Overexpression of the Key Virulence Factor 1,3–1,4-β-d-Glucanase in

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Overexpression of the Key Virulence Factor 1,3−1,4-β‑D‑Glucanase in the Endophytic Bacterium Bacillus halotolerans Y6 To Improve Verticillium Resistance in Cotton Lin Zhang,†,‡,∥ Wenpeng Li,‡,∥ Ye Tao,‡ Suya Zhao,‡ Lunguang Yao,§ Yingfan Cai,*,† and Qiuhong Niu*,‡

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†

State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Sciences, Henan University, Kaifeng, Henan 475001, P. R. China ‡ Department of Life Science and Biotechnology, Nanyang Normal University, Nanyang 473000, P. R. China § China-U.K.-NYNU-RRes Joint Laboratory of Insect Biology, Nanyang Normal University, Nanyang 473000, P.R. China S Supporting Information *

ABSTRACT: Verticillium wilt, caused by Verticillium dahliae, results in a dramatic loss of cotton yields in China. There is great potential for biocontrol to manage this destructive crop disease. In this study, we obtained the endophytic bacterium Bacillus halotolerans Y6 from Verticillium wilt-resistant cotton Gossypium barbadense Xinhai15; this bacterium possesses strong antagonistic abilities that inhibit V. dahliae spore germination and mycelial growth. The results of the enzyme activity assay, heterologous expression, and gene knockdown showed that the key virulence factor of Y6 for antagonizing V. dahliae was βglucanase Bgy6. To facilitate field tests of biological control, we constructed the homologous Bgy6-overexpression strain OY6. Compared with the wild-type Y6 strain, the β-glucanase activity of OY6 was increased by 91.79%, and the inhibition rate of OY6 against V. dahliae V991 exceeded 96.7%. Moreover, the spores of V. dahliae V991 treated with OY6 showed more mucus and larger holes on the surface, as observed by scanning electron microscopy. Potting test results illustrated that both OY6 and Y6 could improve the resistance of upland cotton to Verticillium wilt. With the inoculation of V. dahliae V991 for 45 days, the disease index of G. hirsutum TM-1 treated with OY6 was only 8.33, which was significantly lower than that in plants treated with the wild-type strain Y6 (17.86) or the controls without bacteria (35.94). Our research provides a new idea for the control of Verticillium wilt in upland cotton via transforming endophytic bacteria of Verticillium wilt-resistant cotton and proposes a new solution to prevent and control Verticillium wilt. KEYWORDS: Bacillus halotolerans, cotton, Verticillium wilt, Verticillium dahliae, biocontrol, β-glucanase

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INTRODUCTION

Traditional methods to control cotton Verticillium wilt include intensive plant breeding, cultivar choice, crop rotation, and chemical control.10−12 Biological control methods of V. dahliae have received considerable attention as an alternative disease management tactic due to their potential to provide safe and environmentally friendly disease control.13−15 An increasing number of studies have shown that endophytes can improve plant growth and plant health, playing many beneficial roles in the metabolism and physiology of the host plant.16−18 These roles include degrading toxic compounds,19 inhibiting strong fungal activity, and antagonizing bacterial pathogens.20,21 Among these endophytes, endophytic bacteria may be advantageous for plant survival because they provide protection from environmental stress and microbial competition.3 Endophytic bacteria reside intercellularly or intracellularly within the host tissues and can improve plant growth and plant health. Currently, endophytic bacteria are recognized as

It has been estimated that almost 26−30% of the yield losses for sugar beet, cotton, and wheat are caused by fungal pathogens alone.1 Crop fungal disease control plays an important role in protecting the capacity and quality of crop production.2 The biological control of plant pathogens using microorganisms can be a safe, cost-effective, and efficient method for suppressing plant diseases.3 Cotton (Gossypium hirsutum) is a foundation of the global economy, prized for its role as an important renewable fiber resource.4 Verticillium wilt of cotton caused by V. dahliae is one of the most serious diseases among cotton-producing regions worldwide. Therefore, controlling Verticillium wilt is a priority in cotton breeding and cultivation.5 The prevalence of this disease has progressively increased in many regions, and it has become a serious obstacle to cotton production in China.6 V. dahliae infects cotton by penetrating the roots; then, it spreads across the root cortex and invades the xylem vessels, where it forms the conidia responsible for the colonization of vascular tissues and functional impairment.7,8 The resulting infection will cause cotton yellowing, leaf vein browning and chlorosis, wilt, defoliation, and finally death.9 © 2019 American Chemical Society

