An oomycete effector targets a plant RNA helicase involved in root development and defense.

Oomycete plant pathogens secrete effector proteins to promote disease. The damaging soilborne legume pathogen Aphanomyces euteiches harbors a specific repertoire of Small Secreted Protein effectors (AeSSPs), but their biological functions remain unknown. Here we characterize AeSSP1256. The function of AeSSP1256 is investigated by physiological and molecular characterization of Medicago truncatula roots expressing the effector. A potential protein target of AeSSP1256 is identified by yeast-two hybrid, co-immunoprecipitation, and fluorescent resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) assays, as well as promoter studies and mutant characterization. AeSSP1256 impairs M. truncatula root development and promotes pathogen infection. The effector is localized to the nucleoli rim, triggers nucleoli enlargement and downregulates expression of M. truncatula ribosome-related genes. AeSSP1256 interacts with a functional nucleocytoplasmic plant RNA helicase (MtRH10). AeSSP1256 relocates MtRH10 to the perinucleolar space and hinders its binding to plant RNA. MtRH10 is associated with ribosome-related genes, root development and defense. This work reveals that an oomycete effector targets a plant RNA helicase, possibly to trigger nucleolar stress and thereby promote pathogen infection.

Phyllosphere Colonization by a Soil Streptomyces sp. Promotes Plant Defense Responses Against Fungal Infection.

Streptomycetes are soil-dwelling, filamentous actinobacteria and represent a prominent bacterial clade inside the plant root microbiota. The ability of streptomycetes to produce a broad spectrum of antifungal metabolites suggests that these bacteria could be used to manage plant diseases. Here, we describe the identification of a soil Streptomyces strain named AgN23 which strongly activates a large array of defense responses when applied on Arabidopsis thaliana leaves. AgN23 increased the biosynthesis of salicylic acid, leading to the development of salicylic acid induction deficient 2 (SID2)-dependent necrotic lesions. Size exclusion fractionation of plant elicitors secreted by AgN23 showed that these signals are tethered into high molecular weight complexes. AgN23 mycelium was able to colonize the leaf surface, leading to plant resistance against Alternaria brassicicola infection in wild-type Arabidopsis plants. AgN23-induced resistance was found partially compromised in salicylate, jasmonate, and ethylene mutants. Our data show that Streptomyces soil bacteria can develop at the surface of plant leaves to induce defense responses and protection against foliar fungal pathogens, extending their potential use to manage plant diseases.

MS-CleanR: A Feature-Filtering Workflow for Untargeted LC-MS Based Metabolomics.

Untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) is currently the gold-standard technique to determine the full chemical diversity in biological samples. However, this approach still has many limitations; notably, the difficulty of accurately estimating the number of unique metabolites profiled among the thousands of MS ion signals arising from chromatograms. Here, we describe a new workflow, MS-CleanR, based on the MS-DIAL/MS-FINDER suite, which tackles feature degeneracy and improves annotation rates. We show that implementation of MS-CleanR reduces the number of signals by nearly 80% while retaining 95% of unique metabolite features. Moreover, the annotation results from MS-FINDER can be ranked according to the database chosen by the user, which enhance identification accuracy. Application of MS-CleanR to the analysis of Arabidopsis thaliana grown in three different conditions fostered class separation resulting from multivariate data analysis and led to annotation of 75% of the final features. The full workflow was applied to metabolomic profiles from three strains of the leguminous plant Medicago truncatula that have different susceptibilities to the oomycete pathogen Aphanomyces euteiches. A group of glycosylated triterpenoids overrepresented in resistant lines were identified as candidate compounds conferring pathogen resistance. MS-CleanR is implemented through a Shiny interface for intuitive use by end-users (available at https://github.com/eMetaboHUB/MS-CleanR).

Lipo-chitooligosaccharide signalling blocks a rapid pathogen-induced ROS burst without impeding immunity.

Molecular signals released by microbes at the surface of plant roots and leaves largely determine host responses, notably by triggering either immunity or symbiosis. How these signalling pathways cross-talk upon coincident perception of pathogens and symbionts is poorly described in plants forming symbiosis. Nitrogen fixing symbiotic Rhizobia spp. and arbuscular mycorrhizal fungi produce lipo-chitooligosaccharides (LCOs) to initiate host symbiotic programmes. In Medicago truncatula roots, the perception of LCOs leads to reduced efflux of reactive oxygen species (ROS). By contrast, pathogen perception generally triggers a strong ROS burst and activates defence gene expression. Here we show that incubation of M. truncatula seedlings with culture filtrate (CF) of the legume pathogen Aphanomyces euteiches alone or simultaneously with Sinorhizobium meliloti LCOs, resulted in a strong ROS release. However, this response was completely inhibited if CF was added after pre-incubation of seedlings with LCOs. By contrast, expression of immunity-associated genes in response to CF and disease resistance to A. euteiches remained unaffected by LCO treatment of M. truncatula roots. Our findings suggest that symbiotic plants evolved ROS inhibition response to LCOs to facilitate early steps of symbiosis whilst maintaining a parallel defence mechanisms toward pathogens.

