea or alfalfa and found in different geographical areas. Comparative analysis showed that the core secretome is largely represented by CAZymes and proteases. The specific secretome is mainly composed of a large set of small, secreted proteins (SSP) without any predicted functional domain, suggesting that the legume preference of the pathogen is probably associated with unknown functions. This study forms the basis for further investigations into the mechanisms of interaction of A. euteiches with legumes.

Oomycetes 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.

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, Crinkler's, 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.

Oomycetes are microorganisms that are distantly related to true fungi and many members of this phylum are major plant pathogens. Oomycetes express proteins that are able to interact with plant cell wall polysaccharides, such as cellulose. This interaction is thought to be mediated by carbohydrate-binding modules that are classified into CBM family 1 in the CAZy database. In this study, the two CBMs (1-1 and 1-2) that form part of the cell wall glycoprotein, CBEL, from Phytophthora parasitica have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained indicate that CBEL's CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a fungal family 1 CBM, has produced two variants. The first one lacks its native CBM, whereas the second contains the CBEL CBM1-1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on wheat straw. However, intriguingly the addition of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB variant lacking a CBM1. This suggests that the potentiating effect of CBM1-1 might not require the formation of a covalent linkage to TvXynB.

Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs) and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids.

Oomycetes are a group of filamentous eukaryotic microorganisms that have colonized all terrestrial and oceanic ecosystems, and they include prominent plant pathogens. The Aphanomyces genus is unique in its ability to infect both plant and animal species, and as such exemplifies oomycete versatility in adapting to different hosts and environments. Dissecting the underpinnings of oomycete diversity provides insights into their specificity and pathogenic mechanisms.

Pythium oligandrum is a soil born free living oomycete able to parasitize fungi and oomycetes prey, including important plant and animals pathogens. Pythium oligandrum can colonize endophytically the root tissues of diverse plants where it induces plant defenses. Here we report the first long-read genome sequencing of a P. oligandrum strain sequenced by PacBio technology. Sequencing of genomic DNA loaded onto six SMRT cells permitted the acquisition of 913,728 total reads resulting in 112X genome coverage. The assembly and polishing of the genome sequence yielded180 contigs (N50 = 1.3 Mb; L50 = 12). The size of the genome assembly is 41.9 Mb with a longest contig of 2.7 Mb and 15,007 predicted protein-coding genes among which 95.25% were supported by RNAseq data, thus constituting a new Pythium genome reference. This data will facilitate genomic comparisons of Pythium species that are commensal, beneficial or pathogenic on plant, or parasitic on fungi and oomycete to identify key genetic determinants underpinning their diverse lifestyles. In addition comparison with plant pathogenic or zoopathogenic species will illuminate genomic adaptations for pathogenesis toward widely diverse hosts.

The cellulose binding elicitor lectin (CBEL) of the genus Phytophthora induces necrosis and immune responses in several plant species, including Arabidopsis thaliana. However, the role of CBEL-induced responses in the outcome of the interaction is still unclear. This study shows that some of CBEL-induced defence responses, but not necrosis, required the receptor-like kinase BAK1, a general regulator of basal immunity in Arabidopsis, and the production of a reactive oxygen burst mediated by respiratory burst oxidases homologues (RBOH). Screening of a core collection of 48 Arabidopsis ecotypes using CBEL uncovered a large variability in CBEL-induced necrotic responses. Analysis of non-responsive CBEL lines Ws-4, Oy-0, and Bla-1 revealed that Ws-4 and Oy-0 were also impaired in the production of the oxidative burst and expression of defence genes, whereas Bla-1 was partially affected in these responses. Infection tests using two Phytophthora parasitica strains, Pp310 and Ppn0, virulent and avirulent, respectively, on the Col-0 line showed that BAK1 and RBOH mutants were susceptible to Ppn0, suggesting that some immune responses controlled by these genes, but not CBEL-induced cell death, are required for Phytophthora parasitica resistance. However, Ws-4, Oy-0, and Bla-1 lines were not affected in Ppn0 resistance, showing that natural variability in CBEL responsiveness is not correlated to Phytophthora susceptibility. Overall, the results uncover a BAK1- and RBOH-dependent CBEL-triggered immunity essential for Phytophthora resistance and suggest that natural quantitative variation of basal immunity triggered by conserved general elicitors such as CBEL does not correlate to Phytophthora susceptibility.

Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.