Received: Revised: Accepted: Published: 6828

January 30, 2019 May 25, 2019 May 28, 2019 May 28, 2019 DOI: 10.1021/acs.jafc.9b00728 J. Agric. Food Chem. 2019, 67, 6828−6836

Article

Journal of Agricultural and Food Chemistry The inhibition rate mas measured as follows

important microbial resources and have gradually become a multidisciplinary research hotspot in different fields, including botany, microbiology, plant protection, and plant breeding.22 Mohamad et al. reported that endophytic bacteria associated with medicinal plants possess unique strategies that enhance the growth and survival of host plants.3 In addition, many systematic studies have reported the antagonistic action of cotton endophytic bacteria on plant pathogenic fungi.17,23,24 The main components of the cell wall of fungi are glucan and chitin, and for that reason, many important hydrolytic enzymes, including β-glucanase, chitinase, protease, and lipase, are considered to be important virulence factors.25−28 In the present study, we isolated and screened nine endophytic bacterial strains comprising seven Bacillus, one Enterobacter, and one Klebsiella from Verticillium wilt-resistant cottons with diverse antagonistic activities to V. dahlia. The dual culture test showed that one of the bacterial strains, B. halotolerans Y6, with a β-glucanase identified as a pathogenic factor had the highest antifungal activity. Furthermore, we determined whether overexpressing β-glucanase could increase the inhibition efficiency of B. halotolerans Y6, potentially improving the biological control of Verticillium wilt.

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inhibition rate = (control colony diameter − test colony diameter) × 100%/(control colony diameter − fungus block diameter)

Cloning, Expression, and Knockdown of bgy6. The inhibitory capability of the strain Y6 was found to be positively correlated with β-glucanase activity. Therefore, the gene encoding the purified enzyme was cloned using polymerase chain reaction (PCR) with oligonucleotides based on the conserved sequences of β-glucanase in Bacillus. The sense primer contained a BamHI restriction site, 5-CGC GGA TCC ATG TTT TAT CGT ATG AAA CGA G-3, and the antisense primer contained a HindIII restriction site, 5-CCC AAG CTT TTA TTT TTT TGT ATA GCG CAC CC-3. The product was ligated into the vector pMD19-T (Promega) to create pMD19-T-bgy6 and sequenced. The sequence was analyzed by finding the conserved domain within the SMART software package and BLAST in NCBI to identify the enzyme family. After identification, the product of double enzyme digestion was inserted into the BamHI/HindIII site of the pET-28a plasmid. After sequence confirmation, the plasmid was transformed into E. coli BL21, and the transformants were induced with 0.4 mM isopropyl β-D-thiogalactoside (IPTG) at 30 °C for 12 h to express the enzyme Bgy6. The recombinant protein re-Bgy6 was purified by Ni-NTA affinity chromatography according to the pET system manual (Promega). The purified re-Bgy6 was sent to the Willget Biotechnology Company to produce mouse polyclonal antibodies and measured for β-glucanase and antifungal activities. The plasmid pBD1 was constructed in our lab based on catalytically inactive (“Dead”) Cas9 (dCas9) in Bacillus, which showed high-efficiency knockdown of both the essential and nonessential genes reversibly.31 The “N(C)GG” sequence was found near the initiation codon in the template strand of the target gene bgy6. The 23 nt sequence immediately upstream of the gene was then taken and added to the 3′ end of 5 “TGT-3” to create small guide RNA (sgRNA) oligonucleotide 1 (5′-TGT ATT TGC TTT TTG CCA TAA TC-3′). The reverse complementary sequence of the 23 nt was taken and added to the 3′ end of 5 “AAC-3” to create sgRNA oligonucleotide 2 (5′-AAC GAT TAT GGC AAA AAG CAA AT-3′). After annealing, the two oligonucleotides were cloned into the SapI-digested plasmid pBD1 to produce pBD1-bgy6. The plasmid was transformed into the Y6 strain to obtain the bgy6 knockdown strain. The abundance of Bgy6 was determined by Western blot. Overexpression of β-Glucanase Bgy6. Gene bgy6 was amplified from pMD19-T-bgy6 using an SpeI restriction site sense primer, 5CGG ACT AGT ATG TTT TAT CGT ATG AAA CGA GTC G-3, and an XhoI restriction site antisense primer, 5-CCG CTC GAG TTA TTT TTT TGT ATA GCG CAC CC-3. The PCR-generated fragment was double digested with SpeI and XhoI and then inserted into the bacterial integration vector pAX01, resulting in pAX01-bgy6, which is controlled by the xylose-inducible xylA gene promoter PxylA. After the plasmid pAX01-Bgy6 was propagated in E. coli JM109, it was introduced into B. halotolerans Y6 competent cells according to the recommendations of the supplier for target protein overexpression.30 We verified successful transformants by PCR analysis using upstream primer 5-GAG ACT ACA ACC CCG ATC AGT GG-3, based on the lacA gene N-terminal sequence of the integrated locus, and reverse primer 5-GAA TCC AGT GAC AAG AAG CAG-3, based on the wild-type bgy6 chromosomal gene N-terminal sequence. Overnight cultures of B. halotolerans OY6 transformants were diluted (1:100) in fresh LB medium containing erythromycin. Xylose (2%) was added for the induction of recombinant β-glucanase. The induction culture conditions included shaking at 150 rpm and 37 °C for 60 h. SDSPAGE with Western blotting was performed to examine the expression of the β-glucanase gene by comparing equal loadings of total protein from both wild-type and recombinant strains. Transformant stability was determined by monitoring antibiotic resistance after incubation on erythromycin-free LB plates for at least 10 continuous transfers. β-Glucanase Activity Assay. First, β-glucanase activity was quantitatively determined using Congo red plates. The bacterial

