The leucine-responsive regulatory protein SCAB_Lrp modulates thaxtomin biosynthesis, pathogenicity, and morphological development in Streptomyces scabies
Jing Liu, Yunxia Wang, and Haoyang He contributed equally to this work.
Abstract
Streptomyces scabies is the best-characterized plant-pathogenic streptomycete, which is a special species among the large genus Streptomyces. The pathogenicity of S. scabies relies on the production of the secondary metabolite thaxtomin A. Little is known about the molecular mechanisms underlying the regulation of thaxtomin biosynthesis in S. scabies beyond the pathway-specific activator TxtR and the cellulose utilization repressor CebR. The leucine-responsive regulatory protein (Lrp) family modulates secondary metabolism in nonpathogenic streptomycetes. However, the regulatory relationship between the Lrp and pathogenic streptomycetes remains unknown. In this study, we demonstrated that SCAB_Lrp (SCAB_77931) from S. scabies significantly affects thaxtomin biosynthesis, pathogenicity, and morphological development. SCAB_Lrp deletion resulted in a dramatic decline in thaxtomin A production and a low-virulence phenotype of S. scabies. An in-depth dissection of the regulatory mechanism of SCAB_Lrp revealed that it positively regulates the transcription of the thaxtomin biosynthetic gene cluster by directly binding to the promoter of the cluster-situated regulator gene txtR. SCAB_Lrp also controls the morphological development of S. scabies by directly activating the transcription of amfC, whiB, and ssgB. SCAB_Lrp directly controls the transcription of its own gene by binding a specific sequence (5′-GGACAGTCGCCGTGCTACG-3′). Moreover, phenylalanine and methionine have been characterized as SCAB_Lrp effectors by strengthening the binding affinity and complex status between SCAB_Lrp and DNA. Our findings characterize a multifunctional regulatory protein, SCAB_Lrp, that controls secondary metabolism, pathogenicity, and sporulation in S. scabies and provide new insights into the complex regulatory network that modulates thaxtomin phytotoxins in pathogenic Streptomyces.
1 INTRODUCTION
The genus Streptomyces is best known for producing various natural products with a wide range of biological functions. Nevertheless, rare species of this genus are phytopathogenic and cause pitted or raised scab lesions on root and tuber crops (Lerat et al., 2009; Loria et al., 2006). Streptomyces scabies, S. acidiscabies, and S. turgidiscabies are the best-studied representatives of the species that cause potato common scab. The phytotoxin thaxtomin A has been identified as a principal determinant of the pathogenicity of S. scabies and related species (Loria et al., 2008). Thaxtomin A is a member of a family of nitrated diketopiperazines containing phenylalanine residues and 4-nitrotryptophan (King & Calhoun, 2009). Thaxtomin A was identified as a repressor of cellulose biosynthesis in plants, although its precise target and molecular mode of action remain unclear (Wang et al., 2020). Furthermore, thaxtomin A at the nanomolar level causes serious seedling stunting and plant cell hypertrophy (Bischoff et al., 2009), thus has great potential for use as a natural bioherbicide (Wang et al., 2020).
The biosynthetic pathway of thaxtomin A and its analogues has been investigated through extensive genetic and biochemical studies (Bignell et al., 2014b; Li et al., 2019a, 2019b; Loria et al., 2006). The thaxtomin biosynthetic gene cluster (txt cluster) of thaxtomin is highly conserved and contains seven genes arranged within a region of approximately 18.3 kb (Huguet-Tapia et al., 2016). Among them, six genes (txtA, txtB, txtC, txtD, txtE, and txtH) encode thaxtomin biosynthetic enzymes, whereas txtR encodes the only cluster-situated regulator (CSR) that functions as an activator of thaxtomin biosynthesis (Joshi et al., 2007; Yang et al., 2011). The biosynthesis of thaxtomin A is under strict transcriptional control, involving at least five global regulators belonging to the bld gene family with unresolved mechanism (Bignell et al., 2014a) and a pleiotropic regulator, CebR (Francis et al., 2015), in addition to the CSR protein TxtR (Joshi et al., 2007).
