Sugar beet ( Beta vulgaris L.) is an economically important crop in temperate climates providing nearly 30% of sugar production worldwide. The oomycete Aphanomyces cochlioides is the causative agent of seedling damping-off and root rot disease in sugar beet. The pathogen is responsible for plant degeneration and drastic yield losses in all major sugar beet producing areas. The identification of resistant germplasm is essential to reduce the use of chemical treatments as well as the costs of protective measures and to effectively limit the damage caused by the pathogen. In this study we aimed to establish a qPCR-based method to quantify the pathogen DNA in infected plants and to predict the resistance levels of different sugar beet genotypes in response to A. cochlioides . The difference in the response to A. cochlioides isolates with different geographical origins was investigated. In addition, confocal microscopy was performed in order to observe the spatial and temporal colonization pattern in infected seedlings of susceptible and partially resistant breeding lines. The research presented in this article provides a tool to understand the progress of the infection in infected tissues and to identify the genetic background of resistance to A. cochlioides that can be used to support breeding programs.

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete Aphanomyces euteiches, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional C-terminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in Aphanomyces genus.

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.

Supplementary dataset for chapter 4 of the thesis

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.

Protein–metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein–small molecule interactome in the model yeast Saccharomyces cerevisiae . By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/ . By interpolating PROMIS with the list of predicted protein–metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase ( Pgk1 ). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.

Parasitic Aphanomyces species are a global threat to agri- and aquaculture, causing multimillion USD damage every year. Via the global trade, Aphanomyces has spread across all continents with exception of South America and Antarctica, and has become a major problem in pea, sugar beet, fish and crayfish production. The widespread A. euteiches and A. cochlioides induce root rot diseases in leguminous species and sugar beet respectively. The fish pathogen A. invadans is the causative agent of Epizootic Ulcerative Syndrome in various fish species whilst A. astaci infects crayfishes causing crayfish plague. Aphanomyces have developed an efficient transmission and infection mechanism which allows a rapid colonization and disruption of the host's infected tissues. This review presents an overview on the current research on Aphanomyces genus. We summarise the latest research efforts on four pathogenic Aphanomyces species, shedding light on the biology of these microorganisms, the pathogenicity factors of these parasites, the diseases which they cause, their distribution and finally the strategies to control the diseases.