MATERIALS AND METHODS

Microbial Strains and Plasmids. V. dahlia V991 was preserved in the State Key Laboratory of Cotton Biology, Henan University. The original isolation of B. halotolerans Y6, isolated from Verticilliumwilt-resistant cotton Xinhai15, was deposited in the China Center of Industrial Microbiological Culture Collection (CICC 24636). E. coli JM109 was used as the host to clone the bgy6 gene. The pET-28a expression vectors (Promega) and pBD1 (constructed in this lab) were employed for heterologous expression and knockdown of bgy6, respectively. The vector pAX01 (Bacillus Genetic Stock Center) was used for β-glucanase Bgy6 overexpression. All bacteria were grown on Luria−Bertani (LB) agar plates (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl, and 1.5% agar) with selective antibiotics. All chemical reagents and antibiotics used in this study were purchased from Sigma (St. Louis, MO). Isolation of Endophytic Bacteria Antagonistic against V. dahlia. The screening and isolation of endophytic bacteria were conducted according to the reference with minor modification.29 The root, stem, or leaf of cotton Xinhai15 was collected and washed with phosphate-buffered saline (PBS, pH 7.2) to remove the attached epiphytic bacteria. The samples were further surface-sterilized by sequential immersion in 75% ethanol for 30 s then in 5% sodium hypochlorite for 3 min. The surface sterilization was considered successful when no epiphytic bacteria were cultured from the sterilization solutions when plated onto nutrient agar (NA) plates (5.0 g peptone, 1.5 g yeast extract, 1.5 g beef extract, 5.0 g NaCl, 20 g agar, and 1 L of distilled water; pH 7.2) and incubated at 37 °C overnight. Then, cotton tissue samples were ground to a slurry with sterile water and shaken to mix well. Approximately 50 μL of the suspension was plated onto NA and incubated at 28 °C for 3 days to culture the endophytic bacteria. Pure isolate strains of the endophytic bacteria were classified by phenotypic and genotypic characteristics. We used a dual culture method to test the bacterial antifungal activity.30 First, the fungi and bacteria samples were prepared as follows: The fungus V. dahliae V991 was grown for 7 days at 25 °C on solid potato dextrose agar (PDA) medium. The bacterial strains were cultured in 5 mL of LB liquid medium at 37 °C and 220 rpm for 12 h. From the liquid culture, 200 μL was spread-plated on LB agar and incubated for 12 h at 37 °C. Then, the antagonistic activity assay was assessed according to the following protocol: Four bacterial blocks with a diameter of 1 cm were placed on the PDA plate at an equal distance from the center of the fungal block. A blank LB block was used as a negative control. The growth of the fungal mycelium was observed after culturing at 25 °C for 5 days. 6829

DOI: 10.1021/acs.jafc.9b00728 J. Agric. Food Chem. 2019, 67, 6828−6836

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Journal of Agricultural and Food Chemistry Table 1. Isolation and Identification of Endophytic Bacteria from XinHai15 strain

accession number

closest species in 16S rRNA gene sequences database

similarity (%)

β-glucanasea

A3 H15-12 HT-7 HXC-1 Y4 Y6 M-1 V-1 V-2

AJVF01000043 ASJD01000027 AY603658 JH600280 AYTO01000043 LPVF01000003 JQ070300 FYBF01000083 AJVF01000043

Bacillus siamensis KCTC 13613(T) Bacillus safensis FO-36b(T) Bacillus velezensis CR-502(T) Bacillus mojavensis RO-H-1(T) Bacillus tequilensis KCTC 13622(T) Bacillus halotolerans ATCC 25096(T) Klebsiella michiganensis W14(T) Enterobacter xiangfangensis LMG 27195(T) Bacillus siamensis KCTC 13613(T)

