Construction of plasmid

A target α-MMC protein was generated in plasmid pET28a(+). The DNA sequence encoding α-MMC was obtained from GenBank (CAA40869.1). DNA of which was optimized by the codon optimization tool from Integrated DNA Technologies (IDT) for expression in the system 26, was then synthesized by the Macrogen company (Korea).DNA encoding α-MMC was amplified by PCR using the gene (optimal codons, IDT, USA) as the template and the primer pair ONF/ONR (ONF: 5'-cag cca tat gga tgt tag ctt tcg ttt gtc ggg tgc tga t -3'; ONR: 5'- ggt gct cga gtc agt agc tcg aaa agc cat gtg -3'). The pcr product was inserted into treated pET28a(+) (Merck Millipore, Germany) at the I and I sites by T4 DNA ligase, resulting in the generation of the plasmid pET-MMC.

Amino acid sequence of α-MMC protein:

MGSSHHHHHHHHSSGLVPRGSHMDVSFRL

SGADPRSYGMFIKDLRNALPFREKVYNIPLLL

PSVSGAGRYLLMHLFNYDGKTITVAVDVTNV

YIMGYLADTTSYFFNEPAAELASQYVFRDARR

KITLPYSGNYERLQIAAGKPREKIPIGLPALDSA

ISTLLHYDSTAAAGALLVLIQTTAEAARFKYIEQ

QIQERAYRDEVPSLATISLENSWSGLSKQIQLA

QGNNGIFRTPIVLVDNKGNRVQITNVTSKVVT

SNIQLLLNTRNIAEGDNGDVSTTHGFSSY

Expression and purification of proteins

The plasmid pET-MMC encoding the α-MMC protein was chemically transformed into strain BL21 (DE3). The bacteria were cultured in Luria-Bertani medium (LB) containing 40 mg/L kanamycin, and the cells were grown at 37 °C with shaking at 200 rpm to reach an OD600 of 0.8-1. IPTG (Sigma Aldrich, St. Louis, USA) was subsequently added to a final concentration of 0.75 mM. The cells were continuously incubated for 10 hours at 15 °C with shaking at 200 rpm before being harvested by centrifugation. The pellet was then resuspended in lysis buffer consisting of 20 μg/ml DNase I and 1 mM PMS. The cell wall was broken by sonication, and the insoluble cell debris was then removed by centrifugation at 20000 rpm for 1 hour at 4 °C. The soluble fraction was added to a His-tag column (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the column was washed with 30 volumes of column with 20 mM Tris-HCl, 100 mM NaCl and 10 mM imidazole buffer, pH 7. The proteins were finally eluted with buffer containing 20 mM Tris-HCl, 100 mM NaCl and various imidazole concentrations. The imidazole chemical in the protein solution was removed by centrifugation using an Amicon Ultra15 centrifugal filter (EMD Milipore). The pure α-MMC protein was collected and analyzed by SDS‒PAGE. Total protein expression was analyzed by AlphaEase software. The pure protein was measured by a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

Testing the antifungal activity of α-MMC against pathogens

Initial experiments investigated the antifungal activity against . The α-MMC protein with different concentration ranges and control (water) samples were evenly added to the surface of the PDA-agar plate. Subsequently, the fungal suspension and control water samples were added into the center of the PDA-agar plate and incubated for 24 hours at 28 °C 27.

Construction of plasmid pET-MMC

The DNA sequence of was first optimized for expression in an system by the codon optimization tool from Integrated DNA Technologies (IDT). The gene was provided with the best sequence option by screening and filtering sequences to lower complexity and minimize secondary structures. The gene fragment was amplified by PCR with a pair of specific primers ONF and ONR with additional restriction enzyme cutting sites I (forward primer) and I (reverse primer). PCR products were analyzed by gel electrophoresis with 2% agarose, which showed a single bright band near the 800 bp band of the DNA ladder (Figure 2A), corresponding to the theoretical size of the α-MMC gene fragment of 812 bp.

The pET28 plasmid and the target gene after treatment with 2 restriction enzymes, I and I, were cloned by T4 DNA ligase. The ligated product was transformed into strain DH5α and spread on LB-agar medium containing the antibiotic kanamycin. The colonies grown on the medium were selected to clarify the presence of recombinant vector in strain DH5α by colony PCR. The electrophoresis results showed that 2 colonies (in 7 selected colonies) revealed a band at approximately 1000 bp of the DNA ladder, corresponding to the theoretical size design (1055 bp) (Figure 2B). The recombinant DNA was then sequenced by Macrogen. Inc. (Korea). The sequencing result revealed 100% similarity with the theoretical sequence (data not shown) that clarifies the recombinant vector pET-MMC bearing the gene.

Protein expression and purification

To investigate the expression of the α-MMC protein in a bacterial system, the recombinant plasmid pET-MMC was chemically transformed into the strain BL21(DE3). The IPTG for induction was added after the cells were grown to reach OD0.8-1. The level of protein expression was analyzed by SDS‒PAGE. The results showed that there was a bright band of protein near the 30 kDa band of the ladder, corresponding to the theoretical molecular weight of the α-MMC protein (31.388 Da) under the addition of IPTG. In contrast, the α-MMC protein was not expressed without the addition of IPTG. Using the Software (Alpha Innotech) for calculation of protein expression 28, the α-MMC protein was expressed with a high protein level in of approximately 50% of total protein under induction of IPTG (Figure 3A).

The α-MMC protein was designed with 6xHis for purification. α-MMC fused with a His-tag at the N-terminus was purified by chromatography using the His-tag column. SDS‒PAGE showed that pure α-MMC was collected after elution with different concentrations of imidazole (Figure 3B). The molecular weight of α-MMC is 31.388 Da, which shifted near the 30 kDa band of the ladder. Using the NanoDrop spectrophotometer to measure the protein concentration (total protein fragments with imidazole concentrations of 100 to 200 mM), approximately 70 mg of pure α-MMC was obtained in 1L LB culture medium, which implies that α-MMC can be highly expressed in bacterial systems.

Effect of α-MMC protein on mycelial growth of and

Theα-MMC protein has previously been shown to confer resistance to fungi by blocking the spread of pathogens. In this study, we tested the antifungal activity of the α-MMC protein against (causing blast disease). Compared with the control, the difference between treatments with variant α-MMC protein concentrations was evident in the assays. Treatments with 5 µM, 20 µM, 100 µM and 200 µM α-MMC against (Figure 4) showed that the mycelial growth of fungi was inhibited in a concentration-dependent fashion after 24 hours of incubation. Specifically, for treated with water as the control, the fungal colonies were brown in color and spread over the plate. At low concentrations of α-MMC (5 µM and 20 µM), the inhibition of fungal growth was still weak, with a diameter of fungal colonies of 3.4 cm. Increasing concentrations of α-MMC at 100 µM and 200 µM clearly showed antimicrobial activity against the blast fungus with a colony diameter of 1.75 cm and nonfungal growth, respectively (Figure 4).