As typical transcription regulators, the leucine-responsive regulatory protein (Lrp) regulator family is widespread and well-characterized in bacteria and archaea (Peeters & Charlier, 2010; Ziegler & Freddolino, 2021). Lrps consist of two domains: a helix-turn-helix (HTH) domain at the N-terminus for DNA binding and an αβ sandwich domain at the C-terminus for ligand response (Ziegler & Freddolino, 2021). Studies on Lrps have made significant advances in understanding the regulatory mechanism controlling antibiotic biosynthesis by S. lincolnensis SLCG_Lrp (Xu et al., 2020), S. coelicolor SCO3361 (Liu et al., 2017b), and Saccharopolyspora erythraea SACE_Lrp (Liu et al., 2017a, 2021) and SACE_5717 (Liu et al., 2019) in our laboratory, and S. spiramyceticus SSP_Lrp (Lu et al., 2019).
Among the S. scabies 87.22 genome, 10 Lrp genes were detected by bioinformatic analysis. However, the regulatory function of the Lrps regarding thaxtomin biosynthesis, morphological differentiation, and viability in S. scabies is unclear. In this study, we found that a novel protein SCAB_Lrp (SCAB_77931) among all Lrp homologues from S. scabies 87.22 exhibited the highest amino acid identity with previously reported actinomycete Lrps, implying that SCAB_Lrp may have similar regulatory functions to them. We therefore focused on SCAB_Lrp to investigate its function and mechanism in controlling thaxtomin production, morphological development, and pathogenicity.
2 RESULTS
2.1 Identification of an Lrp family homologue in S. scabies
The genomic sequence of S. scabies predicts that SCAB_77931 is a putative Lrp family homologue named SCAB_Lrp, exhibiting 30%–45% amino acid identity with previously reported actinomycete Lrps (Figure 1a). Specifically, SCAB_Lrp shares high similarities with SACE_5717 (45% identity) (Liu et al., 2019) and SACE_Lrp (36% identity) (Liu et al., 2017a) of S. erythraea, SLCG_Lrp (34% identity) of S. lincolnensis (Xu et al., 2020), SCO3361 (34% identity) of S. coelicolor (Liu et al., 2017b), and SSP_Lrp (30% identity) of S. spiramyceticus (Lu et al., 2019) (Figures 1b and S1). Furthermore, by analysing the amino acid sequences of SCAB_Lrp and the above actinomycete Lrps, we found their N-terminus with the helix-turn-helix DNA-binding motif was more conserved, while the C-terminus with the effector-binding domain was relatively variable (Figure 1b).
2.2 SCAB_Lrp positively regulates the production of thaxtomin A
SCAB_Lrp gene contains 495 nucleotides (nt) and encodes a novel Lrp family protein of 164 amino acids with an unresolved regulatory function in S. scabies. To clarify the relationship between SCAB_Lrp and thaxtomin A production, the SCAB_Lrp gene was disrupted with tsr replacement in S. scabies 87.22 by the method of homologous chromosomal recombination (Figure 2a), and the resulting mutant ΔSCAB_Lrp was confirmed by PCR (Figure 2b). Fermentation and high-performance liquid chromatography (HPLC) analysis showed that the production of thaxtomin A in ΔSCAB_Lrp was only approximately 48% of that in the parent strain 87.22 cultured in the oat bran broth (OBB) liquid medium (Figure 2c). To investigate the effect of SCAB_Lrp deletion on bacterial growth, the biomass curves of mutant ΔSCAB_Lrp and parent strain 87.22 were examined. The results showed that ΔSCAB_Lrp accumulated a lower dry weight of mycelia than that of strain 87.22 (Figure 2d), and the yield of thaxtomin A in ΔSCAB_Lrp relative to the dry weight of mycelia was also decreased by 31%, demonstrating that the decreased thaxtomin yield in ΔSCAB_Lrp partly resulted from changes in cell growth. Thaxtomin A production and bacterial growth of complemented strain ΔSCAB_Lrp/SCAB_Lrp were restored to that of the parent strain 87.22 when a single copy of SCAB_Lrp was introduced into ΔSCAB_Lrp (Figure 2c,d). These results indicate that SCAB_Lrp of S. scabies is an important activator of thaxtomin A production and bacterial growth.
