Common scab disease: structural basis of elicitor recognition in pathogenic Streptomyces species.

peer reviewedIn Streptomyces 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 (KD in the nanomolar range) is the first event for the onset of its pathogenic lifestyle. Here, we report the structure of CebEscab in complex with cellotriose at a resolution of 1.55 Å, adopting a general fold of the B subcluster of SBPs. The interaction between CebEscab and cellotriose involves multiple direct or water-mediated hydrogen bonds and hydrophobic interactions, with 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 CebE-mediated cellotriose transport to the transcriptional activation of thaxtomin phytotoxin biosynthesis is conserved in Streptomyces spp. causing common scab, except for Streptomyces ipomoeae, which specifically colonizes sweet potatoes and responds to other and yet unknown virulence elicitors. Interestingly, strains belonging to the pathogenic species turgidiscabies and caniscabiei have a cellotriose-binding protein orthologous to the CebE protein of the saprophytic species Streptomyces reticuli with lower affinity for its substrate (KD in 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-decomposing Streptomyces species.IMPORTANCECommon scab is a disease caused by a few Streptomyces species 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 of Streptomyces scabiei in 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 except Streptomyces ipomoeae, which causes soft rot disease in sweet potatoes. Our work also provides the structural basis of the uptake of cellobiose and cellotriose in saprophytic Streptomyces species, the first step activating the expression of the enzymatic system degrading the most abundant polysaccharide on earth, cellulose

The virulome of Streptomyces scabiei in response to cello- oligosaccharide elicitors

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

Structure and Function of BcpE2, the Most Promiscuous GH3-Family Glucose Scavenging Beta-Glucosidase.

peer reviewedCellulose 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 revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was 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 covered 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 was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE 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. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material

Adaptations génétiques impliquées dans l'induction de la pathogénicité chez Streptomyces scabies

Streptomyces scabies est l'un des rares pathogènes connus au sein du genre Streptomyces. Considéré comme organisme modèle dans cette catégorie, il est responsable de la maladie de la galle commune affectant les plantes tubéreuses sous forme de lésions cutanées. Ces symptômes sont principalement dus à une phytotoxine nommée thaxtomine. Bien que la voie de signalisation menant à la production de thaxtomine ait récemment été caractérisée, plusieurs interrogations subsistent quant aux mécanismes employés par S. scabies pour percevoir son hôte. En effet, pour distinguer la matière organique vivante de la matière organique morte, il semble que de subtiles changements génétiques aient permis un ajustement au mode de vie pathogène. Nous avons étudié l'importance de l'interaction du régulateur CebR avec ses éléments cis dans la capacité à répondre à différents éliciteurs environnementaux. Les résultats présentés ici nous ont permis de progresser dans notre compréhension de la manière dont ces signaux perçus par S. scabies jouent un rôle dans l'induction de la pathogénicité.Patscab #

New insights into the enzymatic properties and metabolic profile of the phytopathogen Streptomyces scabiei

