Genetic background affects pathogenicity island function and pathogen emergence in Streptomyces

Abstract

With few exceptions, thaxtomin A (ThxA), a nitrated diketopiperazine, is the pathogenicity determinant for plant-pathogenic Streptomyces species. In Streptomyces scabiei (syn. S. scabies), the ThxA biosynthetic cluster is located within a 177-kb mobile pathogenicity island (PAI), called the toxicogenic region (TR). In S. turgidiscabies, the ThxA biosynthetic cluster is located within a 674-kb pathogenicity island (PAIst). The emergence of new plant pathogens occurs in this genus, but not frequently. This raises the question of whether the mobilization of these pathogenicity regions, through mating, is widespread and whether TR and PAIst can confer plant pathogenicity. We showed that ThxA biosynthetic clusters on TR and PAIst were transferred into strains from five non-pathogenic Streptomyces species through mating with S. scabiei and S. turgidiscabies. However, not all of the transconjugants produced ThxA and exhibited the virulence phenotype, indicating that the genetic background of the recipient strains affects the functionality of the ThxA biosynthetic cluster and therefore would be expected to affect the emergence of novel pathogenic Streptomyces species. Thxs have been patented as natural herbicides, but have yet to be commercialized. Our results also demonstrated the potential of the heterologous production of ThxA as a natural and biodegradable herbicide in non-pathogenic Streptomyces species.

Keywords: Streptomyces; background; genetic; pathogenicity island; thaxtomin.

Figure 1

Schematic genetic organization of Streptomyces scabiei ΔlanAB toxicogenic region (TR) and Streptomyces turgidiscabies Car811 pathogenicity island (PAIst) with antibiotic markers. Arrowed boxes represent the location and orientation of the open reading frames (not drawn to scale). Red stars show the locations of the antibiotic markers. hyg^R, hygromycin B resistance; thio^R, thiostrepton resistance; att, attachment site; tomA, tomatinase; fas, fasciation; RDF, recombination directionality factor.

Figure 2

Schematic representation of the mating experiments for the investigation of the mobilization of Streptomyces scabiei toxicogenic region (TR) and Streptomyces turgidiscabies pathogenicity island (PAIst). (1) The mating experiment between non‐pathogenic Streptomyces species with Escherichia coli ET12567/pUZ8002 with pSET152 (apr^R) to create non‐pathogenic Streptomyces species with antibiotic marker; Streptomyces diastatochromogenes ATCC 12309 is naturally streptomycin resistant (str^R). (2) Non‐pathogenic Streptomyces species (apr^R or str^R) were mated with S. scabiei ΔlanAB (hyg^R) to obtain the non‐pathogenic Streptomyces species with TR. (3) Non‐pathogenic Streptomyces species (apr^R or str^R) were mated with S. turgidiscabies Car811 (thio^R) to obtain the non‐pathogenic Streptomyces species with PAIst. apr^R, apramycin resistance; hyg^R, hygromycin B resistance; str^R, streptomycin resistance; thio^R, thiostrepton resistance.

Figure 3

Production of thaxtomins by heterologous hosts in oat bran broth (OBB) medium. (A) Production of thaxtomins by heterologous hosts with the Streptomyces scabiei toxicogenic region (TR) in OBB medium. The average thaxtomin production of S. scabiei 87‐22 is set to 100%. (B) Production of thaxtomins by heterologous hosts with Streptomyces turgidiscabies pathogenicity island (PAIst) in OBB medium. The average thaxtomin production of S. turgidiscabies Car811 is set to 100%. Spores were cultured in tryptic soy broth (TSB) at 30 °C for 48 h. Vegetative culture was diluted to an optical density at 600 nm (OD₆₀₀) = 1 and a 0.5‐mL portion of the diluted culture was inoculated into 50 mL of OBB liquid medium and incubated at 30 °C with shaking (250 rpm) for 6 days. The average percentage production for each strain relative to S. scabiei 87‐22 or S. turgidiscabies Car811 is shown, and the errors bars represent the standard deviation from the mean. Letters represent results of a one‐way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) test; bars not sharing the same letters are significantly different at P < 0.05.