2.3 SCAB_Lrp positively regulates virulence of S. scabies
Because thaxtomin A is the principal virulence determinant of pathogenic streptomycetes, we selected radish and Arabidopsis thaliana (ecotype Col-0) seedlings to evaluate the effect of SCAB_Lrp disruption on the virulence phenotype of S. scabies. Radish (Figure 3a) and A. thaliana (Figure 3b) seedlings were inoculated with the fermentation extract isolated from 87.22 and ΔSCAB_Lrp. After 3 days of growth, radish (Figure 3a) and A. thaliana seedlings (Figure 3b) inoculated with the fermentation broth of ΔSCAB_Lrp displayed reduced root and shoot stunting compared to those inoculated with 87.22. The average length of radish seedlings inoculated with ΔSCAB_Lrp was 3.18 cm, an increaseof 45% (p < 0.05) compared to 2.2 cm of seedlings inoculated with 87.22. Similarly, the average length of A. thaliana seedlings inoculated with ΔSCAB_Lrp was 1.24 cm, an increase of 48% (p < 0.001) compared to seedling length of 0.84 cm when inoculated with 87.22. In addition, a decreased capacity of ΔSCAB_Lrp to induce pitting and necrosis in potato tuber slices in comparison to 87.22 was observed (Figure 3c). Taken together, the disruption of SCAB_Lrp resulted in a low-virulence phenotype of S. scabies, demonstrating that SCAB_Lrp is the primary activator of plant pathogenicity through its effect on the production of thaxtomin and bacterial growth.
2.4 SCAB_Lrp directly controls the expression of the txt cluster
To investigate the relationship between SCAB_Lrp and the txt cluster, transcriptional analyses of ΔSCAB_Lrp and 87.22 were performed during the thaxtomin production process. The txt cluster includes two polycistronic transcriptional units (txtA to txtC and txtE to txtD) and txtR (Figure 4a). txtR encodes only the CSR and is in the opposite orientation to txtA with an intergenic region of 1671 bp (Joshi et al., 2007). Three genes, txtR, txtA, and txtE, were selected for the reverse transcription-quantitative PCR (RT-qPCR) analyses. The results showed that the transcript amounts of txtR, txtA, and txtE were drastically decreased in ΔSCAB_Lrp compared to that in 87.22 (Figure 4b). These results demonstrate that SCAB_Lrp positively controls the transcription of thaxtomin biosynthesis genes in S. scabies.
The txt cluster of S. scabies contains three regulatory regions: putative txtR, txtA, and txtE promoter regions (Li et al., 2021) (Figure 4a). To investigate whether SCAB_Lrp directly binds to the regulatory regions of the txt cluster, electrophoretic mobility shift assays (EMSAs) were performed using purified His6-tagged SCAB_Lrp (Figure 4c) and the corresponding probes (Table S2). The EMSA results showed that SCAB_Lrp could bind to the promoter region of the CSR gene txtR, and the SCAB_Lrp-PtxtR complex formed in a concentration-dependent manner (Figure 4d). However, no protein–DNA complex was formed when PtxtA or PtxtE was incubated with SCAB_Lrp (Figure 4d). We also used a biosensor system with green fluorescent protein (GFP) in Escherichia coli to verify the interaction of SCAB_Lrp with the probe PtxtR in vivo. As shown in Figure 2e, plasmid ptxtR-E in which egfp gene was directly controlled by the promoters of txtR was transformed into E. coli DH5α as control. SCAB_Lrp gene driven by the promoter of the aac(3)IV gene was inserted into ptxtR-E and transformed into E. coli DH5α. When SCAB_Lrp gene was inserted into the ptxtR-E, the green fluorescence was enhanced by 76% compared to that without SCAB_Lrp (Figure 2f). However, when SCAB_Lrp gene was inserted into the ptxtA-E or ptxtE-E, the green fluorescence was almost unchanged compared to that without SCAB_Lrp (Figure 2f).
