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The phytotoxin thaxtomin A (TA) is the key pathogenicity factor synthesized by the bacteria Streptomyces scabiei, the main causal agent of common scab of potato (Solanum tuberosum L.). TA treatment of potato tuber flesh produces a brown color that was attributed to necrosis. The intensity of TA-induced browning was generally thought to correlate with potato sensitivity to the disease. In this study, we found that TA-induced browning was much more intense in the potato tuber flesh of the common scab moderately resistant variety Russet Burbank (RB) than that observed in tubers of the disease-susceptible variety Yukon Gold (YG). However, there was no significant difference in the level of TA-induced cell death detected in both varieties, suggesting that tubers response to TA does not correlate with the level of sensitivity to common scab. TA-treated potato tuber tissues accumulated significantly higher levels of phenolic compounds than untreated controls, with a higher phenol content detected in RB TA-treated tissues than in those of YG. Browning was associated with a significant induction of the expression of genes of the phenylpropanoid pathway in RB tubers, indicating that TA activated this metabolic pathway. These results suggest that tuber flesh browning induced by TA is due to the accumulation of phenolic compounds. These phenolics may play a role in the protection of potato tubers against S. scabiei.

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The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic Streptomyces strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter Pecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal Pecf17 promoter was determined, and a Pecf17 promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S. venezuelae ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 ± 15.3  µ g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S. venezuelae ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces albus J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 µg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse Streptomyces hosts.

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Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 µg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers.

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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.

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BACKGROUND: Thaxtomin A (TA) is a natural cellulose biosynthesis inhibitor (CBI) synthesized by the potato common scab-causing pathogen Streptomyces scabies. Inhibition of cellulose synthesis by TA compromises cell wall organization and integrity, leading to the induction of an atypical program of cell death (PCD). These processes may facilitate S. scabies entry into plant tissues. To study the mechanisms that regulate the induction of cell death in response to inhibition of cellulose synthesis, we used Arabidopsis thaliana cell suspension cultures treated with two structurally different CBIs, TA and the herbicide isoxaben (IXB). RESULTS: The induction of cell death by TA and IXB was abrogated following pretreatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the natural auxin indole-3-acetic acid (IAA). The addition of auxin efflux inhibitors also inhibited the CBI-mediated induction of PCD. This effect may be due to intracellular accumulation of auxin. Auxin has a wide range of effects in plant cells, including a role in the control of cell wall composition and rigidity to facilitate cell elongation. Using Atomic Force Microscopy (AFM)-based force spectroscopy, we found that inhibition of cellulose synthesis by TA and IXB in suspension-cultured cells decreased cell wall stiffness to a level slightly different than that caused by auxin. However, the cell wall stiffness in cells pretreated with auxin prior to CBI treatment was equivalent to that of cells treated with auxin only. CONCLUSIONS: Addition of auxin to Arabidopsis cell suspension cultures prevented the TA- and IXB-mediated induction of cell death. Cell survival was also stimulated by inhibition of polar auxin transport during CBI-treatment. Inhibition of cellulose synthesis perturbed cell wall mechanical properties of Arabidopsis cells. Auxin treatment alone or with CBI also decreased cell wall stiffness, showing that the mechanical properties of the cell wall perturbed by CBIs were not restored by auxin. However, since auxin's effects on the cell wall stiffness apparently overrode those induced by CBIs, we suggest that auxin may limit the impact of CBIs by restoring its own transport and/or by stabilizing the plasma membrane - cell wall - cytoskeleton continuum.

