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

Abstract

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.

The root pathogen Aphanomyces euteiches secretes modular proteases in pea apoplast during host infection

“…The AphanoDB, a database dedicated to the genus Aphanomyces ( https://www.polebio.lrsv.ups-tlse.fr/aphanoDB/ ), contains 518 proteins with a PFAM-based GO Peptidase activity (GO:0008233), with trypsin S1 being the largest family (74 genes) in A. euteiches ATCC201684 strain ( Supplementary Table S1 ). In a previous global analysis of the secretome we reported a large number of secreted proteases compared to the phytopathogen oomycete P. infestans ( Gaulin et al., 2018 ; Kiselev et al., 2022 ). Accordingly, in this work we identified 151 proteins with a predicted signal peptide (+SP) and no transmembrane domain (-TM).…”

“…Samtools (v.1.9) algorithms ‘fixmate’ and ‘markdup’ ( Li et al., 2009 ) were used to clean alignments from duplicated sequences. Reads were counted with HTseq (v.0.9.1) ( Anders et al., 2015 ) using the reference GFF file ( Kiselev et al., 2022 ). The count files were normalized and differentially expressed genes (DEGs) were identified using the DESeq2 algorithm ( Love et al., 2014 ).…”

“…Label-free quantification and match-between-runs was activated ( Cox et al., 2014 ). The MS/MS spectral data were searched against the A. euteiches database ( Kiselev et al., 2022 ) and the P. sativum database ( Kreplak et al., 2019 ). All searches included a contaminants database search (as implemented in MaxQuant, 245 entries).…”

“…Filamentous plant pathogenic oomycetes secrete several types of pathogenicity factors to facilitate infection, such as small secreted proteins (SSP), cellulose-binding proteins (CBELs) or plant DNAdamaging proteins (Gaulin et al, 2006;Ramirez-Garceś et al, 2016;Camborde et al, 2022). Before entering the root cells, these pathogens, including A. euteiches, may pass the apoplast.…”

“…Likewise, the Kazal-like serine protease inhibitors EP1 and EPI10 from P. infestans target tomato P69B subtilase ( Tian et al., 2004 , 2005 ). Interestingly, the A. euteiches genome, in addition to having numerous CWDE, is characterized by a large representation of putative extracellular proteases ( Gaulin et al., 2018 ; Kiselev et al., 2022 ), suggesting a key role in infection.…”