wblE2 transcription factor in Streptomyces griseus S4‐7 plays an important role in plant protection

Abstract Streptomyces griseus S4‐7 was originally isolated from the strawberry rhizosphere as a microbial agent responsible for Fusarium wilt suppressive soils. S. griseus S4‐7 shows specific and pronounced antifungal activity against Fusarium oxysporum f. sp. fragariae. In the Streptomyces genus, the whi transcription factors are regulators of sporulation, cell differentiation, septation, and secondary metabolites production. wblE2 function as a regulator has emerged as a new group in whi transcription factors. In this study, we reveal the involvement of the wblE2 transcription factor in the plant‐protection by S. griseus S4‐7. We generated ΔwblE, ΔwblE2, ΔwhiH, and ΔwhmD gene knock‐out mutants, which showed less antifungal activity both in vitro and in planta. Among the mutants, wblE2 mutant failed to protect the strawberry against the Fusarium wilt pathogen. Transcriptome analyses revealed major differences in the regulation of phenylalanine metabolism, polyketide and siderophore biosynthesis between the S4‐7 and the wblE2 mutant. The results contribute to our understanding of the role of streptomycetes wblE2 genes in a natural disease suppressing system.


| INTRODUCTION
Streptomycetes are Gram-positive, mycelium-forming, soil microorganisms that play important roles in nature. They are of great socio-economic relevance because they produce many secondary metabolites that have been developed into clinical drugs (antibiotics, antitumorals, and immunosuppressants, amongst other) (Hopwood, 2007). Streptomycetes have a complex developmental cycle that resembles filamentous fungi, i.e., forming hyphae, mycelia, and spores.
Traditional research of the streptomycetes developmental cycle mainly focused on sporulation phases in solid culture media (Flärdh & Buttner, 2009). After spore germination, viable vegetative (substrate) mycelium grows on the surface and inside the agar until it differentiates into a reproductive (aerial) mycelium that grows into the air, producing spores at the end of the cycle. Spore germination constitutes the first step of Streptomyces development (Kieser, Bibb, Buttner, Chater, & Hopwood, 2000).
The production of secondary metabolites by streptomycetes generally coincides with, or slightly precedes, the development of aerial hyphae in surface-grown cultures (Guijarro, Suarez, Salas, & Hardisson, 1983). The secondary metabolite production and aerial hyphae formation presumably reflected the need to access multiple nutrients, optimize cellular morphology and metabolic differentiation (Chater & Horinouchi, 2003). These developmental stages govern pathway-specific regulatory genes whose expression, in turn, frequently depends on genes required to produce secondary metabolites (Mikulik, Janda, Weiser, Stastná, & Jiranova, 1984). Some of these genes, notably the whi genes, are necessary for the full differentiation into mature spore chains (Bibb, 2005;Chater & Horinouchi, 2003;Elliot & Talbot, 2004). Remarkably, the whiB-like (wbl) family of regulators (named after the first discovered WhiB protein from Streptomyces coelicolor) is found only in Actinobacteria (Rybniker et al., 2010;Smith et al., 2010). Wbl proteins are generally small (10-15 kDa) and contain a highly-conserved pattern of cysteine residues (Cys-Xn-Cys-X2-Cys-X5-Cys) that bind an iron-sulfur cluster (Jakimowicz et al., 2005). Cluster-free Wbl proteins with disulfide bond play regulatory roles during developmental differentiation of streptomycetes (Crack et al., 2009). Impairment of the Wbl iron-sulfur cluster in bacteria affects Wbl disulfide bond reductase activity (Alam, Garg, & Agrawal, 2007;Beinert, Holm, & Munck, 1997;Kiley & Beinert, 2003). Among wbl regulators, wblE2 is related to bacterial iron-sulfur cluster proteins that wblE2 function as a regulator has emerged as a new group in whi transcription factors (Crack, Green, Hutchings, Thomson, & Le Brun, 2012). The transcription factors are known to involve from in oxidative stress, switching aerial hyphae formation, secondary metabolites production. The results of previous studies suggest that the wbl family of regulator governs various physiological and morphological changes in actinomycetes (Crack et al., 2012).
The Streptomyces griseus S4-7 strain was isolated and characterized from a soil suppressing the strawberry Fusarium wilt disease caused by Fusarium oxysporum f. sp. fragariae (Cha et al., 2016). S4-7 produces bioactive compounds that act to block fungal cell wall biogenesis and stability, resulting in the pathogen growth inhibition. The bioactive compounds and genome sequencing revealed not only a large number of genes dedicated to the secondary metabolite biosynthesis but also a high proportion and diversity of regulatory genes (Cha et al., 2016).
In this study, the function of wblE2 genes of the probiotic plant strain S. griseus S4-7 was evaluated using mutagenesis and biological control experiments, as well as transcriptome analyses. The results contribute to our understanding of the role of streptomycetes wblE2 genes in a natural disease suppressing system.