Streptomyces scabiei (syn. Streptomyces scabies) is a phytopathogenic Gram-positive bacterium known as the causative agent of common scab disease. This microorganism causes, together with about 20 other species in the Streptomyces genus, skin lesions affecting tuber crops and mainly reducing the marketability of potatoes. S. scabiei 87-22 is considered the model strain for the study of virulence-associated mechanisms and has been used to highlight the regulatory network governing the biosynthesis of its main pathogenicity determinant, thaxtomin A. This phytotoxin – acting as an inhibitor of cellulose biosynthesis – is shared by most common scab causing streptomycetes and is the only virulence factor considered to be essential for their success in host colonization. Cellulose oligosaccharides (cello-oligosaccharides) have been shown to be the elicitors of thaxtomin production and, by extension, the triggers of the pathogenic lifestyle. In this work, we reveal the wide effects of these oligosaccharides as inducers of additional genes and specialized metabolites which have thereby been attributed to the so-called ‘virulome’ of S. scabiei. We also provide the first comprehensive and manually curated genome mining analysis of a pathogenic Streptomyces strain, revealing its impressive potential for the biosynthesis of specialized metabolites. Concomitantly to cello-oligosaccharides import, S. scabiei activates a central gene encoding the β-glucosidase BglC involved in the control of the pool of thaxtomin elicitors, and the scopolin phytoalexin which appears to be meaningful in the feedback control of thaxtomin production. However, the loss of this gene leads to major physiological modifications and unexpected phenotypes which indicate yet unknown functions. Studying the adaptations of the bglC null mutant revealed an intriguing phenomenon of genetic compensation causing the transcriptional activation of two other genes, namely bcpE1 and bcpE2. These genes encode two other β-glucosidases which fulfill distinct roles to functionally replace BglC, i.e., BcpE1 is similarly active on cello-oligosaccharides, and BcpE2 hydrolyzes a wide range of plant heterosides including the scopolin phytoalexin. Finally, we provide the first reported characterization of the amylolytic system of S. scabiei which was a surprisingly overlooked aspect for a bacterium that colonizes plant storage organs. We have investigated its ability to consume starch and related carbohydrates to find that maltose was poorly utilized by S. scabiei. This unexpected phenotype was attributed to a strong repressive control mediated by the MalR transcriptional regulator which was also shown to be involved in the maltose-dependent control of the secreted amylase enzymes. Overall, the present work provides insights into the understanding of i) the diversity of the virulome of S. scabiei, ii) the molecular mechanisms associated with the multiple roles of bglC/BglC, and iii) the primary metabolism dedicated to the utilization of the most abundant storage carbohydrates of tuber hosts.Streptomyces scabiei (syn. Streptomyces scabies) est une bactérie Gram-positive phytopathogène bien connue comme étant l’agent responsable de la gale commune. Aux côtés d’autres espèces du genre Streptomyces, ce microorganisme cause la formation de lésions superficielles qui affectent les plantes tubéreuses et en particulier la pomme de terre dont la valeur marchande se voit réduite. Considérée comme modèle pour l’étude des mécanismes de virulence, la souche 87-22 de S. scabiei a notamment permis de mettre en évidence la cascade de signalisation menant à la biosynthèse de la thaxtomine A. Cette phytotoxine, qui agit comme inhibiteur de la biosynthèse de cellulose, est considérée comme le déterminant majeur de la pathogénicité. Il s’agit également du seul facteur de virulence dont la présence est essentielle pour assurer la colonisation des hôtes. Les oligosaccharides de cellulose, ou cello-oligosaccharides, sont les éliciteurs requis pour la production de thaxtomine et donc, par extension, pour le développement du mode de vie pathogène. Ce travail nous a permis de révéler l’étendue du rôle des cello-oligosaccharides qui permettent l’induction de nombreux gènes et la production de métabolites spécialisés qui ont dès lors été attribués au « virulome » de S. scabiei. De plus, nous proposons la première analyse approfondie du potentiel biosynthétique au sein du génome d’un Streptomyces phytopathogène, démontrant l’impressionnant potentiel du métabolisme spécialisé de S. scabiei 87-22. Simultanément à l’import des cello-oligosaccharides, un gène essentiel codant pour la β-glucosidase BglC est activé pour contrôler l’abondance des éliciteurs de la thaxtomine. BglC est également capable d’hydrolyser la scopoline qui pourrait jouer un rôle majeur dans le rétrocontrôle de la production de thaxtomine. La perte de ce gène cause des changements physiologiques inattendus et entraîne un étonnant phénomène de compensation génétique. L’étude de ce dernier a permis de révéler l’activation transcriptionnelle de deux gènes, bcpE1 et bcpE2, qui codent chacun pour des β-glucosidases aux fonctions bien distinctes. BcpE1 a la capacité de remplacer BglC pour l’hydrolyse des cello-oligosaccharides, tandis que BcpE2 clive de nombreux hétérosides originaires des plantes dont la scopoline. Enfin, nous nous sommes intéressés au système amylolytique de S. scabiei qui avait été, jusqu’ici, étonnamment négligé malgré son importance suspectée dans la colonisation des plantes tubéreuses. Nous avons notamment découvert que S. scabiei consomme très mal le maltose alors qu’il utilise assez bien les autres sources de carbone provenant de l’amidon. Ce phénotype a pu être attribué au répresseur MalR qui exerce un contrôle très fort sur le système de consommation de maltose, et qui intervient également dans la production d’amylases en fonction de la concentration en maltose. D’un point de vue global, ce travail amène des avancées au sujet de la compréhension du mode de vie de S. scabiei, à savoir (i) la diversité de son virulome, (ii) les mécanismes moléculaires en lien avec les différents rôles joués par bglC/BglC et (iii) son métabolisme primaire dédié à la consommation de la source de carbone la plus abondante trouvée chez ses hôtes.Patscab#

Old Enzyme, New Role: The β-Glucosidase BglC of Streptomyces scabiei Interferes with the Plant Defense Mechanism by Hydrolyzing Scopolin

peer reviewedThe beta-glucosidase BglC fulfills multiple functions in both primary metabolism and induction of pathogenicity of Streptomyces scabiei, the causative agent of common scab in root and tuber crops. Indeed, this enzyme hydrolyzes cellobiose and cellotriose to feed glycolysis with glucose directly and modifies the intracellular concentration of these cello-oligosaccharides, which are the virulence elicitors. The inactivation of bglC led to unexpected phenotypes such as the constitutive overproduction of thaxtomin A, the main virulence determinant of S. scabiei. In this work, we reveal a new target substrate of BglC, the phytoalexin scopolin. Removal of the glucose moiety of scopolin generates scopoletin, a potent inhibitor of thaxtomin A production. The hydrolysis of scopolin by BglC displayed substrate inhibition kinetics, which contrasts with the typical Michaelis–Menten saturation curve previously observed for the degradation of its natural substrate cellobiose. Our work, therefore, reveals that BglC targets both cello-oligosaccharide elicitors emanating from the hosts of S. scabiei, and the scopolin phytoalexin generated by the host defense mechanisms, thereby occupying a key position to fine-tune the production of the main virulence determinant thaxtomin A

