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

The 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.

iTRAQ-Based Proteomics Analysis of Response to Solanum tuberosum Leaves Treated with the Plant Phytotoxin Thaxtomin A

Thaxtomin A (TA) is a phytotoxin secreted by Streptomyces scabies that causes common scab in potatoes. However, the mechanism of potato proteomic changes in response to TA is barely known. In this study, the proteomic changes in potato leaves treated with TA were determined using the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique. A total of 693 proteins were considered as differentially expressed proteins (DEPs) following a comparison of leaves treated with TA and sterile water (as a control). Among the identified DEPs, 460 and 233 were upregulated and downregulated, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, many DEPs were found to be involved in defense and stress responses. Most DEPs were grouped in carbohydrate metabolism, amino acid metabolism, energy metabolism, and secondary metabolism including oxidation–reduction process, response to stress, plant–pathogen interaction, and plant hormone signal transduction. In this study, we analyzed the changes in proteins to elucidate the mechanism of potato response to TA, and we provided a molecular basis to further study the interaction between plant and TA. These results also offer the option for potato breeding through analysis of the resistant common scab.

Comparative Genomics of Potato Common Scab-Causing Streptomyces spp. Displaying Varying Virulence

Common scab of potato causes important economic losses worldwide following the development of necrotic lesions on tubers. In this study, the genomes of 14 prevalent scab-causing Streptomyces spp. isolated from Prince Edward Island, one of the most important Canadian potato production areas, were sequenced and annotated. Their phylogenomic affiliation was determined, their pan-genome was characterized, and pathogenic determinants involved in their virulence, ranging from weak to aggressive, were compared. 13 out of 14 strains clustered with Streptomyces scabiei, while the last strain clustered with Streptomyces acidiscabies. The toxicogenic and colonization genomic regions were compared, and while some atypical gene organizations were observed, no clear correlation with virulence was observed. The production of the phytotoxin thaxtomin A was also quantified and again, contrary to previous reports in the literature, no clear correlation was found between the amount of thaxtomin A secreted, and the virulence observed. Although no significant differences were observed when comparing the presence/absence of the main virulence factors among the strains of S. scabiei, a distinct profile was observed for S. acidiscabies. Several mutations predicted to affect the functionality of some virulence factors were identified, including one in the bldA gene that correlates with the absence of thaxtomin A production despite the presence of the corresponding biosynthetic gene cluster in S. scabiei LBUM 1485. These novel findings obtained using a large number of scab-causing Streptomyces strains are challenging some assumptions made so far on Streptomyces’ virulence and suggest that other factors, yet to be characterized, are also key contributors.

Habituation to thaxtomin A increases resistance to common scab in ‘Russet Burbank’ potato

Common scab is a potato disease characterized by the formation of scab-like lesions on the surface of potato tubers. The actinobacterium Streptomyces scabiei is the main causal agent of common scab. During infection, this bacterium synthesizes the phytotoxin thaxtomin A which is essential for the production of disease symptoms. While thaxtomin A can activate an atypical programmed cell death in plant cell suspensions, it is possible to gradually habituate plant cells to thaxtomin A to provide resistance to lethal phytotoxin concentrations. Potato ‘Russet Burbank’ calli were habituated to thaxtomin A to regenerate the somaclone RB9 that produced tubers more resistant to common scab than those obtained from the original cultivar. Compared to the Russet Burbank cultivar, somaclone RB9 generated up to 22% more marketable tubers with an infected tuber area below the 5% threshold. Enhanced resistance was maintained over at least two years of cultivation in the field. However, average size of tubers was significantly reduced in somaclone RB9 compared to the parent cultivar. Small RB9 tubers had a thicker phellem than Russet Burbank tubers, which may contribute to improving resistance to common scab. These results show that thaxtomin A-habituation in potato is efficient to produce somaclones with increased and durable resistance to common scab.

The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

<p>TxtE is a cytochome P450 (CYP) homolog that mediates a nitric oxide (NO)-dependent direct nitration of l-tryptophan (l-Trp) to form 4-nitrotryptophan (4-NO₂-l-Trp). This nitrated product is a precursor for thaxtomin A, a virulence factor produced by plant-pathogenic bacteria that causes the disease potato scab. A recent study provided the first characterization of intermediates along the TxtE nitration pathway.¹ The authors’ accumulated evidence supported a mechanism in which O₂ binds to Fe^II TxtE to form an {FeO₂}⁸ intermediate, which subsequently reacted with NO to ultimately form Fe^III TxtE and 4-NO₂-l-Trp. Typical CYP mechanisms reduce and protonate the {FeO₂}⁸ intermediate to form a ferric-hydroperoxo species (Fe^III–OOH) en route to formation of the active oxidant compound I. The previously reported lack of hydroxylated tryptophan resulting from TxtE turnover suggests that the TxtE cycle must stall at the {FeO₂}⁸ intermediate to avoid hydroxylation. Here we present LC-MS experiments showing suggesting that TxtE can hydroxylate l-Trp by the peroxide shunt but not via reduction of the {FeO₂}⁸ intermediate. Comparison of stopped-flow time courses in the presence and absence of excess reducing equivalents and common CYP electron transfer partners shown no spectral or kinetic evidence for reduction of the {FeO₂}⁸ intermediate. Furthermore, the electron coupling efficiency of TB14—a self-sufficient TxtE variant with C-terminal reductase domain—to form 4-NO₂-l-Trp exhibits a 3% electron coupling efficiency when it is loaded with one reducing equivalent. This efficiency <i>increases</i> by 2-fold when TB14 is loaded with two or four reducing equivalents. This observation provides further evidence for our key conclusion that the TxtE {FeO₂}⁸ intermediate resists reduction. The resistance of the {FeO₂}⁸ intermediate to reduction is a key feature of TxtE, enabling reaction with NO and efficient nitration turnover.<b></b></p>