| Bacterial strains, plasmids, and culture conditions
Strains and plasmids used in this study are listed in Table 1. S. griseus S4-7 was precultured in 25 ml of YEME medium (yeast extract-malt extract: 3 g of yeast extract, 3 g of malt extract, 5 g of bacteriological peptone, 10 g of glucose, 170 g of sucrose per 1 L). S4-7 and mutants were grown for 40 hr. The mycelium was harvested and washed twice with distilled water, then ground in 5 ml of 10.3% (w/v) sucrose with YT (16 g of Bacto tryptone, 10 g of Bacto yeast extract, and 5 g of NaCl per 1 L), incubated at 50°C for 10 min to induce germination, then cooled to room temperature. E. coli cells (0.5 ml) were added to 0.5 ml of heat-shocked S4-7 spores. The mixture was centrifuged for a few seconds (a pulse), the supernatant was discarded, and the cell pellet was resuspended in the residual liquid (~100 μl). The suspension was spread on a MS agar plate supplemented with 10 mmol/L MgCl 2 , which had been dried in a laminar flow cabinet for 1 hr prior to plating. After incubation at 28°C for 16-20 hr, the plate was overlaid with 0.5 ml of distilled water containing 25 μl of nalidixic acid (Ndx; final concentration 20 μg/ml, assuming 25 ml of agar per plate), to eliminate the E. coli donor, and the appropriate antibiotic for plasmid selection. The overlay was distributed evenly using a glass spreader.
The plate was left in a laminar flow cabinet until all the liquid had been absorbed. Incubation was then continued at 28°C.

| Overexpression of the wblE2 gene
To overexpress the function of the WhiB-type transcriptional regulator (wblE2) in the S. griseus S4-7, the gene was cloned into pIJ10257 (Novotna, Hill, Vincent, Liu, & Hong, 2012)  An initial denaturation step at 98°C for 2 min was followed by 30 cycles consisting of denaturation at 98°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s.
The cloning and conjugation protocols were followed as described above. For transformant selection, following incubation for 6 hr at 30°C, 500 μl of antibiotic solution [20 μl of Ndx (stock solution, 25 mg/ml) and 80 μg/ml hygromycin] was spread on the conjugation plate and airdried for 10-20 min in a sterile hood. Plates were incubated for 3 days at 30°C. Bacterial genomic DNA was purified using a Solg ™ genomic DNA PrepKit (Solgent LTD, Korea), and PCR was performed with hygromycin detection primers Hyg-det5 (CGGCTCATCACCAGGTAGGG) and Hyg-det3 (TCCGCTGTGACACAAGAATC) to verify the integration of pIJ10257 into the S. griseus S4-7 genome. Overexpressed S. griseus S4-7 colonies were randomly picked and checked for their ability to inhibit F. oxysporum growth.

| In vitro and in planta antagonistic and biocontrol activity assays
Loss of function of the member of the whi type transcription regulators in S4-7 mutants were assessed by evaluating their activity against the Fusarium wilt pathogen both in vitro and in planta. Fungal discs (5 mm diameter) with cultures pre-grown for 7 days on PDA at 27°C were placed at the center of PDK plates (24 g of potato dextrose, 10 g of peptone, and 18 g of agar per 1 L). The wild-type and mutant strains, pre-grown for 5 days on TSB at 28°C, were transferred to the opposite sides of plates, 2.5 cm away from the fungal disc. PDK plates were then incubated for 7 days at 28°C. Inhibition zones were measured every day, and the antifungal activity of the wild-type and mutants was evaluated after 7 days.
To evaluate the biological control activity of the whi-type transcription regulators, S4-7 and mutant strains were grown in PDK broth for 7 days at 28°C, following which the cell densities were adjusted to 4.2 × 10 7 cfu/ml. Strawberry roots were dipped in bacterial sus- , and the incidence and severity of disease were observed every 7 days up to 45 days after planting. The incidence and severity of the disease were evaluated using a 0-5 scale, every week: 0, healthy; 1, 1 to 3 leaves rolled and yellowed; 2, 3 to 4 leaves rolled and deformed; 3, chlorosis and early plant wilting; 4, necrosis and entire plant wilting; and 5, dead or nearly so. The results were statistically analyzed by Tukey's HSD test (with significance set at p = .05) using SigmaPlot ver.

