Construction of PET-32a-PtDef and Recombinant Plasmid Expression

Primers (Supplementary Table S1) were designed and synthesized based on the BamHI and NotI restriction sites in the PET-32a plasmid. The restriction enzymes BamHI and NotI (TaKaRa) were used to digest the PCR product and PET-32a plasmid, respectively, followed by ligation using T4 ligase (TaKaRa). Positive clones were electrophoresed in 1% agarose gel and sequenced.

The plasmid pET32a-PtDef was transformed into E. coli BL21 (DE3) (Solarbio, China). A single colony was selected on a plate containing 50 μg/ml ampicillin and cultured in liquid medium containing 50 μg/ml ampicillin. The expression of pET32a-PtDef in E. coli BL21 (DE3) was induced by 1 mmol/L isopropyl-β-D-1-thiogalactopyranoside (IPTG) in 100 ml Luria-Bertani (LB) medium for 4 h at 30°C. Cells were harvested by centrifugation at 5000 rpm for 15 min, resuspended in 10 ml binding buffer (20 mM Tris-base, 500 mM NaCl, and 20 mM imidazole), frozen at −20°C, and thawed in an ice bath; lysozyme was then added for a final volume of 1 mg/ml Next, the mixture was placed on ice for 30 min, Triton X-100 was added, and the concentration of the solution was diluted to 0.1%. DNase and RNase (5 μg/ml) were added and the mixture was incubated at 4°C for 10 min. Finally, DL-dithiothreitol (DTT) was added to a concentration of 1 mmol/L, the mixture was centrifuged, and the supernatant was decanted and stored at 4°C.

His-PtDef was purified by His-label affinity chromatography at a flow rate of 0.5 ml/min and a pressure of 0.25 MPa; the UV280 signal was set to zero. Samples were added after the baseline had stabilized. Next, wash buffer (20 mM Tris-base, 500 mM NaCl, and 50 mM imidazole) was added until the baseline returned to zero and elution buffer (20 mM Tris-base, 500 mM NaCl, and 250 mM imidazole) was applied to elute the target protein. The fractions recovered were evaluated by 12% SDS-PAGE.

Aspergillus niger, Alternaria Nees, Mucor corymbifer, Marssonina populi, Rhizopus sp., and Neurospora crassa were inoculated on PDA and activated in a 25°C incubator. The fungi were resuspended in 0.9% NaCl (pH 7.0) and transferred to liquid PDA, which was poured into Petri dishes. After setting, holes were made in the PDA. We dissolved 120 μl PtDef in 0.9% NaCl (pH 7.0) to 40, 20, 10, 5, 2.5, 1.25, and 0.625 μg/ml and added the solutions to PDA in triplicate. PDA containing 0.9% NaCl (pH 7.0) was used as a negative control. The plates were incubated at 25°C for 72 h until observation of mycelia.