99.95 99.93 100 99.80 99.73 99.93 99.52 99.80 99.93

− − ++ + ++ +++ ++ − −

−, no production; +, weak hydrolytic circle around the colony (0.50 to 1.50 cm); ++, clear hydrolytic circle around the colony (1.50 to 2.00 cm); +++, strong hydrolytic circle around the colony (2.00 to 3.00 cm). a

Figure 1. β-glucanase activity assay and inhibition of V. dahlia. (A) B. halotolerans Y6 β-glucanase activity on a plate containing 0.1% Congo red solution for 30 min, followed by washing with 5 M NaCl. (B) Antifungal activity against V. dahlia. (C) Negative control E. coli showed no antifungal activity.

Figure 2. Evolutionary relationship between Bgy6 and related proteins shown by a neighbor-joining phylogenetic tree. Bootstrap values of 70% or greater are shown at the nodes. The scale bar corresponds to a genetic distance of 0.2 substitutions/site. The enzyme activity of β-glucanase was calculated as described.32,33 The amount of enzyme required to catalyze 1.0 mmol substrate per min was defined as one enzyme activity unit. We measured the enzyme activity of the mixture by the absorbance at 540 nm. The mixture in the absence of enzymes constituted the negative control. We performed three assays per experiment to determine the mean and standard deviation.

strains were plated onto the isolation medium that included 0.20 g βD-glucan (Sigma), 0.003 g Congo red, 0.15 g KNO3, 0.12 g NaH2PO4, 0.03 g MgSO4, 0.01 g CaCO3, 1.8 g agar, and 100 mL of sterile water. Plates were incubated for 4 days at 28 °C. The ability of these strains to degrade β-D-glucan was assayed by incubation with a 0.1% Congo red solution for 0.5 h, followed by washing with 5.0 M NaCl. The diameter of the hydrolyzing ring on the plate showed comparatively high glucanase production. 6830

DOI: 10.1021/acs.jafc.9b00728 J. Agric. Food Chem. 2019, 67, 6828−6836

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Journal of Agricultural and Food Chemistry Western Blot Assay of β-Glucanase. Western blot analysis was performed according to established protocols.34 The OD600nm values of both the wild-type and recombinant cultures were measured and adjusted to the same level. The proteins from equal culture volumes were separated by SDS-PAGE35 and electrotransferred onto immunoblot polyvinylidene difluoride (PVDF) membranes (BioRad, Hercules, CA). The membranes were blocked with a solution of 10% skim milk, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.02% Tween-20 (TBST buffer) before incubation with anti-Bgy6 polyclonal antibody (1:800). After three washes with TBST, membranes were incubated with goat antimouse IgG alkaline phosphatase-conjugated secondary antibody (1:1000; Bio-Rad) and washed again. Colorimetric detection was performed with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Antagonistic Activity Analysis. The antagonistic activity of the overexpressing strains was assayed as previously described. The morphological changes of fungal spores were also examined by SEM. The spores from these assays were washed three times in phosphate buffer (pH 7.0) prior to fixation with 4% glutaraldehyde for 120 min, followed by 2% osmic acid for 40−60 min. After dehydration in a graded ethanol series, the samples were dried, gold sputter-coated, and examined under a Zeiss Supra55 SEM in environmental mode at 0.7 kV. Potting Experiments. Potting tests were carried out as follows: V. dahliae V991 was first cultured in a PDA dish at 25 °C for 7 days and then inoculated in Czapek Dox liquid medium and cultured for 5 days at 25 °C and 180 rpm. The spore solution was obtained through gauze filtration, and the number of spores was counted with a hemocytometer. The spores were poured into the soil (loess/black soil/vermiculite 1:1:1) and mixed to obtain 1 × 106 spores/g of spore soil. The wild-type strain Y6 and the overexpressing strain OY6-1 were cultured in LB liquid medium at 37 °C and 220 rpm overnight. The bacteria were collected by centrifugation for 5 min at 8000 rpm and adjusted to OD600nm = 1.0 by sterile water for use in the following experiments. The spore soil was loaded into a square bowl measuring 7 cm × 7 cm × 7 cm. Spore soil was separately poured in 100 mL of sterile water (1st group), 10 mL of Y6 suspension (OD600nm = 1.0) + 90 mL of sterile water (2nd group), and 10 mL of OY6 suspension (OD600nm = 1.0) + 90 mL of sterile water (3rd group). The cotton seeds were planted in the three groups of soil and cultured at 25 °C in a light incubator with a light/dark 16/8 h photoperiod. After 30 days of incubation, the disease indices (DIs) were statistically counted every 5 days.