The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

<p>TxtE is a cytochome P450 (CYP) homolog that mediates a nitric oxide (NO)-dependent direct nitration of l-tryptophan (l-Trp) to form 4-nitrotryptophan (4-NO₂-l-Trp). This nitrated product is a precursor for thaxtomin A, a virulence factor produced by plant-pathogenic bacteria that causes the disease potato scab. A recent study provided the first characterization of intermediates along the TxtE nitration pathway.¹ The authors’ accumulated evidence supported a mechanism in which O₂ binds to Fe^II TxtE to form an {FeO₂}⁸ intermediate, which subsequently reacted with NO to ultimately form Fe^III TxtE and 4-NO₂-l-Trp. Typical CYP mechanisms reduce and protonate the {FeO₂}⁸ intermediate to form a ferric-hydroperoxo species (Fe^III–OOH) en route to formation of the active oxidant compound I. The previously reported lack of hydroxylated tryptophan resulting from TxtE turnover suggests that the TxtE cycle must stall at the {FeO₂}⁸ intermediate to avoid hydroxylation. Here we present LC-MS experiments showing suggesting that TxtE can hydroxylate l-Trp by the peroxide shunt but not via reduction of the {FeO₂}⁸ intermediate. Comparison of stopped-flow time courses in the presence and absence of excess reducing equivalents and common CYP electron transfer partners shown no spectral or kinetic evidence for reduction of the {FeO₂}⁸ intermediate. Furthermore, the electron coupling efficiency of TB14—a self-sufficient TxtE variant with C-terminal reductase domain—to form 4-NO₂-l-Trp exhibits a 3% electron coupling efficiency when it is loaded with one reducing equivalent. This efficiency <i>increases</i> by 2-fold when TB14 is loaded with two or four reducing equivalents. This observation provides further evidence for our key conclusion that the TxtE {FeO₂}⁸ intermediate resists reduction. The resistance of the {FeO₂}⁸ intermediate to reduction is a key feature of TxtE, enabling reaction with NO and efficient nitration turnover.<b></b></p>

Interactive regulation between aliphatic hydroxylation and aromatic hydroxylation of thaxtomin D in TxtC: a theoretical investigation

ABSTRACTTxtC is an unusual bifunctional cytochrome P450 that is able to perform sequential aliphatic and aromatic hydroxylation of the diketopiperazine substrate thaxtomin D in two remote sites to produce thaxtomin A. Though the X-ray structure of TxtC complexed with thaxtomin D revealed a binding mode for its aromatic hydroxylation, the preferential hydroxylation site is aliphatic C14. It is thus intriguing to unravel how TxtC accomplishes such two-step catalytic hydroxylation on distinct aliphatic and aromatic carbons and why the aliphatic site is preferred in the hydroxylation step. In this work, by employing molecular docking and molecular dynamics (MD) simulation, we revealed that thaxtomin D could adopt two different conformations in the TxtC active site, which were equal in energy with either the aromatic C-H or aliphatic C14-H laying towards the active Cpd I oxyferryl moiety. Further ONIOM calculations indicated that the energy barrier for the rate-limiting hydroxylation step on the aliphatic C14 site was 8.9 kcal/mol more favorable than that on the aromatic C20 site. The hydroxyl group on the monohydroxylated intermediate thaxtomin B C14 site formed hydrogen bonds with Ser280 and Thr385, which induced the L-Phe moiety to rotate around the Cβ−Cγ bond of the 4-nitrotryptophan moiety. Thus, it adopted an energy favorable conformation with aromatic C20 adjacent to the oxyferryl moiety. In addition, the hydroxyl group induced solvent water molecules to enter the active site, which propelled thaxtomin B towards the heme plane and resulted in heme distortion. Based on this geometrical layout, the rate-limiting aromatic hydroxylation energy barrier decreased to 15.4 kcal/mol, which was comparable to that of the thaxtomin D aliphatic hydroxylation process. Our calculations indicated that heme distortion lowered the energy level of the lowest Cpd I α-vacant orbital, which promoted electron transfer in the rate-limiting thaxtomin B aromatic hydroxylation step in TxtC.

A Novel Species-Level Group of Streptomyces Exhibits Variation in Phytopathogenicity Despite Conservation of Virulence Loci

The genus Streptomyces includes several phytopathogenic species that cause common scab, a devastating disease of tuber and root crops, in particular potato. The diversity of species that cause common scab is unknown. Likewise, the genomic context necessary for bacteria to incite common scab symptom development is not fully characterized. Here, we phenotyped and sequenced the genomes of five strains from a poorly studied Streptomyces lineage. These strains form a new species-level group. When genome sequences within just these five strains are compared, there are no polymorphisms of loci implicated in virulence. Each genome contains the pathogenicity island that encodes for the production of thaxtomin A, a phytotoxin necessary for common scab. Yet, not all sequenced strains produced thaxtomin A. Strains varied from nonpathogenic to highly virulent on two hosts. Unexpectedly, one strain that produced thaxtomin A and was pathogenic on radish was not aggressively pathogenic on potato. Therefore, while thaxtomin A biosynthetic genes and production of thaxtomin A are necessary, they are not sufficient for causing common scab of potato. Additionally, results show that even within a species-level group of Streptomyces strains, there can be aggressively pathogenic and nonpathogenic strains despite conservation of virulence genes.