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Thaxtomin A (TA) is a cellulose biosynthesis inhibitor synthesized by the soil actinobacterium Streptomyces scabies, which is the main causal agent of potato common scab. TA is essential for the induction of scab lesions on potato tubers. When added to Arabidopsis thaliana cell cultures, TA induces an atypical programmed cell death (PCD). Although production of reactive oxygen species (ROS) often correlates with the induction of PCD, we observed a decrease in ROS levels following TA treatment. We show that this decrease in ROS accumulation in TA-treated cells is not due to the activation of antioxidant enzymes. Moreover, Arabidopsis cell cultures treated with hydrogen peroxide (H2O2) prior to TA treatment had significantly fewer dead cells than cultures treated with TA alone. This suggests that H2O2 induces biochemical or molecular changes in cell cultures that alleviate the activation of PCD by TA. Investigation of the cell wall mechanics using atomic force microscopy showed that H2O2 treatment can prevent the decrease in cell wall rigidity observed after TA exposure. While we cannot exclude the possibility that H2O2 may promote cell survival by altering the cellular redox environment or signaling pathways, our results suggest that H2O2 may inhibit cell death, at least partially, by reinforcing the cell wall to prevent or compensate for damages induced by TA.

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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.

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The biosynthesis of phytotoxin thaxtomin A (TXT) constitutes the major pathogenicity determinant in Streptomyces scabies, the most widely studied phytopathogen causing scab disease in potato and other root crops. It is recognized that S. scabies regulates its pathogenicity via γ-butyrolactone (GBL)-dependent quorum sensing (QS) signalling. AttM, from Agrobacterium tumefaciens C58 strain, has recently been proposed to have GBL-assimilative capacity. Here, we presented the introduction of A. tumefaciens-derived attM gene into S. scabies using the Escherichia coli-Streptomyces shuttle vector pIJ8600 via intergeneric conjugation, followed by the investigation of secondary metabolism (mycelium growth, TXT production and pathogenicity) in S. scabies attM exconjugants (S.s/attM) in comparison with their wild-type parent strain (S.s/WT). Among the resultant S.s/attM exconjugants, attM was found to be integrated into S. scabies chromosome as analysed by Southern blotting. Moreover, S.s/attM failed to evoke the disease symptoms in planta and displayed altered morphological differentiation in contrast to S.s/WT. The abolishment of TXT production in S.s/attM substantiated the loss of pathogenicity and also implied that attM, when constitutively expressed in S. scabies, could paralyse its GBL signalling pathway. Altogether, lactonase-coding gene attM would be useful in a quorum quenching strategy for plant protection via suppressing TXT production and pathogenicity of S. scabies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an efficient means to introduce the lactonase gene attM from Agrobacterium tumefaciens into Streptomyces scabies for evaluating the role of γ-butyrolactone (GBL) in thaxtomin A production and pathogenicity, etc. Our results showed that pathogenicity was abrogated in attM-expressing S. scabies exconjugants. Although there are gene knockout approaches to inactivating GBL signalling and thus pathogenicity in S. scabies, they are not only time consuming due to refractory host but also possibly incomplete in view of gene redundancy. Our work is the first report for a kind of lactonase affecting pathogenicity and/or virulence of scab-causing Streptomyces species.

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To induce potato variants with enhanced resistance to common scab disease that retain the desirable agronomic traits of the original cultivars, we used a cell culture technique that employs thaxtomin A, the primary phytotoxin that induces scab symptoms. We induced 24 variants from the potato cultivar 'Saya-akane', developed in Japan, and selected two with enhanced resistance to the disease by growing them in planters with bacteriainoculated soil and in a field infested with the disease. We also examined toxin tolerance in micro-tubers of variants that showed a lower degree or percentage of infection in the glasshouse screening, and found no significant difference relative to the original cultivar. To clarify the effect of using thaxtomin A, we examined the efficiency of induction of the potential enhanced resistance by comparing the degree of infection among variants grown in planters with inoculated soil. We observed no significant difference between variants induced on culture medium with and without the toxin. These results suggest that the effect of using the toxin as a positive selection agent is restrictive and that most resistance-enhancing mutations are induced by the cell culture procedure itself.

