The cellobiose sensor CebR is the gatekeeper of Streptomyces scabies pathogenicity

Abstract

A relatively small number of species in the large genus Streptomyces are pathogenic; the best characterized of these is Streptomyces scabies. The pathogenicity of S. scabies strains is dependent on the production of the nitrated diketopiperazine thaxtomin A, which is a potent plant cellulose synthesis inhibitor. Much is known about the genetic loci associated with plant virulence; however, the molecular mechanisms by which S. scabies triggers expression of thaxtomin biosynthetic genes, beyond the pathway-specific activator TxtR, are not well understood. In this study, we demonstrate that binding sites for the cellulose utilization repressor CebR occur and function within the thaxtomin biosynthetic cluster. This was an unexpected result, as CebR is devoted to primary metabolism and nutritive functions in nonpathogenic streptomycetes. In S. scabies, cellobiose and cellotriose inhibit the DNA-binding ability of CebR, leading to an increased expression of the thaxtomin biosynthetic and regulatory genes txtA, txtB, and txtR. Deletion of cebR results in constitutive thaxtomin A production and hypervirulence of S. scabies. The pathogenicity of S. scabies is thus under dual direct positive and negative transcriptional control where CebR is the cellobiose-sensing key that locks the expression of txtR, the key necessary to unlock the production of the phytotoxin. Interestingly, CebR-binding sites also lie upstream of and within the thaxtomin biosynthetic clusters in Streptomyces turgidiscabies and Streptomyces acidiscabies, suggesting that CebR is most likely an important regulator of virulence in these plant-pathogenic species as well.

Importance: What makes a microorganism pathogenic is not limited to the genes acquired for virulence. Using the main causative agent of scab lesions on root and tuber crops as an example, our work identified the subtle but essential genetic changes that generate the cis-acting elements necessary for proper timing of the expression of the cluster of genes responsible for the biosynthesis of thaxtomin A, the primary virulence factor in plant-pathogenic streptomycetes. These data illustrate a situation in which a regulator associated with primary metabolism in nonpathogens, CebR, has been coopted as a master regulator of virulence in pathogenic species. Furthermore, the manipulation of CebR-mediated control of thaxtomin production will facilitate overproduction of this natural and biodegradable herbicide for commercial purposes. Our work thus provides a concrete example of how a strictly theoretical and computational work was able to elucidate a regulatory mechanism associated with the virulence of a plant pathogen and to generate solutions to purely agro-industrial concerns.

FIG 1

CebR binding to cbs associated with thaxtomin biosynthetic genes is relieved by cellobiose. (A) Sequences and positions of CebR-binding sites identified in the chromosome region of the thaxtomin biosynthetic genes in S. scabies. Numbers associated with genes/ORFs are SCAB numbers from the annotated genome of S. scabies 87-22. The lowercase letters indicate nucleotides that do not match with the cbs consensus sequence. (B) EMSA demonstrating CebR binding to DNA motifs identified in the txtRA intergenic region (cbs^txtR-A) and in txtB (cbs^txtB). Numbers 1 to 14 refer to increasing concentrations of pure CebR-His₆, i.e., 0 (free probe, 30 nM), 80, 160, 240, 320, 400, 480, 560, 640, 720, 960, 1,200, 1,600, and 3,200 nM, respectively. (C) EMSAs demonstrating cellobiose as the best allosteric effector of CebR. Numbers 1 and 2 refer to EMSAs with free probes (6 nM) and with probes incubated with CebR-His₆, respectively. Numbers 3 to 7 refer to EMSAs with CebR-His₆ preincubated with oligosaccharides, i.e., cellobiose (lane 3), cellotriose (lane 4), cellotetraose (lane 5), cellopentaose (lane 6), or cellohexaose (lane 7). Quantification of the shifted bands revealed that cellobiose (lane 3), cellotriose (lane 4), and cellohexaose (lane 7) were able to impair the CebR DNA-binding ability by approximately 56, 20, and 12%, respectively.

FIG 2

Effect of cebR deletion in S. scabies on the transcription levels of the thaxtomin biosynthetic and regulatory genes. qPCR analysis of gene expression levels in S. scabies 87-22 and in the ΔcebR strain. Data were normalized using the gyrA, murX, and hrdB genes as internal controls. Mean normalized expression levels (± standard deviations) from three biological replicates analyzed in triplicate are shown.

FIG 3

Effect of the cebR deletion on thaxtomin production in S. scabies. (A) Deletion of cebR in S. scabies resulted in higher thaxtomin production (yellow pigmentation) than in the wild type on OBA and even in its production under conditions which do not trigger thaxtomin production in the wild-type strain 87-22 (on ISP-4). (B) HPLC analysis of thaxtomin A extracted from plates shown in panel A.

FIG 4

Effect of the deletion of cebR on the virulence of S. scabies. (A) Phenotype of A. thaliana grown for 8 days in the presence of S. scabies 87-22 (wild type) and its cebR null mutant; (B) closeup of representative plants grown in the MS plates shown in panel A; (C) potato tuber slice assay.