Taken together, these results demonstrated that SCAB_Lrp affects thaxtomin biosynthesis by directly activating the expression of the CSR gene txtR in S. scabies.
2.5 SCAB_Lrp is directly involved in the morphological differentiation of S. scabies
To investigate the role of SCAB_Lrp disruption on morphological differentiation, 87.22 and ΔSCAB_Lrp spores were grown on a soy flour mannitol (SFM) agar for phenotypic observation. Compared with 87.22, ΔSCAB_Lrp showed significantly delayed spore formation, and the morphological 87.22 phenotype in ΔSCAB_Lrp/SCAB_Lrp was recovered (Figure 5a), indicating that SCAB_Lrp has a significant effect on the morphological development of S. scabies.
RT-qPCR assays were performed on three genes related to morphological differentiation of Streptomyces: amfC (SCAB_49711), whiB (SCAB_55081), and ssgB (SCAB_53351). The expression levels of amfC, whiB, and ssgB were dramatically decreased in ΔSCAB_Lrp compared to that in 87.22 (Figure 5b). Moreover, the binding affinity of SCAB_Lrp to the promoter regions of amfC, whiB, or ssgB was assessed by EMSAs, and SCAB_Lrp could directly bind to the promoters of these three genes to different extents (Figure 5c). In particular, there were two shifted bands in SCAB_Lrp incubated with the probes PamfC and PwhiB, while only one shifted band appeared after SCAB_Lrp was incubated with PssgB (Figure 5c). Results from GFP reporter assays showed that when the SCAB_Lrp gene was expressed in the presence of pamfC-E, pwhiB-E and pssgB-E, the green fluorescence was enhanced by 111%, 58%, and 82% compared to that without SCAB_Lrp (Figure 5d). These results suggest that SCAB_Lrp positively controls sporulation by directly activating the expression of the genes amfC, whiB, and ssgB.
Based on the above findings, SCAB_Lrp was identified to directly regulate thaxtomin biosynthesis and control the morphological development of S. scabies.
2.6 Identification of the binding site and the amino acid effector of SCAB_Lrp
Based on the regulatory model of the Lrp, we hypothesized that SCAB_Lrp might regulate the transcription of its own gene, so the expression level of SCAB_Lrp in ΔSCAB_Lrp was examined. Through measuring the remaining 99-bp 5′-region of SCAB_Lrp gene, the transcript amount of SCAB_Lrp in ΔSCAB_Lrp was found to be slightly increased compared to that in 87.22 at 24 h but dramatically decreased at 48 h (Figure 6a). Furthermore, in vitro, EMSAs results showed that an evidently shifted band appeared after SCAB_Lrp incubation with probe P77931 (the promoter region of SCAB_Lrp), confirming that SCAB_Lrp could specifically bind to the promoter of its own gene with the highest affinity compared to the other probes mentioned above (Figure 6b).
SCAB_Lrp shares the highest similarity with SACE_5717 of S. erythraea. The DNA-binding site of SACE_5717 (5′-GAACGTTC GCCGTCACGCC-3′) has previously been reported (Liu et al., 2019) and this was used to search for a SCAB_Lrp potential binding site. In sequence BLAST analysis, a 19 bp highly similar sequence (5′-GGACAGTCGCCGTGCTACG-3′) was identified in P77931, which lay in the SCAB_Lrp promoter region (named as site OP) (Figure 6b). To detect whether SCAB_Lrp directly interacts with the site OP, EMSAs were performed using SCAB_Lrp and the mutated probe Pd(siteOP) derived from P77931 with the site OP deleted. The SCAB_Lrp-P77931 complex completely disappeared when SCAB_Lrp was incubated with Pd(siteOP), indicating that SCAB_Lrp specifically binds to its own promoter by directly interacting with the OP site (Figure 6b).