| S. griseus S4-7 whi genes and mutant phenotypes
The genomic sequence of S. griseus S4-7 has been deposited (GenBank Accession: GCA_000932225.1) and analyzed in RAST server (http:// rast.nmpdr.org). Genes encoding ten members of whi transcription factors or related genes have been identified in the S4-7 genome based on functional annotation by RAST (Table S1). To further reveal the function of these genes, we obtained four mutant stains, following ΔwblE, ΔwblE2, ΔwhiH, and ΔwhmD ( Figure S1). Their functions in streptomycetes have not been clearly verified, except for the whiH gene. whiH participates cell division in sporulation stage (McCormick & Flȁrdh, 2012). The loci were disrupted by homologous knockout mutagenesis using pKC1132 suicide vector system. The four mutants showed reduced differentiation and aerial mycelium/spore formation compared with the wild type (Table 2 and Figure 1). The color of the wild-type aerial mycelium was gray because of spore development, while the mutant mycelia were yellow-white. The ΔwblE and ΔwblE2 strains showed similar phenotypical characteristics to S4-7 at the early stage of aerial mycelium formation. However, ΔwhiH and ΔwhmD were significantly less gray and spore development (Figure 1).
The result suggests that wblE and wblE2 gene may less involve cell development and sporulation in S. griseus S4-7.

| Plant protection ability of member of the whi transcription family
The antifungal activity and plant protection ability of the mutants were determined both in vitro and in planta. In vitro, the antifungal activity against the wilt disease pathogen of ΔwblE, ΔwhmD, and ΔwblE2 strains was decreased in comparison with the wild type ( Figure 2).
The ΔwhiH antifungal activity was not diminished compared with that of the wild type ( Figure 2). This was especially evident for the ΔwblE2 mutants, which lost their antifungal activity completely. To verify wblE2 gene function, a wblE2 overexpression strain was constructed, where a single copy of wblE2 was expressed under a strong constitutive ermE promoter. The overexpression wblE2 strain fully suppressed F. oxysporum (Figure 2).

Regarding the mutants' disease suppressive activity in planta,
ΔwblE2, ΔwhmD, ΔwblE, and ΔwhiH mutants failed to fully protect the strawberry against the Fusarium wilt pathogen (Figure 3 and Figure   S2). The disease progression and severity of ΔwblE2-assisted plants were similar to those of pathogen-only treated plants (Figure 3b). The mutant significantly differed from the S. griseus S4-7 with respect antifungal/plant protection ability. The results indicated that wblE2 transcription factor may be a responsible factor of S. griseus S4-7 in suppressing the wilt disease occurrence.

| Transcriptome of the ΔwblE2 mutant
To begin to reveal the difference between S4-7 and ΔwblE2 mutant in secondary metabolite production, we generated six RNA samples fold-change >2 and by edgeR (p < .005) as significant, we identified 414 genes differentially expressed in S4-7 and the mutant (Table S3).
Among the differently expressed genes, only 33 were significantly upregulated in the ΔwblE2 strain compared with the wild type (Table   S3 and Figure S3). By contrast, 381 genes were dramatically downregulated in the ΔwblE2 strain. Based on GO functional assignment, carbohydrate transport and metabolism (40 genes), transcription (32 genes), inorganic ion transport and metabolism (20 genes), amino acid transport and metabolism (15 genes), signal transduction (15 genes), and energy production and conversion (10 genes) were differently expressed in the ΔwblE2 strain ( Figure S3).
The differently expressed genes were categorized into four groups based on their expression patterns (Table 3 and (Table S3). Transcriptome data presented that majority of the down regulated genes in ΔwblE2 strain are related antibiotic precursor or synthesis pathway.