A local score approach improves GWAS resolution and detects minor QTL: application to Medicago truncatula quantitative disease resistance to multiple Aphanomyces euteiches isolates.

Quantitative trait loci (QTL) with small effects, which are pervasive in quantitative phenotypic variation, are difficult to detect in genome-wide association studies (GWAS). To improve their detection, we propose to use a local score approach that accounts for the surrounding signal due to linkage disequilibrium, by accumulating association signals from contiguous single markers. Simulations revealed that, in a GWAS context with high marker density, the local score approach outperforms single SNP p-value-based tests for detecting minor QTL (heritability of 5-10%) and is competitive with regard to alternative methods, which also aggregate p-values. Using more than five million SNPs, this approach was applied to identify loci involved in Quantitative Disease Resistance (QDR) to different isolates of the plant root rot pathogen Aphanomyces euteiches, from a GWAS performed on a collection of 174 accessions of the model legume Medicago truncatula. We refined the position of a previously reported major locus, underlying MYB/NB-ARC/tyrosine kinase candidate genes conferring resistance to two closely related A. euteiches isolates belonging to pea pathotype I. We also discovered a diversity of minor resistance QTL, not detected using p-value-based tests, some of which being putatively shared in response to pea (pathotype I and III) and/or alfalfa (race 1 and 2) isolates. Candidate genes underlying these QTL suggest pathogen effector recognition and plant proteasome as key functions associated with M. truncatula resistance to A. euteiches. GWAS on any organism can benefit from the local score approach to uncover many weak-effect QTL.

Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation.

BACKGROUND: 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. RESULTS: By carrying out genomic analyses of the plant pathogen A. euteiches and the crustacean pathogen A. astaci, we show that host specialization is correlated with specialized secretomes that are adapted to the deconstruction of the plant cell wall in A. euteiches and protein degradation in A. astaci. The A. euteiches genome is characterized by a large repertoire of small secreted protein (SSP)-encoding genes that are highly induced during plant infection, and are not detected in other oomycetes. Functional analysis revealed an SSP from A. euteiches containing a predicted nuclear-localization signal which shuttles to the plant nucleus and increases plant susceptibility to infection. CONCLUSION: Collectively, our results show that Aphanomyces host adaptation is associated with evolution of specialized secretomes and identify SSPs as a new class of putative oomycete effectors.

High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays.

BACKGROUND: The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment. RESULTS: We have developed a pioneering tool ("NeoViGen96" chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4 h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the "NeoViGen96" chip as well as their efficacy against downy mildew. BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway. FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes. CONCLUSIONS: The NeoViGen96" chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4 h and 1,500 €), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270 h and 9,000 €) thus a throughput 60-70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.

Genomic Signature of Selective Sweeps Illuminates Adaptation of Medicago truncatula to Root-Associated Microorganisms.

Medicago truncatula is a model legume species used to investigate plant-microorganism interactions, notably root symbioses. Massive population genomic and transcriptomic data now available for this species open the way for a comprehensive investigation of genomic variations associated with adaptation of M. truncatula to its environment. Here we performed a fine-scale genome scan of selective sweep signatures in M. truncatula using more than 15 million single nucleotide polymorphisms identified on 283 accessions from two populations (Circum and Far West), and exploited annotation and published transcriptomic data to identify biological processes associated with molecular adaptation. We identified 58 swept genomic regions with a 15 kb average length and comprising 3.3 gene models on average. The unimodal sweep state probability distribution in these regions enabled us to focus on the best single candidate gene per region. We detected two unambiguous species-wide selective sweeps, one of which appears to underlie morphological adaptation. Population genomic analyses of the remaining 56 sweep signatures indicate that sweeps identified in the Far West population are less population-specific and probably more ancient than those identified in the Circum population. Functional annotation revealed a predominance of immunity-related adaptations in the Circum population. Transcriptomic data from accessions of the Far West population allowed inference of four clusters of coregulated genes putatively involved in the adaptive control of symbiotic carbon flow and nodule senescence, as well as in other root adaptations upon infection with soil microorganisms. We demonstrate that molecular adaptations in M. truncatula were primarily triggered by selective pressures from root-associated microorganisms.

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. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-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.

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, 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.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. • A high-density (≈5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. • GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (σ(A)²= 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. • These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

Root-associated Streptomyces produce galbonolides to modulate plant immunity and promote rhizosphere colonization.

The rhizosphere, which serves as the primary interface between plant roots and the soil, constitutes an ecological niche for a huge diversity of microbial communities. Currently, there is little knowledge on the nature and the function of the different metabolites released by rhizospheric microbes to facilitate colonization of this highly competitive environment. Here, we demonstrate how the production of galbonolides, a group of polyene macrolides that inhibit plant and fungal inositol phosphorylceramide synthase (IPCS), empowers the rhizospheric Streptomyces strain AgN23, to thrive in the rhizosphere by triggering the plant's defence mechanisms. Metabolomic analysis of AgN23-inoculated Arabidopsis roots revealed a strong induction in the production of an indole alkaloid, camalexin, which is a major phytoalexin in Arabidopsis. By using a plant mutant compromized in camalexin synthesis, we show that camalexin production is necessary for the successful colonization of the rhizosphere by AgN23. Conversely, hindering galbonolides biosynthesis in AgN23 knock-out mutant resulted in loss of inhibition of IPCS, a deficiency in plant defence activation, notably the production of camalexin, and a strongly reduced development of the mutant bacteria in the rhizosphere. Together, our results identified galbonolides as important metabolites mediating rhizosphere colonization by Streptomyces.