Lrp transcriptional regulators respond to various amino acids (Ziegler & Freddolino, 2021). To investigate the potential effectors of SCAB_Lrp, a series of EMSAs was conducted using SCAB_Lrp and P77931 present with different protein amino acids (Figure S2). The results showed that adding methionine (Figure 6c) and phenylalanine (Figure 6c) could enhance the binding affinity between the SCAB_Lrp and P77931 probes, resulting in a new upper-shifted band of the protein–DNA complex. However, we did not identify any amino acids that could reduce the binding activity of SCAB_Lrp to P77931 (Figure S2). We also used the above biosensor system with GFP to assess the interaction of SCAB_Lrp with its effectors in vivo. Results showed that the green fluorescence was significantly enhanced by 36% and 59% in DH5α/pLrp-77931-E when 10 mM methionine and phenylalanine was added to the culture medium, respectively (Figure 6d). As the control, the green fluorescence showed no difference in DH5α/p77931-E after the addition of 10 mM methionine or phenylalanine (Figure 6d). Moreover, we found that similar results occurred in DH5α/pLrp-txtR-E, DH5α/pLrp-amfC-E, DH5α/pLrp-whiB-E, and DH5α/pLrp-ssgB-E, and the green fluorescence was enhanced to different degrees when 10 mM methionine and phenylalanine were added to the culture medium (Figures 6e and S3). Taken together, these findings suggest that methionine and phenylalanine are indeed the effectors of SCAB_Lrp.
3 DISCUSSION
The most important regulator of thaxtomin production is the TxtR protein, which acts as a transcriptional activator and induces transcription of the txt cluster (Joshi et al., 2007). TxtR is the primary regulator of thaxtomin production and virulence (Joshi et al., 2007). Another important regulator, CebR, has been characterized as a primary inhibitor of thaxtomin A biosynthesis via direct binding to two sites in the txt cluster (Francis et al., 2015). Cellobiose and cellotriose have been identified as ligands for CebR and mediate its binding affinity to DNA, thereby improving the expression of the genes in txt cluster (Francis et al., 2015). In this study, the third important regulator, SCAB_Lrp, of the Lrp family protein from S. scabies, was characterized as a master activator of thaxtomin biosynthesis and morphological development by directly modulating the transcription of the CSR gene txtR and sporulation-associated genes amfC, whiB, and ssgB, as summarized in Figure 7. Our findings broaden our limited insight into the molecular mechanisms underlying the role of transcriptional regulators in thaxtomin biosynthesis and morphogenesis in S. scabies.
Previous studies have revealed that Lrp family proteins are crucial regulators of diverse biological processes, such as amino acid metabolism, and respond to various amino acids in various bacterial species (Ziegler & Freddolino, 2021). Recently, significant advances in understanding the regulatory mechanism controlling antibiotic biosynthesis by Lrp regulators have been made (Liu et al., 2017a, 2017b, 2019, 2021; Lu et al., 2019; Xu et al., 2020). Furthermore, this study demonstrated that a novel Lrp family protein, SCAB_Lrp, from plant-pathogenic S. scabies, is directly involved in thaxtomin biosynthesis, pathogenicity, and sporulation. Thus far, this study has established a concrete regulatory relationship between Lrp family regulators and thaxtomin phytotoxins for the first time, expanding the function of the Lrp on secondary metabolism and morphogenesis from nonpathogenic to pathogenic in the large genus Streptomyces.
The plant-pathogenic bacterium Erwinia amylovora causes the devastating fire blight disease in apple and pear trees (Geier & Geider, 1993; Pusey, 2000). Lrp from E. amylovora Ea1189 is involved in the virulence expression and several virulence-associated traits, including biofilm formation, levansucrase activity, and production of the exopolysaccharide amylovoran (Schachterle & Sundin, 2019). In our study, SCAB_Lrp from S. scabies 87.22 is implicated in bacterial growth and production of thaxtomin A. These findings indicated that the role of regulatory mechanisms of Lrp regulators on pathogenicity and virulence might be numerous and complex in different pathogenic bacteria. More interestingly, Lrp is directly regulated at the posttranscriptional level by the small RNA ArcZ through destabilization of Lrp mRNA in E. amylovora Ea1189 (Schachterle & Sundin, 2019), prompting us to explore further the topology of Lrp in the virulence regulatory network in S. scabies.