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Common scab, a globally important potato disease, is caused by infection of tubers with pathogenic Streptomyces spp. Previously, disease-resistant potato somaclones were obtained through cell selections against the pathogen's toxin, known to be essential for disease. Further testing revealed that these clones had broad-spectrum resistance to diverse tuber-invading pathogens, and that resistance was restricted to tuber tissues. The mechanism of enhanced disease resistance was not known. Tuber periderm tissues from disease-resistant clones and their susceptible parent were examined histologically following challenge with the pathogen and its purified toxin. Relative expression of genes associated with tuber suberin biosynthesis and innate defense pathways within these tissues were also examined. The disease-resistant somaclones reacted to both pathogen and toxin by producing more phellem cell layers in the tuber periderm, and accumulating greater suberin polyphenols in these tissues. Furthermore, they had greater expression of genes associated with suberin biosynthesis. In contrast, signaling genes associated with innate defense responses were not differentially expressed between resistant and susceptible clones. The resistance phenotype is due to induction of increased periderm cell layers and suberization of the tuber periderm preventing infection. The somaclones provide a valuable resource for further examination of suberization responses and its genetic control.

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KEY MESSAGE: The Arabidopsis mutant ( ucu2 - 2/gi - 2 ) is thaxtomin A, isoxaben and NPA-sensitive indicated by root growth and ion flux responses providing new insights into these compounds mode of action and interactions. Thaxtomin A (TA) is a cellulose biosynthetic inhibitor (CBI) that promotes plant cell hypertrophy and cell death. Electrophysiological analysis of steady-state K(+) and Ca(2+) fluxes in Arabidopsis thaliana roots pretreated with TA for 24 h indicated a disturbance in the regulation of ion movement across the plant cell membrane. The observed inability to control solute movement, recorded in rapidly growing meristematic and elongation root zones, may partly explain typical root toxicity responses to TA treatment. Of note, the TA-sensitive mutant (ucu2-2/gi-2) was more susceptible with K(+) and Ca(2+) fluxes altered between 1.3 and eightfold compared to the wild-type control where fluxes altered between 1.2 and threefold. Root growth inhibition assays showed that the ucu2-2/gi-2 mutant had an increased sensitivity to the auxin 2,4-D, but not IAA or NAA; it also had increased sensitivity to the auxin efflux transport inhibitor, 1-naphthylphthalamic acid (NPA), but not 2,3,5- Triiodobenzoic acid (TIBA), when compared to the WT. The NPA sensitivity data were supported by electrophysiological analysis of H(+) fluxes in the mature (but not elongation) root zone. Increased sensitivity to the CBI, isoxaben (IXB), but not dichlobenil was recorded. Increased sensitivity to both TA and IXB corresponded with higher levels of accumulation of these toxins in the root tissue, compared to the WT. Further root growth inhibition assays showed no altered sensitivity of ucu2-2/gi-2 to two other plant pathogen toxins, alternariol and fusaric acid. Identification of a TA-sensitive Arabidopsis mutant provides further insight into how this CBI toxin interacts with plant cells.

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Thaxtomin A (TXT) is a phytotoxin produced by all plant-pathogenic Streptomyces scabies involved in the potato scab disease. Their pathogenicity was previously correlated with the production of TXT. Calcium is known to be an essential second messenger associated with pathogen-induced plant responses and cell death. We have effectively shown that in Arabidopsis thaliana cell suspensions, TXT induces an early short lived Ca(2+) influx which is involved in the cell death process and other TXT-induced responses. We extended our study to Nicotiana tabacum BY2 by monitoring cell death and changes in cytosolic calcium concentration on cells expressing the apoaequorine Ca(2+) reporter protein to compare the responses to TXT of the two model plants, tobacco and A. thaliana. Our investigations show that cell death in BY2 appeared to be dose dependent with a lag of sensitivity comparing to A. thaliana. Moreover, pathway leading to cell death in BY2 does not involve calcium signaling. Our results suggest that different pathways are engaged in A. thaliana and N. tabacum BY2 to achieve the same response to TXT.