Kanamycin

RPMI 1640 medium, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco, USA. Minimum Eagle’s medium (MEM) was obtained from HyClone, USA. Antibiotic/antimycotic solution, kanamycin, puromycin, and dimethyl sulfoxide (DMSO) were obtained from SolarBio, China. DSF was purchased from Selleck, China, whereas CuCl₂ was obtained from J&K, China. Phenylmethanesulfonyl fluoride (PMSF), radioimmunoprecipitation assay (RIPA) buffer, and bicinchoninic acid (BCA) reagent were obtained from Beyotime, China. Cell counting kit-8 (CCK-8) reagent was purchased from Dojindo, Japan, whereas AnnexinV-FITC (AV-FITC), propidium iodide (PI) and PI kit were purchased from Roche, Germany. Electrochemiluminescence (ECL) was obtained from Biosharp, China. Lipofectamine 2000 (Lip2000) was purchased from Invitrogen, USA, whereas ROS assay kit was obtained from NJJCBIO, China. Seahorse XF Glycolysis Stress Test and XF Cell Mito Stress Test kits were purchased from Agilent, USA. DeadEnd™ Fluorometric TUNEL System was obtained from Promega, USA, whereas antifade mounting medium with DAPI was purchased from SolarBio, China.

The promoter sequences came from B. subtilis 168 (College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China). The cloning and expression hosts were E. coli JM109 (College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China) and B. subtilis RIK 1285 (TaKaRa, Dalian, China), respectively. The empty plasmid pP43NMK (a gift from Dr. Yunbin Lv, Jiangnan University, Wuxi, China) was utilized as an expression vector for L-asparaginase. BlAase expression was previously achieved using the vector pP43NMK-BlA-His (College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China). L-asparagine was purchased from Aladdin (Shanghai, China). Kanamycin was obtained from Solarbio (Beijing, China). All other chemicals were of analytical grade.

The red-fleshed apple callus was transformed as described previously^{19 (link)}. The coding sequences (CDSs) of MdMYB6 and MdTMT1 were ligated into the pRI-101 vector containing GFP and the 35S CaMV promoter, respectively, to generate the 35S:MdMYB6-GFP and 35 S:MdTMT1-GFP plasmids (Supplementary Fig. S1A). The CDS of MdMYB6 was ligated into the pCAMBIA1301 vector, which contained a sequence encoding a His tag as well as the 35S CaMV promoter, to construct the 35S:MdMYB6-His recombinant plasmid (Supplementary Fig. S1E). The recombinant plasmids were transformed into Agrobacterium tumefaciens LBA4404. We then infected the red-fleshed calli with the transformed Agrobacterium and cocultured them on MS medium in the dark at 24 °C for 48 h. The resulting calli were then moved onto a screening medium containing 250 mg/L carbenicillin and 50 mg/L kanamycin (Solarbio, Beijing, China) for the MdMYB6- or MdTMT1-overexpressing calli. The screening medium for cultivating the calli cotransfected with MdMYB6 and MdTMT1 contained 250 mg/L carbenicillin, 20 mg/L hygromycin and 50 mg/L kanamycin.

Escherichia coli BL21(DE3) (F^–ompT hsdSB (r_B^–m_B^–) gal dcm) (Shanghai Weidi Biotechnology Co., Ltd., Shanghai, China) and E. coli DH5α (dlacZ Delta M15 Delta (lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1) were used for protein expression and cloning, respectively (Studier and Moffatt, 1986 (link); Taylor et al., 1993 (link)). A prepacked desalting column and Ni Sepharose column were purchased from Smart Lifesciences (Changzhou China). The pET28a+ vector from Personalbio (Shanghai, China) was used to express recombinant proteins. Isopropylthio-β-D-galactoside (IPTG) and kanamycin were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Ethylenediaminetetraacetic acid (EDTA), 1,4-dithiothreitol (DTT), 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB), acetyl coenzyme A trilithium salt (AcCoA), and enzyme substrates including 2-aminofluorene (2-AF), hydralazine (HDZ), 5-aminosalicylate (5-AS), 4-amino salicylic (4-AS), sulfamethoxazole (SMX), INH, 4-chloro-3-methylaniline (4-C3ME) and 4-aminobenzoic acid (pABA) were purchased from Aladdin (Shanghai, China).

Aflatoxin B₁ (AFB₁), zearalenone (ZEN), deoxynivalenol (DON), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,6-dimethoxy phenol (DMP), syringaldazine (SGZ), and methyl syringate were purchased from Sigma-Aldrich (St. Louis, MO, USA). FB₁ (fumonisin B₁) and OTA (ocharatoxin A) were purchased from Pribolab (Beijing, China). DNA polymerase, T4 ligase, acetonitrile, and trifluoroacetic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Vanillin, p-coumaric, syringic acid, syringaldehyde, caffeic acid, 1-hydroxybenzotriazole (HBT), gallic acid, isopropyl-β-D-thiogalactoside (IPTG), and kanamycin were purchased from Solarbio (Beijing, China). Ni-NTA agarose was purchased from QIAGEN (Hilden, Germany). The fungal laccase from Ganoderma sp. was purchased from Sunson (Yinchuan, Ningxia, China). Plant extracts from E. brevicornu, C. sativus L., L. angustifolia, A. officinalis, and S. tenuifolia were purchased from Ciyuan Biotech (Xi’an, Shanxi, China). All other chemicals were of analytical grade or chromatographically pure, and were commercially available.

S. coelicolor A3(2) was cultivated at 30°C in TSB medium composed of 1.5% (w/v) tryptone, 0.5% soy peptone, and 0.5% NaCl. The Escherichia coli strains were grown in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) at 37°C with 50 μg/ml kanamycin (Solarbio, China) when required.

After infection, the cells were detached from the dish using a cell scraper and centrifuged to remove the supernatant. Then, the cells were treated with PBS containing 0.01% Triton X-100 (Solarbio, Beijing, China) and vigorously agitated to lyse the cells and release Mm. The cell lysate was diluted and plated onto Middlebrook 7H10 (BD) agar plates and incubated at 30 °C for approximately 14 days. Infected zebrafish were immersed in PBS containing 3% kanamycin (Solarbio) and incubated for 45 min at a temperature of 27 °C. Thereafter, a cell tissue crusher was employed to lyse the infected zebrafish and release Mm. The zebrafish lysate was subsequently coated onto 7H10 agar plates and incubated for 2 weeks at a temperature of 30 °C.