Previous studies have shown that the secondary or hierarchical regulatory function of Lrp, which directly controls other regulators, is rare. Our previously reported Lrp SCO3361 directly regulated the actII-ORF4 of CSR gene to control the biosynthesis of actinorhodin in S. coelicolor (Liu et al., 2017b), which is very similar to the case of SCAB_Lrp in S. scabies (Figure 4). SSP_Lrp directly modulates three regulatory genes to control the biosynthesis of spiramycin and bitespiramycin in S. spiramyceticus (Lu et al., 2019). Recently, we reported that SACE_Lrp directly controls the MarR family protein SACE_6745, which plays an essential regulatory role in erythromycin biosynthesis and export (Liu et al., 2021).
Regarding morphological development, SCAB_Lrp directly activated the expression of amfC, whiB, and ssgB, thereby affecting the morphological development of S. scabies (Figure 5). In contrast, in S. coelicolor, SCO3361 activates the transcription of amfC, whiB, and ssgB but only directly binds to the amfC promoter region (Liu et al., 2017b). Furthermore, two shifted bands appeared after SCAB_Lrp incubation with the amfC promoter region (Figure 5c), whereas only one shifted band appeared after SCO3361 incubation with the amfC promoter region (Liu et al., 2017b). These findings indicate that the regulatory model of the role of Lrps in morphological development may vary in the large genus Streptomyces.
The Lrp family proteins generally utilize amino acids as structural effectors (Ziegler & Freddolino, 2021). In our previous studies, SLCG_Lrp (Xu et al., 2020), SCO3361 (Liu et al., 2017b), and SACE_Lrp (Liu et al., 2017a) responded to bidirectional amino acid effectors, either increasing or decreasing the DNA-binding affinity of the protein. SACE_5717 (Liu et al., 2019) and SCAB_Lrp exhibited only a one-directional response to their respective effectors. Notably, tryptophan, tyrosine, and arginine decreased the DNA-binding affinity of SACE_5717 (Liu et al., 2019), whereas methionine and phenylalanine increased the DNA-binding affinity of SCAB_Lrp, resulting in a new protein–DNA complex (Figure 6c). Although SCAB_Lrp shares the highest similarity to SACE_5717 (45% identity), it responds to different amino acids, probably because the ligand-binding domain in its C-terminal is not conserved to interact with a wide variety of effectors (Figure 1). These findings demonstrate the sophisticated mechanism by which Lrps respond to different amino acid effectors. The response of SCAB_Lrp to methionine and phenylalanine may have more important biological significance, and the plant pathogenicity of S. scabies could be altered by changing the concentration of methionine and phenylalanine in the environment to coordinate the affinity between SCAB_Lrp and its target.
4 EXPERIMENTAL PROCEDURES
4.1 Bacterial strains and cultivation conditions
The plasmids, E. coli strains, S. scabies, and its derivatives used in present work are listed in Table S1. E. coli strains were cultured in liquid or on solid Luria Bertani (LB) medium at 37°C. E. coli DH5α was used for plasmid construction, E. coli ET12567 (pUZ8002) was used as the donor host for plasmid conjugation to S. scabies, and E. coli BL21 (DE3) was used for overproduction of SCAB_Lrp protein. S. scabies and its derivatives were cultured at 28°C on SFM solid medium for sporulation and phenotypic observation, or in tryptic soy broth (TSB) liquid medium for growth of mycelia and seed culture (Kieser et al., 2000). In addition, OBB liquid medium was used for thaxtomin production (Francis et al., 2015). The antibiotics were added at the following concentrations where required: ampicillin 100 μg/ml, apramycin 100 μg/ml, kanamycin 50 μg/ml, and chloramphenicol 25 μg/ml for E. coli strains; thiostrepton 25 μg/ml, nalidixic acid 50 μg/ml, and apramycin 100 μg/ml for S. scabies strains.
