Production of thaxtomin a by two species ofStreptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five of nine isolates were pathogenic on potato minitubers. Four of the pathogenic isolates produced thaxtomin A (ThxA) in infected tuber tissues. The lesion surface areas inducing ThxA were highest in treatment of the minitubers with an extract of OMB inoculated with S-66 and S-67, intermediate with that inoculated with S-64 and lowest with S-63. The pathogenic isolates were identified by gray aerial mycelia, melanin pigment productivity, the type of spore chain morphology and carbon utilization asS. scabies strains S-63, S-64 and S-68, andS. acidiscabies strains S-66 and S-67. Strains S-63 and S-64 produced 0.65 and 1.60 mg ThxA per L of OMB, respectively, strains S-66 and S-67 producing similar amounts,viz. 2.36 and 2.10 mg/L, respectively. The optimal temperature for production (by both species) was 28 °C. Production of ThxA byS. scabies strain S-64 andS. acidiscabies strain S-66 was suppressed at least 50-fold at 0.5 and 0.3 % of glucose, respectively. Fructose enhanced the production by both species.     

Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.     

<Emphasis Type="Italic">Streptomyces scabiei</Emphasis> and its toxin thaxtomin A induce scopoletin biosynthesis in tobacco and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Streptomyces scabiei is the predominant causal agent of common scab of potato in North America. The virulence of common scab-causing streptomycetes relies on their capacity to synthesize thaxtomins. In this study, the effects of S. scabiei infection and of thaxtomin A, the main toxin produced by S. scabiei, were tested for the elicitation of plant defense molecules in the model plants tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Tobacco leaves infected with spores of S. scabiei strain EF-35 or infiltrated with purified thaxtomin A produced a blue fluorescent compound that was not detected in leaves infiltrated with spores of a S. scabiei mutant deficient in thaxtomin A biosynthesis. Thin layer chromatography and high performance liquid chromatography identified this fluorescent compound as scopoletin, a plant defense phytoalexin. Arabidopsis seedlings grown in liquid medium also excreted scopoletin as a reaction to S. scabiei and thaxtomin A. The effects of the presence of scopoletin on S. scabiei were also investigated. The phytoalexin scopoletin caused a slight reduction of bacterial growth and a severe decrease of thaxtomin A production. Scopoletin was shown to inhibit thaxtomin A production by repression of a gene involved in the toxin biosynthesis.     

Genetic background affects pathogenicity island function and pathogen emergence in Streptomyces

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.     

Nitric oxide synthase inhibitors and nitric oxide donors modulate the biosynthesis of thaxtomin A, a nitrated phytotoxin produced by Streptomyces spp.

Evidence for the involvement of a bacterial nitric oxide synthase (NOS) in the biosynthesis of a phytotoxin is presented. Several species of Streptomyces bacteria produce secondary metabolites with unusual nitrogen groups, such as thaxtomin A (ThxA), which contains a nitroindole moiety. ThxA is a phytotoxin made by three pathogenic Streptomyces species that cause common scab of potato. All three species possess a gene homologous to the oxygenase domain of murine inducible NOS, and this gene, nos, is essential for normal levels of ThxA production. We grew Streptomyces turgidiscabies in the presence of several known NOS inhibitors and a nitric oxide (NO) scavenger to determine their effect on ThxA production. The NO scavenger (CPTIO) and four NOS inhibitors (NAME, NMMA, AG, and 7-NI) reduced ThxA production without affecting bacterial growth. A strain of S. turgidiscabies from which the nos gene had been deleted was grown in the presence of three NO donors (DEANO, SIN, and SNAP), and all three partially restored ThxA production. Our data suggest that bacterial nitric oxide synthases may, at least in part, produce NO for biosynthetic purposes, rather than for cellular signaling, as they do in mammals.     

TxtH is a key component of the thaxtomin biosynthetic machinery in the potato common scab pathogen Streptomyces scabies