Figure 3. Expression of the fusion protein re-Bgy6 in E. coli transformant and the identification of its β-glucanase and antifungal activities. (A) SDS-PAGE (12%) of re-Bgy6. Lanes: M, marker; 1, total protein of the uninduced transformant; 2, total protein of the induced transformant; 3, culture supernatant of the induced transformant; 4, the purified re-Bgy6. (B) β-glucanase activity of reBgy6 on Congo red plate: 1. purified re-Bgy6 solution (0.39 mg/mL); 2. diluted re-Bgy6 solution (0.13 mg/mL); 3. diluted re-Bgy6 solution (0.04 mg/mL); and 4. control (heat-denatured re-Bgy6). (C) Antifungal activity of re-Bgy6 against V. dahlia: 1. V. dahlia incubated with re-Bgy6 (0.39 mg/mL) and 2. V. dahlia incubated with boiled reBgy6. (D) V. dahlia incubated with re-Bgy6 at 25 °C, 180 rpm, 20 h. Bar, 5 μm. (E) V. dahlia incubated with boiled re-Bgy6 at 25 °C, 180 rpm, 20 h. Bar, 5 μm.

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RESULTS AND DISCUSSION Isolation and Identification of the Strain Y6 That Is Antagonistic against V. dahlia. A total of 107 endophytic bacteria were isolated from Gossypium barbadense Xinhai15, representing 8 genera and 23 species. The isolates were screened for their ability to inhibit V. dahlia, including the Gram-positive phylum Firmicutes and the Gram-negative phylum Proteobacteria. Among the 107 isolates examined, nine isolates displayed comparatively strong inhibitory activity. These nine strains contained seven Bacillus, one Enterobacter, and one Klebsiella (Table 1), which showed that the genus Bacillus showed the highest antifungal activity against V. dahlia. Among the nine strains, Y6 showed the maximum β-glucanase and antifungal activities (Figure 1). Members of the genus Bacillus, in particular, B. amyloliquefaciens, B. atrophaeus, B. velezensis, and B. mojavensis, were the most effective biocontrol agents, with most strains exhibiting broad antibacterial and antifungal activities.22,36,37 Most of these bacteria contain extracellular digestive enzymes that may destroy or neutralize a variety of pathogens, including chitinases, cellulases, lipases, and proteases.38 Moreover,

Bacillus species are known to have high secretion capacities and have long been used for the production of various industrial enzymes, mainly amylase and proteases.39 Chitinase and β-1,3-glucanase are considered to be important hydrolytic enzymes in the lysis of fungal cell walls, for example, the cell walls of Fusarium oxysporum, Sclerotinia minor, and Sclerotium rolfsii.25−27 Moreover, Xu et al. reported a β-1,3−1,4-glucanase gene from B. velezensis ZJ20 exerting an antifungal effect on plant pathogenic fungi.28 Here we obtained seven Bacillus species that antagonized the pathogenic fungi V. dahlia. We first reported that B. halotolerans exhibited biological control activities against cotton Verticillium wilt, mainly depending on the extracellular β-glucanase. Cloning and Phylogenetic Analysis of bgy6. The 732 bp gene bgy6 was amplified by PCR from B. halotolerans Y6 and sequenced. The gene encoding the enzyme was submitted to GenBank (MH643779). The mature enzyme had 243 residues containing a catalytic domain belonging to glycoside hydrolase family 3 (GH3). The whole amino acid sequence 6831

DOI: 10.1021/acs.jafc.9b00728 J. Agric. Food Chem. 2019, 67, 6828−6836

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Journal of Agricultural and Food Chemistry