Tracking the Subtle Mutations Driving Host Sensing by the Plant Pathogen Streptomyces scabies

The acquisition of genetic material conferring the arsenal necessary for host virulence is a prerequisite on the path to becoming a plant pathogen. More subtle mutations are also required for the perception of cues signifying the presence of the target host and optimal conditions for colonization. The decision to activate the pathogenic lifestyle is not “taken lightly” and involves efficient systems monitoring environmental conditions. But how can a pathogen trigger the expression of virulence genes in a timely manner if the main signal inducing its pathogenic behavior originates from cellulose, the most abundant polysaccharide on earth? This situation is encountered by Streptomyces scabies, which is responsible for common scab disease on tuber and root crops. We propose here a series of hypotheses of how S. scabies could optimally distinguish whether cello-oligosaccharides originate from decomposing lignocellulose (nutrient sources, saprophyte) or, instead, emanate from living and expanding plant tissue (virulence signals, pathogen) and accordingly adapt its physiological response

Thaxtomin A : une alternative prometteuse aux herbicides synthétiques

In times of trouble for the world’s most popular herbicide – namely Bayer / Monsanto’s Roundup – finding alternatives to glyphosate has become a priority for some companies willing to capture this highly valuable market. Besides synthetic herbicides and their derivatives that are being developed in the industry, interest is growing for natural and ‘green’ active molecules. The plant pathogen Streptomyces scabies provides an interesting clue, as its main virulence factor is a phytotoxin called thaxtomin A. This filamentous Gram + bacteria is known as the causing agent of the common scab disease affecting tuber crops. While it was discovered in 1989, thaxtomin’s action mechanisms are still partly unknown. It appears that it targets the plant cell wall, with reports highlighting similarities to the herbicide isoxaben. Despite being highly active in herbicide assays, the phytotoxin is not being used on a large scale for a simple reason: its cost. The low levels of production and high price of culture media components make it too expensive to be commercialized. Studying and engineering genetic regulation networks controlling thaxtomin A production in S. scabies helped us improving production yields while reducing costs. With improvements still to come, thaxtomin A could become economically-viable and thus be a sustainable alternative herbicide.Patscab #

A la poursuite des mutations subtiles permettant la perception de l'hôte chez le pathogène végétal Streptomyces scabies

The model plant pathogen Streptomyces scabies causes the common scab disease. Thaxtomin A is the phytotoxin responsible for this disease. Recently, its production pathway has been unveiled, highlighting a double regulation system. However, there are still some unclear aspects about how the pathogen senses his potential host. We propose here some hypothesis to explain the features responsible for the induction of pathogenicity.Le modèle de la pathogénicité végétale chez les Streptomyces, S. scabies est responsable de la maladie de la galle commune. La thaxtomine est la phytotoxine qui cause cette maladie. Il y a peu, la voie de production de la toxine a été découverte : celle-ci met en oeuvre un double mécanisme de régulation génétique. Cependant, certains aspects de la perception de son hôte par le pathogène sont toujours inconnus. Dans ce poster, nous proposons plusieurs hypothèses pour expliquer comment quelques éléments subtiles peuvent être à l'origine de ce mécanisme de pathogénicité

Proteomic Response to Thaxtomin Phytotoxin Elicitor Cellobiose and to Deletion of Cellulose Utilization Regulator CebR in Streptomyces scabies

Streptomyces scabies is responsible for common scab disease on root and tuber vegetables. Production of its main phytotoxin thaxtomin A is triggered upon transport of cellulose byproducts cellotriose and cellobiose, which disable the repression of the thaxtomin biosynthesis activator gene txtR by the cellulose utilization regulator CebR. To assess the intracellular response under conditions where S. scabies develops a virulent behavior, we performed a comparative proteomic analysis of wild-type S. scabies 87-22 and its cebR null mutant (hyper-virulent phenotype) grown in the absence or presence of cellobiose. Our study revealed significant changes in abundance of proteins belonging to metabolic pathways known or predicted to be involved in pathogenicity of S. scabies. Among these, we identified proteins of the cello-oligosaccharide-mediated induction of thaxtomin production, the starch utilization system required for utilization of the carbohydrate stored in S. scabies's hosts, and siderophore synthesis utilization systems, which are key features of pathogens to acquire iron once they colonized the host. Thus, proteomic analysis supported by targeted mass spectrometry-based metabolite quantitative analysis revealed the central role of CebR as a regulator of virulence of S. scabies