| DISCUSSION
The genus Streptomyces is considered a source of various secondary metabolites, especially antibiotics. Nearly 70% of clinical antibiotics were developed from the products of this genus (Bibb, 2013).
However, the knowledge of the role of Streptomyces in microbe-plant interactions and in agriculture is relatively limited. S. griseus S4-7 was isolated from the strawberry rhizosphere in a 15-year continuous monoculture field highly suppressive to Fusarium wilt disease (Cha et al., 2016). This strain exerts its antifungal effect by disturbing fungal cell wall remodeling and RNA polymerase activity (Cha et al., 2016). Different secondary metabolic gene clusters respond to various environmental and physiological signals and stress conditions (Bibb, 2005(Bibb, , 2013 elongates into long branching hyphae that grow into the substrate and form vegetative mycelium. Upon differentiation, aerial mycelium is formed, which then develops into chains of spores (Soliveri, Gomez, Bishai, & Chater, 2000). Cell differentiation is triggered by nutrient depletion and other signals, and both the production of secondary metabolites and morphological differentiation are initiated.
whi mutants defective in different sporulation stages are employed in genetic and biochemical analyses of these developmental stages (Yagüe, López-Garcia, Rioseras, Sánchez, & Manteca, 2013). The regulation of secondary metabolism in streptomycetes is diverse and complex. Antibiotic production and antimicrobial activity observed in the laboratory are coordinated with the morphological development in surface-grown cultures (Bibb, 2005). Developmental regulatory genes, members of the wbl transcription factor, are only found in Actinobacteria (Soliveri et al., 2000). indicated that the wblE2 gene regulates the production of secondary metabolites that strongly contribute to the strawberry-protecting property of S4-7.
From 7,501 expressed genes, 5.5% (414 genes) were detected as differently regulated in the ΔwblE2 mutant. The differently expressed genes were divided into four groups based on their expression patterns and expression intensities. Genes in group A, only weakly expressed in the mutant, suggested that the ΔwblE2 mutant may produce fewer antibiotic-related secondary metabolites but with increased bacterial volatile emission compared with the wild type. A reduction of antibiotic production after loss-of-function mutation of whi transcription factors in Streptomyces has been well-documented in previous studies (Bibb, 2005(Bibb, , 2013Burian et al., 2012;Rybniker et al., 2010). However, the involvement or regulatory role of wblE2 transcription factor in secondary metabolite and antibiotic production was not described.
Transcriptome analysis revealed that sulfur metabolism and expression of related genes were correlated with antifungal activity in S4-7.
However, the ΔwblE2 mutant had defective sulfur metabolism. In this mutant, not only was sulfur metabolism decreased but also polyketide, siderophore. The data clearly indicated that the wblE2 transcription factor governs and contributes to specific metabolite production and activities of S. griseus S4-7, including protecting the strawberry against the Fusarium wilt pathogen. Conprimycin (a novel thiopeptide) was highlighted in our previous study as the key antifungal polypeptide of the suppressive system (Cha et al., 2016). However, the expression levels of conprimycin or other thiopeptide biosynthesis genes in both the ΔwblE2 mutant and the wild type were similar (data not shown). This result suggests two possible reasons, the first is that S4-7 cells produce several antibiotics and each pathway may have an independent regulatory system. And second is that the conprimycin, which ribosomally synthesized a small peptide, may regulate post-translational modification to be activation, such as cleavage prepeptide, azoline formation, dehydration of Ser/Thr residues, i.e. (Zheng, Fang, & Liu, 2017).
In conclusion, the plant probiotic strain, S. griseus S4-7, protects the strawberry against Fusarium wilt disease and wblE2 transcription factor is extensively involved in the regulation of antifungal secondary metabolites in the strain. The identified wblE2 transcription factor regulates antibiotic, polyketide, xenobiotic, and siderophore metabolites in S. griseus S4-7. The wblE2 transcription factor plays the role of a master regulator in antifungal metabolite production in S. griseus S4-7. Further studies of S. griseus S4-7 will enable the development of better-integrated models of Streptomyces responses to particular conditions and the involvement of this transcription factor in plant protection phenomena.