Streptomyces scabies causes potato common scab disease, which reduces the quality and market value of affected tubers. The predominant pathogenicity determinant produced by S. scabies is the thaxtomin A phytotoxin, which is essential for common scab disease development. Production of thaxtomin A involves the nonribosomal peptide synthetases (NRPSs) TxtA and TxtB, both of which contain an adenylation (A-) domain for selecting and activating the appropriate amino acid during thaxtomin biosynthesis. The genome of S. scabies 87.22 contains three small MbtH-like protein (MLP)-coding genes, one of which (txtH) is present in the thaxtomin biosynthesis gene cluster. MLP family members are typically required for the proper folding of NRPS A-domains and/or stimulating their activities. This study investigated the importance of TxtH during thaxtomin biosynthesis in S. scabies. Biochemical studies showed that TxtH is required for promoting the soluble expression of both the TxtA and TxtB A-domains in Escherichia coli, and amino acid residues essential for this activity were identified. Deletion of txtH in S. scabies significantly reduced thaxtomin A production, and deletion of one of the two additional MLP homologues in S. scabies completely abolished production. Engineered expression of all three S. scabies MLPs could restore thaxtomin A production in a triple MLP-deficient strain, while engineered expression of MLPs from other Streptomyces spp. could not. Furthermore, the constructed MLP mutants were reduced in virulence compared to wild-type S. scabies. The results of our study confirm that TxtH plays a key role in thaxtomin A biosynthesis and plant pathogenicity in S. scabies.     

Variation of xylanosomal subunit composition of <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> by nitrogen sources

Besides affecting the xylanases production, different nitrogen sources present in the media also caused changes in the xylanosomal subunit composition of Streptomyces olivaceoviridis E-86. Four xylanosome fractions, purified from the culture supernatant of S. olivaceoviridis E-86 grown on different nitrogen sources, exhibited high specificity towards different xylans and were composed of different subunits. Thus, S. olivaceoviridis E-86 regulates the expression of xylanase activity and varies the xylanosome composition according to the nitrogen sources possibly through the action of the secreted proteases.     

The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa

Valdensinia heterodoxa (Sclerotiniacae) is a potential fungal bioherbicide for control of salal (Gaultheria shallon). The effect of culture media, substrates and relative humidity (RH) on growth, sporulation and conidial discharge of V. heterodoxa was determined for two isolates PFC2761 and PFC3027 in vitro. Culture media significantly affected the growth, sporulation, and conidial discharge of V. heterodoxa. Of eight agar media used, colony radial growth was optimal on salal oatmeal agar and salal potato dextrose agar for isolates PFC2761 and PFC3027, respectively; whereas sporulation was at an optimum on salal oatmeal agar for both isolates. Of the eight liquid media tested, mycelial production was highest on wheat bran–salal–potato dextrose broth. Growth on solid substrates greatly stimulated sporulation and conidial discharge of V. heterodoxa. Of the 12 solid substrates used, the greatest numbers of discharged conidia were observed from wheat bran and wheat bran–salal within 14 d of sporulation. Sporulation on solid substrates continued for 42 d. RH significantly affected the sporulation and conidial discharge for both isolates across all solid substrates tested. No conidia were produced or discharged below 93 % RH on wheat bran–salal and millet. With an increase of the RH from 93 to 97 %, sporulation and the number of discharged conidia increased significantly for both isolates on wheat bran–salal, but not on millet.     

Identification of the <Emphasis Type="Italic">bkdAB</Emphasis> gene cluster,a plausible source of the starter-unit for virginiamycin M production in <Emphasis Type="Italic">Streptomyces virginiae</Emphasis>

The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for virginiamycin production. Gene disruption of bkdA completely abolished the production of virginiamycin M (a polyketide-peptide antibiotic), while the production of virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the virginiamycin M production, indicating that the bkdAB cluster is essential for virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli. Supply of an additional bkdA gene under the strong and constitutive promoter ermE* in the wild-type strain of S. virginiae resulted in enhanced production of virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of virginiamycin M.     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Stimulation of in vitro root and shoot growth of potato by increasing sucrose concentration in the presence of fluridone,an inhibitor of abscisic acid synthesis

Fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, strongly stimulated rooting of nodal stem segments of potato (Solanum tuberosum L.) cultivar Arran Banner cultured in darkness on tuberisation medium. Inclusion of 10^-6 M ABA in the culture medium prevented this rooting response, indicating that root proliferation in the presence of fluridone could be due to inhibition of ABA synthesis. The rooting response to fluridone (increased total root number and root fresh weight) was obtained only at high sucrose concentrations (0.175 and 0.234 M) and was demonstrated with two potato cultivars and two culture media; one which favoured tuberisation and one which did not. Shoot numbers were also increased, but to a lesser extent than root numbers, and total fresh weight of plant material per culture was greatly increased by inclusion of both fluridone (10^-6 or 10^-5 M) and 0.234 M sucrose in the culture medium. The role of sucrose was not simply osmotic because when the osmolarity of fluridone medium was increased using mixtures of mannitol and sucrose, no root proliferation occurred unless sucrose predominated in the mixture.     