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.

Pathogen-associated molecular pattern-triggered immunity and resistance to the root pathogen Phytophthora parasitica in Arabidopsis.

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.

The mycoparasite Pythium oligandrum induces legume pathogen resistance and shapes rhizosphere microbiota without impacting mutualistic interactions.

Pythium oligandrum is a soil-borne oomycete associated with rhizosphere and root tissues. Its ability to enhance plant growth, stimulate plant immunity and parasitize fungal and oomycete preys has led to the development of agricultural biocontrol products. Meanwhile, the effect of P. oligandrum on mutualistic interactions and more generally on root microbial communities has not been investigated. Here, we developed a biological system comprising P. oligandrum interacting with two legume plants, Medicago truncatula and Pisum sativum. P. oligandrum activity was investigated at the transcriptomics level through an RNAseq approach, metabolomics and finally metagenomics to investigate the impact of P. oligandrum on root microbiota. We found that P. oligandrum promotes plant growth in these two species and protects them against infection by the oomycete Aphanomyces euteiches, a devastating legume root pathogen. In addition, P. oligandrum up-regulated more than 1000 genes in M. truncatula roots including genes involved in plant defense and notably in the biosynthesis of antimicrobial compounds and validated the enhanced production of M. truncatula phytoalexins, medicarpin and formononetin. Despite this activation of plant immunity, we found that root colonization by P. oligandrum did not impaired symbiotic interactions, promoting the formation of large and multilobed symbiotic nodules with Ensifer meliloti and did not negatively affect the formation of arbuscular mycorrhizal symbiosis. Finally, metagenomic analyses showed the oomycete modifies the composition of fungal and bacterial communities. Together, our results provide novel insights regarding the involvement of P. oligandrum in the functioning of plant root microbiota.

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

BACKGROUND: 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. RESULTS: To 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. CONCLUSIONS: This 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.

A Comprehensive Assessment of the Secretome Responsible for Host Adaptation of the Legume Root Pathogen Aphanomyces euteiches.

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.

Comparative Transcriptomics Suggests Early Modifications by Vintec® in Grapevine Trunk of Hormonal Signaling and Secondary Metabolism Biosynthesis in Response to Phaeomoniella chlamydospora and Phaeoacremonium minimum.

Given their well-known antifungal abilities, species of the genus Trichoderma are of significant interest in modern agriculture. Recent studies have shown that Trichoderma species can induce plant resistance against different phytopathogens. To further extend this line of investigation, we investigate herein the transcriptomic response of grapevine trunk to Vintec®, which is a Trichoderma atroviride SC1-based commercial formulation for biological control of grapevine trunk diseases and which reduces wood colonization. The aim of the study is to understand whether the biocontrol agent Vintec® modifies the trunk response to Phaeoacremonium minimum and Phaeomoniella chlamydospora, which are two esca-associated fungal pathogens. An analysis of transcriptional regulation identifies clusters of co-regulated genes whose transcriptomic reprogramming in response to infection depends on the absence or presence of Vintec®. On one hand, the results show that Vintec® differentially modulates the expression of putative genes involved in hormonal signaling, especially those involved in auxin signaling. On the other hand, most significant gene expression modifications occur among secondary-metabolism-related genes, especially regarding phenylpropanoid metabolism and stilbene biosynthesis. Taken together, these results suggest that the biocontrol agent Vintec® induces wood responses that counteract disease development.

Modification of Early Response of Vitis vinifera to Pathogens Relating to Esca Disease and Biocontrol Agent Vintec® Revealed By Untargeted Metabolomics on Woody Tissues.

Esca disease is one of the most destructive grapevine trunk diseases. Phaeoacremonium minimum and Phaeomoniella chlamydospora are two of the known fungal pathogens associated with this disease. Today, biocontrol agents against Esca are mainly based on the use of the strain of the mycoparasite fungal genus Trichoderma such as the Vintec® product. The aim of this study was to investigate early response of woody tissues to Esca pathogens and identify metabolites that could be correlated with a biocontrol activity within a complex woody matrix. An untargeted liquid chromatography-high-resolution mass spectrometry metabolomic approach coupled to a spectral similarity network was used to highlight clusters of compounds associated with the plant response to pathogens and biocontrol. Dereplication highlighted the possible role of glycerophospholipids and polyphenol compounds, the latest mainly belonging to stilbenoids. Antifungal activity of some relevant biomarkers, evaluated in vitro on Phaeomoniella chlamydospora and Botrytis cinerea, suggests that some of these compounds can play a role to limit the development of Esca pathogens in planta.

Expression and purification of a biologically active Phytophthora parasitica cellulose binding elicitor lectin in Pichia pastoris.

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.