4.2 Deletion mutant construction
To generate the SCAB_Lrp deletion mutant in S. scabies 87.22, two 1500 bp flanking fragments of SCAB_Lrp were prepared by PCR with the corresponding primers (Table S2). The two fragments were treated with KpnI/EcoRI and XbaI/HindIII, respectively, and then cloned into the vector pUCTSR (Han et al., 2011) to obtain recombinant plasmid pUC-ΔSCAB_Lrp. Subsequently, pUC-ΔSCAB_Lrp was transferred into S. scabies 87.22 by conjugation with E. coli ET12567 (pUZ8002). By the homologous chromosomic recombination, a 303-bp fragment of the SCAB_Lrp gene was replaced by the thiostrepton resistance gene (tsr) in S. scabies 87.22, with 99-bp initial part of SCAB_Lrp gene remained. The mutant ΔSCAB_Lrp with thiostrepton resistance was confirmed by PCR with the corresponding primers (Table S2).
4.3 Gene complementation
To construct the complementation strain ΔSCAB_Lrp/pIB-SCAB_Lrp, full-length SCAB_Lrp gene of 495-bp was prepared by PCR from the 87.22 genomic DNA with the corresponding primers (Table S2). The obtained fragment was inserted into the vector pIB139 (Wilkinson et al., 2002) by NdeI/XbaI sites to construct plasmid pIB-SCAB_Lrp. By conjugation, pIB-SCAB_Lrp and pIB139 were introduced into the mutant ΔSCAB_Lrp, generating the complemented strains ΔSACE_Lrp/SACE_Lrp and ΔSACE_Lrp/pIB139, respectively, by apramycin-screening and PCR with the corresponding primers (Table S2).
4.4 Determination of thaxtomin A production
Quantified 107 spores of S. scabies 87.22 and its derivatives were inoculated into 50 ml of TSB liquid medium with shaking at 28°C, 220 rpm for 2 days as a seed culture, and then 5 ml of seed culture was transferred into 50 ml of OBB liquid medium at 28°C with shaking at 220 rpm for 7 days. For thaxtomin A analysis, the extracts were isolated and analysed by an HPLC system with a Wondasil C18 Superb column (4.6 × 150 mm, 5 μm; GC Sciences) as previously described (Francis et al., 2015; Jourdan et al., 2016). The production of thaxtomin A was quantified by a standard curve generated using an authentic thaxtomin A standard (AbMole BioScience).
4.5 Determination of cell growth
The biomass of S. scabies 87.22 and mutant ΔSCAB_Lrp were measured as previously described (Wu et al., 2014) to determine the effect of SCAB_Lrp deletion on cell growth. Cultivation of 87.22 and ΔSCAB_Lrp was as described in Section 4.4. One millilitre of culture samples was collected once a day and followed by centrifugation. Cell pellets were washed twice with sterile water and dried until constant weight. The dry weight of cell pellets was measured with a precise analytical balance.
4.6 RNA extraction and RT-qPCR experiment
Total cells from S. scabies 87.22 and mutant ΔSCAB_Lrp were prepared by centrifugation, and total RNA was extracted using TransZol reagent (TransGen Biotech) at 24 and 48 h of growth in OBB liquid medium. Briefly, isolated RNA was treated with DNase I (TransGen Biotech) and cDNA synthesis was achieved by reverse transcription with a cDNA synthesis kit (TransGen Biotech). Quantitative real-time PCR was performed on the Applied Biosystems QuantStudio 6 Flex system, and all operation procedures were performed in accord with the manufacturer's instructions (TransGen Biotech). The 16S rRNA gene from S. scabies served as an internal control to normalize samples. All primers used for RT-qPCR assay are listed in Table S2.
4.7 SCAB_Lrp protein overproduction and purification
The coding sequence of the SCAB_Lrp (SCAB77931) gene was amplified from the S. scabies 87.22 genome by PCR using the corresponding primers (Table S2), and inserted into the HindIII/NdeI sites of pET28a to construct pET-SCAB_Lrp plasmid. pET-SCAB_Lrp was transferred into E. coli BL21 (DE3) for SCAB_Lrp overproduction. Overproduction and purification of His6-tagged SCAB_Lrp were carried out as previously described (Liu et al., 2017a).
