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.

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.

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 ribosomerelated 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.

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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.

26 Microbial effectors from plant pathogens are molecules that target host components to 27 facilitate colonization. While eukaryotic pathogens are virtually able to produce hundreds of 28 effectors, the molecular mechanisms allowing effectors to promote infection are still largely 29 unexplored. Here we show that the effector AeSSP1256 from the soilborne oomycete 30 pathogen Aphanomyces euteiches is able to interact with plant RNA. Heterologous expression 31 of AeSSP1256 delays Medicago truncatula host roots development and facilitate pathogen 32 colonization. Transcriptomic analyses of AeSSP1256-expressing roots show a downregulation 33 of genes implicated in ribosome biogenesis pathway. A yeast-two hybrid approach reveals 34 that AeSSP1256 associates with a nucleolar L7 ribosomal protein and a M. truncatula RNA 35 helicase (MtRH10) orthologous to the Arabidopsis RNA helicase RH10. Association of 36 AeSSP1256 with MtRH10 impaired the capacity of MtRH10 to bind nucleic acids. 37 Promoter:GUS composite plants revealed that MtRH10 is expressed preferentially in the 38 meristematic root cells. Missense MtRH10 plants displayed shorter roots with developmental 39 delay and are more susceptible to A. euteiches infection. These results show that the effector 40 AeSSP1256 facilitates pathogen infection by causing stress on plant ribosome biogenesis and 41 by hijacking a host RNA helicase involved in root development and resistance to root 42 pathogens. 43 105 ribosomal protein as interactors of AeSSP1256. By FRET-FLIM analyses we reveal that 106 AeSSP1256 co-opts MtRH10 to abolish its nucleic acid binding capacity. We provide a 107 mechanistic explanation of this observation by demonstrating the implication of MtRH10 in 108 roots development by generating missense and overexpressing Medicago lines. Finally we 109 5 observed that silenced-MtRH10 roots are highly susceptible to A. euteiches infection like 110 AeSSP1256-expressing roots, showing that MtRH10 as AeSSP1256 activities modify the 111 outcome of the infection. We now present results supporting effector-mediated manipulation 112 of a nuclear RNA helicase as a virulence mechanism during plant-eukaryotic pathogens 113 interactions.114 115 6 Results 116 117 AeSSP1256 contains RGG/RG domains and binds RNA in planta 118 AeSSP1256 is a member of a large family of A. euteiches effectors devoid of any predicted 119 functional domain, except the presence of a signal peptide at the N-terminus (Gaulin et al., 120 2018). As showed in Figure 1A, AeSSP1256 protein is enriched in glycine residues (30% of 121 the amino acid sequence). Analysis using the Eukaryotic Linear Motif database (Gouw et al., 122 2018) revealed 3 GGRGG motifs (positions 81-85; 95-99 and 99-103). These motifs are 123 variant arginine methylation site from arginine-glycine(-glycine) (RGG/RG) domains, 124 presents in many ribonucleoproteins and involved in RNA binding (Thandapani et al., 2013; 125 Bourgeois et al., 2020). We then noticed the presence of two di-RGG domains (RGG(X 0-126 5 )RGG) (position 75-85...

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