The development of spots or lesions symptomatic of common scab on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87–22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87–22 show a production and/or a transcriptional response to cello-oligosaccharides. Comparative metabolomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include (i) thaxtomin and concanamycin phytotoxins, (ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, (iii) ectoine for protection against osmotic shock once inside the host, and (iv) bottromycin and concanamycin antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cello-oligosaccharides reduced the production of the spore germination inhibitors germicidins thereby giving the ‘green light’ to escape dormancy and trigger the onset of the pathogenic lifestyle. For most metabolites - either with induced or reduced production - cellotriose was revealed to be a slightly stronger elicitor compared to cellobiose, supporting an earlier hypothesis which suggested the trisaccharide was the real trigger for virulence released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs’ expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of a yet unknown mechanism for switching on the virulome. Finally, a transcriptomic analysis revealed nine additional cryptic BGCs that have their expression awakened by cello-oligosaccharides, suggesting that other and yet to be discovered metabolites could be part of the virulome of S. scabiei .

Plant colonization by Streptomyces scabiei, the main cause of common scab disease on root and tuber crops, is triggered by cello-oligosaccharides, cellotriose being the most efficient elicitor. The import of cello-oligosaccharides via the ATP-binding cassette (ABC) transporter CebEFG-MsiK induces the production of thaxtomin phytotoxins, the central virulence determinants of this species, as well as many other metabolites that compose the ‘virulome’ of S. scabiei. Homology searches revealed paralogues of the CebEFG proteins, encoded by the cebEFG2 cluster, while another ABC-type transporter, PitEFG, is encoded on the pathogenicity island (PAI). We investigated the gene expression of these candidate alternative elicitor importers in S. scabiei 87-22 upon cello-oligosaccharide supply by transcriptomic analysis, which revealed that cebEFG2 expression is highly activated by both cellobiose and cellotriose, while pitEFG expression was barely induced. Accordingly, deletion of pitE had no impact on virulence and thaxtomin production under the conditions tested, while the deletion of cebEFG2 reduced virulence and thaxtomin production, though not as strong as the mutants of the main cello-oligosaccharide transporter cebEFG1. Our results thus suggest that both ceb clusters participate, at different levels, in importing the virulence elicitors, while PitEFG plays no role in this process under the conditions tested. Interestingly, under more complex culture conditions, the addition of cellobiose restored thaxtomin production when both ceb clusters were disabled, suggesting the existence of an additional mechanism that is involved in sensing or importing the elicitor of the onset of the pathogenic lifestyle of S. scabiei.

Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides.

InStreptomyces scabiei, the main causative agent of common scab disease of root and tuber crops, the interaction between the substrate-binding protein (SBP) CebE (CebEscab) and cellotriose released by the plant host (KDin the nanomolar range) is the first event for the onset of its pathogenic lifestyle. Here we report the structure of CebEscabin complex with cellotriose at a 1.55 Å resolution, adopting a general fold of the B subcluster of SBPs. The interaction between CebEscaband cellotriose involves multiple direct or water-mediated hydrogen bonds and hydrophobic interactions, the glucose monomer at the non-reducing end occupying the most conserved part of the substrate-binding cleft. As main interactions between the two domains of CebE involve cellotriose itself, the closed conformational state of CebE is performed via an induced-fit ligand binding mechanism where cellotriose binding triggers the domain movement. Analysis of regulon predictions revealed that the signaling pathway from the CebE-mediated cellotriose transport to the transcriptional activation of thaxtomin phytotoxin biosynthesis is conserved inStreptomycesspp causing common scab, except forStreptomyces ipomoeaethat specifically colonizes sweet potatoes and responds to other and yet unknown virulence elicitors. Interestingly, strains belonging to pathogenic speciesturgidiscabiesandcaniscabieshave a cellotriose-binding protein orthologous to the CebE protein of the saprophytic speciesStreptomyces reticuliwith lower affinity for its substrate (KDin the micromolar range), suggesting higher cellotriose concentrations for perception of their host. Our work also provides the structural basis for the uptake of cellobiose and cellotriose by non-pathogenic cellulose-decomposingStreptomycesspecies.ImportanceCommon scab is a disease caused by fewStreptomycesspecies that affects important root and tuber crops including potato, beet, radish, and parsnip, resulting in major economic losses worldwide. In this work we unveiled the molecular basis of host recognition by these pathogens by solving the structure of the sugar-binding protein CebE ofS.scabieiin complex with cellotriose, the main elicitor of the pathogenic lifestyle of these bacteria. We further revealed that the signaling pathway from CebE-mediated transport of cellotriose is conserved in all pathogenic species exceptS.ipomoeaethat causes soft rot disease on sweet potatoes. Our work also provides the structural basis of the uptake of cellobiose and cellotriose in saprophyticStreptomycesspecies, the first step activating the expression of the enzymatic system degrading the most abundant polysaccharide on earth, cellulose.Graphical abstractHighlightsCellotriose uptake triggers common scab in tuber/root crops byStreptomyces scabieiCrystal structure of CebE ofS.scabieiinteracting with cellotriose is solvedCellotriose triggers the closed conformational state of CebEThe CebE/cellotriose route to pathogenicity is conserved inStreptomycesspeciesCebE-type background may affect the cellotriose concentration eliciting virulence

Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here we reveal that the BglC compensating enzyme BcpE2 is the GH3-family beta-glucosidase that displays the highest reported substrate promiscuity able to release the glucose moiety of all tested types of plant-derived heterosides (aryl β-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covers the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin is the only substrate commonly hydrolyzed by both BglC and BcpE2 thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes thus also interfere with the plant defense mechanisms to fine-tune the strength of virulence.