diverse organisms were chosen from the NCBI, CAZY, and PDB databases to perform phylogenetic analysis. It can be seen from the phylogenetic tree that Bgy6 is not closely related to the other β-glucanase family members, indicating that it could be a novel isoform (Figure 2). Heterologous Expression and Enzyme Activity Analysis of Bgy6. The Bgy6 was heterologously expressed in E. coli BL21. The SDS-PAGE analysis showed that recombinant protein re-Bgy6 was ∼32 kDa in the culture supernatant (Figure 3A), which corresponds to mature Bgy6-His-fused protein. The micro BCA kit (Pierce, Rockford, IL) was used to measure the purified re-Bgy6 solution, and the concentration was measured as 0.39 mg/mL. The re-Bgy6 protein solution was diluted separately to 0.13 and 0.04 mg/mL with PBS (pH 7.4) to detect the β-glucanase activities using Congo red plates. The results indicated transparent rings around the filter paper, even when the solution was diluted to 0.04 mg/mL (Figure 3B-1−3). There was no clear zone of inhibition around the control treated with boiled re-Bgy6 (Figure 3B-4). The recombinant enzyme Bgy6 showed significant antifungal activities. After 7 days of incubation, the hypha growth of the fungi V. dahlia treated with the re-Bgy6 solution (0.39 mg/mL) was negligible (1.0 cm colony diameter); however, the mean colony diameter of V. dahlia treated with PBS buffer alone obviously grew and reached 3.4 cm (Figure 3C). The inhibition rate of Bgy6 was up to 70.59%. Microscopic observation showed that the enzyme could strongly inhibit spore germination after coculture with the V. dahliae spore solution at 25 °C for 20 h (Figure 3D,E). After infecting the host vascular tissue, V. dahliae produces a large number of conidia. The germinated spores flow with the body fluid and colonize in the duct. During this process, the germination of the pathogen and the elimination of the host occur simultaneously. However, in susceptible plants, pathogens can overcome the elimination of the host and continue to germinate.40 Our results showed that Bgy6 strongly inhibits spore germination, which may be the major antagonistic factor of B. halotolerans Y6 on V. dahlia. Knockdown of bgy6 and Antifungal Activity Analysis of Knockdown Strain. An efficient, specific, and reversible system for gene knockdown in Bacillus was successfully constructed in our lab based on the type II clustered regularly interspaced short palindromic repeat (CRISPR)/dead cas9 (dCas9).31 We successfully silenced and complemented the bgy6 gene in B. halotolerans Y6 using this system because its effect was inducible and reversible. A sgRNA was designed to bind to the translational start site of the gene. The results showed that in the presence of 0.3 mM IPTG, knockdown strains of YKD6 showed dramatic protein reduction based on Western blot analysis (Figure 4A) and β-glucanases activity levels (Figure 4B). Considering that there was almost no effect on the growth of the fungi V. dahlia at comparatively low IPTG concentrations, we decided to use 0.3 mM IPTG induction to measure the biological control effect, although the Bgy6 protein expression decreased more at 1.0 mM IPTG induction. The knockdown strains almost lost the inhibitory capabilities against V. dahlia, but the complementary strains without IPTG showed obvious inhibitory effects, which were similar to those of wild type strain Y6 (Figure 4C). These results confirmed that B. halotolerans Y6 inhibits V. dahlia mainly with the β-glucanase Bgy6 as the key pathogenic factor.

Figure 4. Knockdown of the gene bgy6 in B. halotolerans Y6 and identification of its β-glucanase and antifungal activities. (A) Bgy6 protein levels in the knockdown strain YKD6 with or without IPTG induction. (B) β-glucanase activity of knockdown and control strains with or without IPTG induction was determined by Congo red plate assay. (C) Antagonism of B. halotolerans Y6 and its mutant derivatives against V. dahlia.

Figure 5. Expression assays of the overexpression strain OY6: (A) βglucanase activity was measured on plates. (B) β-glucanase activity was evaluated with β-glucan as a substrate by the absorbance value of the mixture at 540 nm. (C) Western blot analysis of Bgy6 production. Lanes 1−3 represent samples from the wild-type strain Y6 and the transformant strains OY6-2 and OY6-1, respectively.

demonstrated 88.89% similarity with the β-1,3-1,4-glucanase from B. amyloliquefaciens (ACX55805) and 50−89% identity with other glucanases from bacteria but comparatively low homology with the enzyme produced by several fungi such as Streptomyces sp. and Halothermothrix orenii H168. We calculated the theoretical molecular mass of Bgy6 as 27.6 kDa and the pI as 8.6 using the ExPASy program (http://cn. expasy.org/tools/pi_tool.html). According to phylogenetic analysis, Bgy6 belongs to the known GH3 domain family and β-glucanase members. Twenty-two β-glucanases from 6832

DOI: 10.1021/acs.jafc.9b00728 J. Agric. Food Chem. 2019, 67, 6828−6836

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Journal of Agricultural and Food Chemistry

Figure 6. (a) Antifungal effects of the wild-type and Bgy6 overexpression strains on V. dahlia V991: (A) negative control, (B) treatment with wildtype strain Y6, and (C) treatment with overexpressing strain OY6-1. (b) SEM examination of the effects of the B. halotolerans strains on the spores of V. dahlia V991: (A,D) normal spores were regular and neat, (B,E) spores treated with the wild-type strain Y6, and (C,F) spores treated with the overexpressing strain OY6-1.