A xylan-degrading strain of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>: isolation and characterization of the xylanase activity

Two strains (O and X₂) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O and X₂, grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100°C, and exhibited an optimal temperature and pH of 90°C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.Communicated by G. Antranikian     

Anther culture of Solanum genotypes

Anther culture response was examined in five Solanum genotypes. Large genotypic differences exist in response to culture and liquid culture media were found to be superior to agar solidified media systems. Pre-treatment effects on embryoid induction were also investigated and two of the genotypes displayed differential response to temperature stress pretreatment. In general the beneficial effect of charcoal in the media was confirmed. Successful embryoid production and plantlet regeneration was obtained from the wild potato species, Solanum papita. The influence of sucrose concentration on embryoid production was also investigated. Large genotype by sucrose interactions were detected and this was mainly due to the differential response of the tuberosum clones to increasing sucrose concentration when compared with S. papita. Embryoids were produced from this species in media containing 15% sucrose although the optimum concentration of sucrose for embryoid production was 9%. The possible role of anther culture techniques in gene introgression and potato improvement is discussed.     

Influence of selected soil saprotrophes on gas exchange,growth and yield of <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

The influence of saprotrophic soil fungi: Aspergillus versicolor, Penicillium frequentans, Penicillium verrucosum var. cyclopium, and Trichocladium asperum on development, wholesomeness, gas exchange and yield of potato cv. Mila was studied in pot experiments during 1998–2000. The presence of each of the mentioned fungi species in soil accelerated potato germination and stimulated growth of overground plant parts in comparison with control plants. Additionally, the presence of the tested saprotrophes in soil prologed the potato growing period by inhibiting chloroses of necroses. The tested saprotrophic fungi also modified plant physiological processes, such as transpiration and assimilation. The contact of plant root system with soil saprotrophes diminished significantly assimilation and transpiration intensity of overground parts in comparison with the control plants, in all the years of the experiment. However, this response did not reduce the yield of tubers.     

The role of fungal proteinases in pathophysiology of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis>

The adverse health effects of Stachybotrys chartarum have often been linked to exposure to the trichothecene mycotoxins. Recent studies have shown that in addition to mycotoxins this fungus is capable of producing and secreting in vivo proteins such as hemolysins and proteinases. Spore extracts obtained from a high trichothecene producing isolate JS 58-17 exhibited a significantly lower proteolytic activity compared to the low trichothecene producer, JS 58-06. Growing isolates on rice or potato dextrose agar results in higher proteolytic activity of the spores compared to those grown on drywall. Proteinases in the spore extracts can hydrolyze gelatin and collagen I and IV. Analysis of zymograms shows the presence of several proteins with proteolytic activity in the spores of S. chartarum. Human tracheal epithelial cells exposed to spore extracts produced significantly higher levels of IL-6, IL-8, and TNF-α than control cells. This stimulation of cytokine production was completely abolished by Pefabloc, a serine protease inhibitor. Neutrophil numbers and proinflammatory cytokine (IL1-β and TNF-α) concentrations were highly elevated in the lungs of 7 day old rat pups exposed intratracheally to 4 × 10⁴ spores/gm body weight compared to control. No significant differences in those inflammatory indices in vivo were noted between the treatments with the high trichothecene producer, isolate JS 58-17 and JS 58-06, which does not produce macrocyclic trichothecenes. Immunohistochemistry revealed reduced collagen IV labeling in spore-induced lung granulomas in rat pups exposed to both isolates. These results suggest that proteinases from S. chartarum spores significantly contribute to lung inflammation and injury.     

Accumulation of vitamin E in potato (Solanum tuberosum) tubers

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). α-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in α-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less α-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.     

Enzymatic synthesis of α-2-deoxyglucosyl derivatives catalyzed by organic solvent-resistant α-glucosidase

Organic solvent-resistant Aspergillus niger α-glucosidase (ANGase) can synthesize α-2-deoxyglucosyl derivatives (2DDs) in water-organic solvent media by a trans-addition reaction from d-glucal to various acceptors. Herein, we studied the influence of four different solvents on ANGase stability and activity. ANGase exhibited 47 or 43% residual activity following incubation in 50% (v/v) or in 70% (v/v) acetone for 4 h, respectively. When various carbohydrates were used as acceptor molecules, ANGase catalyzed the addition reaction of four different sugar alcohols, glucose, sucrose, or trehalose to d-glucal. Among the acceptor molecules tested, xylitol was the best acceptor by producing the highest yield (87% addition). The concentration of acetone/acceptor influenced the formation of 2DDs and the yields. We confirmed the molecular weight of five kinds of products by mass spectrometry and enzymatic hydrolysis. Current method is useful for the production of carbohydrates containing 2-deoxyglucose moiety.