Figure 4

Virulence assays of non‐pathogenic Streptomyces species with Streptomyces scabiei toxicogenic region (TR) and Streptomyces turgidiscabies pathogenicity island (PAIst). Surface‐sterilized radish seedlings (six per treatment) were inoculated with mycelia from tryptic soy broth (TSB)‐grown cultures of Streptomyces strains. Plants were incubated at room temperature under a 12‐h photoperiod for 4 days. Representative plants for each treatment are shown.

Figure 5

Production of thaxtomins by heterologous hosts in Thx defined medium (TDM) supplemented with 1% cellobiose (TDMc). (A) Production of thaxtomins by heterologous hosts with Streptomyces scabiei toxicogenic region (TR) in TDMc. The average thaxtomin production of S. scabiei 87‐22 is set to 100%. (B) Production of thaxtomins by heterologous hosts with Streptomyces turgidiscabies pathogenicity island (PAIst) in TDMc. The average thaxtomin production of S. turgidiscabies Car811 is set to 100%. Spores were cultured in tryptic soy broth (TSB) at 30 °C for 48 h. Vegetative culture was diluted to an optical density at 600 nm (OD₆₀₀) = 1 and a 0.5‐mL portion of the diluted culture was inoculated into 50 mL of TDMc liquid medium and incubated at 30 °C with shaking (250 rpm) for 6 days. The average percentage production for each strain relative to S. scabiei 87‐22 or S. turgidiscabies Car811 is shown, and the errors bars represent the standard deviation from the mean. Letters represent results of a one‐way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) test; bars not sharing the same letters are significantly different at P < 0.05.

Figure 6

Production of thaxtomins by Streptomyces albus/TR on an International Streptomyces Project medium 4 (ISP‐4) plate. Streptomyces albus/TR (middle) produced thaxtomin (yellow pigmentation) on the ISP‐4 plate, which did not trigger thaxtomin production in Streptomyces scabiei 87‐22 (left) or S. albus J1074 wild‐type (right). TR, Streptomyces scabiei toxicogenic region.

Figure 7

Thaxtomin production of Streptomyces albus/TR and Streptomyces scabiei 87‐22 in liquid Thx defined medium (TDM) supplemented with 1% cellobiose (TDMc) at seven time points (1, 2, 3, 4, 5, 6 and 7 days post‐inoculation). The final yield of thaxtomins of S. scabiei 87‐22 is set to 100%. The average percentage production of thaxtomin for the two strains at each time point relative to the final yield of S. scabiei 87‐22 is shown, and the errors bars represent the standard deviation from the mean. Spores were cultured in tryptic soy broth (TSB) at 30 °C for 48 h. Vegetative culture was diluted to an optical density at 600 nm (OD₆₀₀) = 1 and a 1‐mL portion of the diluted culture was inoculated into 100 mL of liquid TDMc and incubated at 30 °C with shaking (250 rpm). Samples (3 mL) of the cultures were collected every 24 h to determine the concentration of thaxtomins. TR, Streptomyces scabiei toxicogenic region.

Figure 8

Nitrated precursors of Streptomyces albus/TR and Streptomyces scabiei 87‐22 in liquid Thx defined medium (TDM) supplemented with 1% cellobiose (TDMc). (A) High‐performance liquid chromatography (HPLC) traces of C18 Solid Phase Extraction (SPE) 25% methanol washes of TDMc medium for the analysis of nitrated precursors of thaxtomins. The top chromatogram (pink) is from the S. albus/TR extract at 7 days after inoculation, and the bottom chromatogram (black) is from the S. scabiei 87‐22 extract at 7 days after inoculation. (B) Accumulation of 4‐nitrotryptophan of S. albus/TR and S. scabiei 87‐22 in liquid TDMc. The final yield of N‐methyl‐4‐ nitrotryptophan of S. scabiei 87‐22 is set to 100%. The average percentage production of nitrated precursors for the two strains at each time point relative to the S. scabiei 87‐22 final yield of N‐methyl‐4‐nitrotryptophan is shown, and the errors bars represent the standard deviation from the mean. Spores were cultured in tryptic soy broth (TSB) at 30 °C for 48 h. Vegetative culture was diluted to an optical density at 600 nm (OD₆₀₀) = 1 and a 1‐mL portion of the diluted culture was inoculated into 100 mL of liquid TDMc and incubated at 30 °C with shaking (250 rpm). Samples (3 mL) of cultures were collected every 24 h to determine the concentration of nitrated precursors. TR, Streptomyces scabiei toxicogenic region.