Overexpressing β-Glucanase in Vivo. Similar to our previous study, we used the ectopic integration vector pAX01 from BGSC to overexpress the bgy6 gene via double recombination at the lacA chromosomal locus.41,42 A double crossover between pAX01, which contains the “front” and “back” regions of the lacA gene, results in heterologous DNA from the plasmid, replacing the interlocus sequences.43 The βglucanase gene bgy6 was inserted into the multiple cloning sites between the SpeI and XhoI restriction sites to create the plasmid pAX01-Bgy6. Colony PCR and double enzyme digestion were used to confirm the success of the plasmid construction (Figure S1A,B). Colony PCR showed four positive transformants, whereas only two transformants produced the target 732 bp fragments after digestion. The successfully constructed plasmids were extracted from one of the positive transformants, confirmed by DNA sequencing, and transformed into B. halotolerans Y6. Candidate transformants were selected on plates containing erythromycin (1 μg/mL). Five transformants with the lacA::bgy6 genotype were chosen and verified by PCR. Three transformants produced the expected fragment of ∼1.6 kb (Figure S1C, lanes 3−5),

whereas the wild-type Y6 strain did not produce a product of the proper size (Figure S1C, lane 1), nor did the two other candidate transformants (Figure S1C, lanes 2 and 6). This result suggests that the bgy6 gene was successfully inserted into the genomic target site. Among the three successful transformants, one mutant strain, B. halotolerans OY6-1 (lacA::bgy6 genotype), showed greater stability than the other two strains. The OY6-1 strain showed stable erythromycin resistance after being transferred and cultured for 30 generations on agar media without antibiotic selection pressure. All three overexpression mutant strains showed relatively slow growth compared with the wildtype Y6 strain (data not shown), although there were no obvious morphological changes when compared with the original strain B. halotolerans Y6. The overexpression of virulence factors is a common strategy to enhance pathogenicity in hosts, including plants and microbes; it has been shown that the overexpression of a 32 kDa recombinant chitinase in an Escherichia coli host improved the antifungal activity of barley.44 The transformant overexpressing a serine protease in Paecilomyces lilacinus 6833

DOI: 10.1021/acs.jafc.9b00728 J. Agric. Food Chem. 2019, 67, 6828−6836

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Journal of Agricultural and Food Chemistry

Figure 7. Results of potting test observations: (A) control cotton group without inoculation of biocontrol bacteria; (B) cotton group inoculated with the Y6 strain; and (C) cotton group inoculated with OY6-1 strain. (D) Statistical analysis of disease index (DI) results at different days past inoculation (DPI).

Under optimized conditions, the β-glucanase production of the overexpression strain increased by 91.79%, which is inconsistent with the 2.8-fold improvement observed in Western blot analysis results. We inferred that the overexpressed β-glucanase was not completely transported to the extracellular media due to a limited transport system or the lack of other factors to accomplish secretion. The reasons for the limited increase in β-glucanase activity are that (1) the conditions of induction remain to be further optimized, (2) the high expression of the β-glucanase bgy6 gene results in cumulative feedback inhibition, or (3) other unknown genes may be affected as a result of the integration of plasmid pAX01 into the chromosome of the host bacteria B. halotolerans Y6. Bioassay and Potting Experiment Results. Bioassay experiment results showed that the overexpressing strain OY61 had a significantly higher inhibition rate on V. dahliae V991 than the wild-type Y6 strain. In detail, the inhibition rate upon OY6-1 treatment was ∼96.7% higher within 5 days than that of the treatment with wild-type strain Y6 (Figure 6a). The normal spores of the fungi V. dahliae V991 are smooth, orderly, and germinated (Figure 6b-A,D); however, there were obvious holes on the surface of spores after treatment with the wild-type strain B. halotolerans Y6 (Figure 6b-B,E), which might be due to the action of β-glucanase on spores. The spores treated with the overexpressing strain OY-6 changed more obviously; more mucus and larger holes were observed on the surfaces of spores (Figure 6b-C,F). Potting test results showed that the leaves of the control cotton group appeared yellow after 45 days of infection; some of the leaves showed atrophy and abscission to different degrees, and cotton stalks showed obvious black symptoms