4.8 EMSA
The promoter regions of txtR (SCAB_31801), txtA (SCAB_31791), txtE (SCAB_31831), amfC (SCAB_49711), whiB (SCAB_55081), ssgB (SCAB_53351), and SACB_Lrp (SCAB_77,931) were amplified from 87.22 genomic DNA by PCR with the respective primers (Table S2). The probes of PCR products were purified, quantified, and then used for EMSA (Hellman & Fried, 2007). All probes were individually incubated with His6-tagged SCAB_Lrp in binding buffer as described previously (Liu et al., 2017a).
For analysis of the potential amino acid-effectors of SCAB_Lrp, 20 natural amino acids were individually incubated with His6-tagged SCAB_Lrp protein and the probe P77931 as described previously (Liu et al., 2017a).
4.9 Plant virulence bioassays
Virulence assays of S. scabies strains on radish seedlings were prepared as described previously (Jourdan et al., 2016). Germinated radish seeds were placed into six agar wells formed in a deep 1.5% water agar plate and were inoculated each with 200 μl of fermentation extract, which was isolated from S. scabies 87.22 or mutant ΔSCAB_Lrp as previously described (Francis et al., 2015, Jourdan et al., 2016). The plates were incubated at 21 ± 2°C under a 16 h photoperiod for 6–7 days. Virulence assays of S. scabies strains on Arabidopsis thaliana (ecotype Col-0) seedlings were prepared as described previously (Francis et al., 2015). A. thaliana seedlings were transferred and inoculated with 200 μl of fermentation samples of 87.22 or ΔSCAB_Lrp. The plates were incubated at 21 ± 2°C, with a 12 h photoperiod, for 3–4 days. The virulence phenotypes of S. scabies strains on potato tuber slices were assessed as described previously (Loria et al., 1995). Quantified 107 spores of S. scabies 87.22 and mutant ΔSCAB_Lrp were cultured on SFM plates until well sporulated. Agar plugs with mature spores were prepared and then inverted onto the potato tuber slices. The tuber slices were then incubated at 23 ± 2°C in a dark and moist incubator for 7–8 days.
4.10 GFP reporter assay
For construction of the GFP reporter plasmids, a fragment containing the putative promoter region of txtR was amplified from the 87.22 genome by PCR with PtxtR-F and PtxtR-egfp-R primers (Table S2), and the enhanced green fluorescent protein gene (egfp) fragment was amplified from pUPW-EGFP (Liu et al., 2017a) by PCR with PtxtR-egfp-F and egfp-R primers. The two fragments were together used as templates for an overlapping PCR with PtxtR-F and egfp-R primers (Table S2) to obtain the PtxtR-egfp fragment, then digested with BamHI/EcoRI and joined into pKC1139 to create the control plasmid ptxtR-E. To evaluate the regulatory effect of SCAB_Lrp on txtR gene, Paac(3)IV promoter was amplified from pIB139 (Wilkinson et al., 2002) using primers Paac(3)IV -F and Paac(3)IV-Lrp-R (Table S2) and the SCAB_Lrp gene was amplified from the 87.22 genome using primers Paac(3)IV-Lrp-F and Lrp-R (Table S2), respectively. The two fragments were together used as templates for an overlapping PCR with Paac(3)IV -F and Lrp-R primers (Table S2) to obtain the Paac(3)IV-SCAB_Lrp fragment, then digested with XbaI/HindIII and joined into ptxtR-E to create the plasmid pLrp-txtR-E. The above method was also used to obtain the other reporter plasmids with corresponding primers listed in Table S2. These plasmids were transformed into E. coli DH5α. Green fluorescence was detected by excitation at 485 nm, and emission at 510 nm (Molecular Devices). All fluorescence values were normalized to growth rates (OD600).
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (31800057, 31972930), the Initial Foundation of Scientific Research in Anhui Agricultural University (yj2018-08), and the University Synergy Innovation Program of Anhui Province (GXXT-2019-035).
CONFLICT OF INTEREST
All authors declare they have no conflict of interest.
Open Research
DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are available from the corresponding author on reasonable request.