enhanced virulence by 20% against Meloidogyne incognita eggs.45 The simultaneous overexpression of two virulence enzymes (protease CDEP1 and a Chitinase Bbchite1) in another entomopathogenic fungus, Beauveria bassiana, showed synergy and resulted in greater pathogenicity than either mutant alone.46 The overexpression of a mucinase, TagA, a virulence factor in Vibrio cholerae, significantly increased attachment to the intestinal mucosa of its host.47 Therefore, increasing the antifungal activities of B. halotolerans Y6 by overexpressing the key virulence factor β-glucanase could potentially enhance the value of this species as an effective biocontrol agent. Activities of the Overexpression Transformant OY61. First, we measured the transparent ring sizes on Congo red plates to quantitatively estimate the β-glucanase activity of the overexpression transformants. The results showed that the expression of Bgy6 in the overexpression transformant strain OY6-1 was obviously higher than that of OY6-2 or the wildtype strain Y6 (Figure 5A). We therefore quantitatively examined the β-glucanase activity of transformant OY6-1 colorimetrically. In detail, samples taken at different times (from 24 to 120 h) were collected from cultures of Y6 and OY6-1 to measure the β-glucanase activity (Figure 5B). The OY6-1 supernatant showed a maximum activity of 350.2 U/ mL at 96 h, whereas the maximum activity of the wild-type strain Y6 was 182.6 U/mL when cultivated at 72 h. This result revealed that the overexpression transformants increased βglucanase production by 91.79% over the wild-type strain. Furthermore, Western blot results indicated ∼2.8 times more protein compared with the wild type (Figure 5C). 6834

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(Figure 7A). The cotton group treated with Y6 showed minor symptoms of Verticillium wilt, and a few leaves were withered (Figure 7B). The group treated with the overexpressing strain OY6-1 grew best, with almost no disease symptoms, and only a few leaves showed partial chlorosis (Figure 7C). The results indicated that the overexpressing strain had a better control effect against V. dahliae V991 than the wild biocontrol strain B. halotolerans Y6. Moreover, the disease indices were consistent with the cotton symptoms. The group inoculated with the overexpressing strain B. halotolerans OY6-1 had the best control effect, and the disease indices for 30 DPI (days past inoculation), 35 DPI, 40 DPI, and 45 DPI were 0, 0, 1.19, and 8.33, respectively, which were much lower than the group inoculated with the wild-type strain Y6, for which the DI was 0, 2.38, 7.17, and 17.86, respectively, at the same time points. The control group without inoculation of a biocontrol strain showed the most serious morbidity, and the DI ranged from 15.62 at 30 DPI up to 35.93 at 45 DPI (Figure 7D). Verticillium wilt has become the major factor restricting cotton production in China, and as the main cultivar, upland cotton is sensitive to it. Because there is no high-yielding and anti-Verticillium wilt cultivar, using antagonistic bacteria to control Verticillium wilt has become one of the effective means to ensure cotton production. Sea Island cotton is highly resistant to Verticillium wilt due to its own traits and endophytes. Identifying island cotton anti-Verticillium endophytes and applying them to upland cotton has broad applications. The results of our study reveal that this B. halotolerans strain is a promising biocontrol agent for conferring Verticillium wilt resistance in cotton by overexpressing the most important pathogenic factor, β-glucanase Bgy6. The homologous overexpression of pathogenic factors of endophytes from natural populations of disease-resistant plants could effectively improve the antifungal effect of endophytes. Our results also demonstrate that antimicrobial activities can be modulated by regulating primary virulence factor expression in candidate strains.

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Funding

This work was supported by the projects from the National Natural Science Foundation Program of the People’s Republic of China (31570120, 31100104, 31571724), from program for Science & Technology Innovation Talents in Universities of Henan province, HASTIT (17HASTIT041), and from Innovation Scientists and Technicians Troop Construction Projects of Henan Province. Notes

The authors declare no competing financial interest.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b00728. Figure S1. Construction of β-glucanase bgy6 overexpression mutants (PDF)

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L.Z. and W.L. contributed equally to this work.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.C.). *E-mail: [email protected] (Q.N.). ORCID

Qiuhong Niu: 0000-0003-1695-7117 Author Contributions

Q.N. and Y.C. conceived and designed the experiments. L.Z. and W.L. performed the isolation and overexpression experiments. Y.T. and S.Z. assayed the enzyme activities. L.Y. performed and analyzed the pot experimental data. Q.N. and L.Z. drafted the manuscript. N.L. revised and polished the English expression. W.L., Y.C., Y.T., and S.Z. participated in the verification experiments and helped draft the manuscript. All authors read and approved the final manuscript. 6835

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