Strain and fermentation culture
Metarhizium anisopliae (BNCC114445), obtained from Research Institude of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China, was inoculated onto Sabouraud medium (SDAcontaining 50 µg/mL chloramphenicol and cultured at 30 °C for 10 days. Six 5 mm plugs of mycelium were transferred into 200 mL SDA liquid culture media and fermented at 28 °C, 180 rpm, for 1 d, 3 d, 5 d, or 7 d. The mycelium and the fermentation broth were filtered through three layers of Miracloth (EMD Millipore Corp, Billerica, MA, USA) to collect the mycelium. No conidia were detected from the cultures. The mycelium was dried at room temperature and stored at 4 °C for use.
RT-qPCR analysis of genes in the SW biosynthesis pathway of M. anisopliae
Fungal RNA was extracted using the E.Z.N.A. Fungal RNA Kit (Omega). RNA was reverse transcribed into DNA by using PrimeScriptTMRT reagent Kit (Takara). Primers for amplification of SwnN, SwnT, SwnK, SwnH1, SwnH2, SwnR, and 18S rRNA are shown in Table S1 of supporting information. All of the genes were amplified using RT-qPCR with the following conditions: 1 cycle of 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 90 s, and an extension at 72 °C for 32 s. Cultures were tested using RT-qPCR at 1 d, 3 d, 5 d, and 7 d.
Identification of swnR gene of M. anisopliae
Fungal DNA was extracted using the CTAB method. Primers for amplification of swnR were designed from the swnR sequence from Cook et al. , GeneBankKID61009 of M. anisopliae ARSEF 549. The swnR gene (GeneBank MN892388) was amplified from M. anisopliae DNA using L1/R1 primers(Additional file 2: Table S2).
The upstream and downstream fragments of the swnR gene (Additional file 1: Figure S3) and the benomyl (fungicide) resistance gene (ben) (Additional file 1: Figure S4) were inserted into pUC19 (Takara) digested with EcoR I/BamH I (Takara) (Additional file 1: Figure S5) using the In-Fusion® HD Cloning System (Takara) to construct a knockout construct targeting the swnR gene (Additional file 1: Figures S6 and S7 and Additional file 3: Text S1).
The primers L3 and R3 (Additional file 2: Table S2) were used to amplify the ben resistance gene from pBARGPE1-BenA (Wuhan Jingxiu Scientific Biotechnology Co., Ltd., China) as a template. The primers L2/R2 and L4/R4 (Table S1, supporting information) were used to amplify the upstream target fragment (swnR-I) and the downstream target fragment (swnR-II), respectively, of the swnR gene from the genomic DNA of M. anisopliae. The swnR-I, ben, swnR-II and the double-cut pUC19 vector were ligated using In-Fusion cloning. The swnR gene fragment was amplified using primers L1/R1 (Additional file 2: Table S2) from the genomic DNA of M. anisopliae. To produce a complementation vector, the swnR was inserted between trpC promoter and trpC terminator of pBARGPE1 vector, which contains the glufosinate (herbicide) resistance (bar) gene as a marker, using In-Fusion cloning (Fig. 4).
Preparation of protoplasts
Six 5 mm plugs from 10-day cultures of M. anisopliae grown on SDA media were transferred into each 200 mL flask of SDA liquid culture media, and incubated at 28 °C, 180 rpm for 1 d, 2 d, 3 d, 4 d and 5 d. The resulting mycelia were filtered through sterile miracloth. To the collected hyphae were added different concentrations of enzymatic hydrolysate (Sigma Aldrich) prepared with 1.2 M KCl, and hydrolyzed at 30 °C, 100 rpm, for 1 h, 3 h, 5 h, 7 h, and 10 h. The optimal combination of enzymes and conditions were determined based on protoplast yield. Yield from different enzymes, including 1% snail enzyme, 1% cellulase, and 1% lysing enzymes, and combinations of the enzymes were also tested. The enzymatically digested mixtures were filtered through a layer of sterile miracloth and two layers of filter paper into a sterile 50 mL centrifuge tube, and the protoplasts were washed extensively with 1.2 M KCl and centrifuged at 4000 rpm for 6 min at room temperature. After discarding the supernatant, 10 mL of STC Buffer (0.6 M Sorbitol; 10 mM Tris-HCl; 10 mM CaCl2, pH 6.5) was added and the protoplasts were gently resuspended. The mixture was centrifuged at 4000 rpm for 6 minutes. After discarding the supernatant, 1 mL of STC Buffer was added. The protoplasts were then centrifuged at room temperature at 3500 rpm for 6 min, which was repeated. Finally, protoplasts were adjusted to 2-5×107/mL for subsequent experiments.
PEG mediated DNA transformation
Transformation of the protoplasts were done as in Proctor et al.  Approximately 5-10 µg of the linearized swnR knockout vector was added to a 50 mL centrifuge tube containing 2-5×107/mL protoplasts, and allowed to stand at room temperature for 20 min without shaking. Then 1-1.25 mL of 40% PTC (40% PEG 8000, 20% sucrose, 50 mM CaCl2, 10 mM Tris-HCl) was added to the tube (mixed thoroughly by inversion), and let stand at room temperature for 20 min without shaking. Thereafter, 5 mL of TB3 (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose) containing 50 g/mL ampicillin (Sigma Aldrich) was added and shaken at room temperature overnight. The overnight protoplasts were centrifuged at 4000 rpm for 6 min, the supernatant was discarded, and about 1 mL of the remaining liquid was used to suspend the remainder. The regenerated protoplasts were added to 10 mL of Bottom Agar (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose, 1% Agar) containing 100 µg/mL benomyl (Additional file 1: Figure S6). After incubation at 30 °C for 10 hours, Top Agar (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose, 1.5% Agar) containing 200 µg/mL benomyl was added. After 3-5 d, a single colony transformant grew on the plate, which was transferred to SDA medium containing 200 µg/mL benomyl. The wild type M. anisopliae was used as a control. The swnR gene mutant strain of M. anisopliae was named MT. The transformation of the complement vector was the same as described above, and 2 mg/mL of glufosinate (Additional file 1: Figure S8) was used for screening of the complement (CT).
PCR identification of MT and CT
The M. anisopliae strain carrying the benomyl resistance gene was used as a template, and PCR amplification was carried out using primer L1/R1. Subsequently, the complemented strain was subjected to the same PCR amplification using primers L1/R1 (Additional file 1: Table S2).
Phenotypic observation and growth rate determination of MT, CT and WT
Colonies of MT and WT of the same size were inoculated into the same position on the SDA medium and grown at 28 °C for 3 d, 5 d and 10 d, after which they were measured for diameter and photographed.
SW content detection of fermentation broth of WT, MT, CT in M. anisopliae
The WT, MT, CT strains were inoculated into SDA medium containing 50 µg/ml chloramphenicol and cultured at 28 °C for 10 days. Then six 5 mm plugs of each strain were transferred into 200 mL flasks of SDA (without agar) culture medium and grown at 28 °C, 180 rpm, for 3 d. The flasks of fermentation broth of WT, MT, and CT were combined by strain and filtered to obtain 500 mL of fermentation broth. The SW (control from Sigma Aldrich) in each extract was analyzed using Q Exactive Mass Spectrometer (Thermo Fisher) using the methods of Song et al. . SW concentration was tested three times for each strain.
In this study, each measurement was tested three times. Statistical analysis was performed on the measured data using SPSS 20.0 software. The results were expressed as mean ± SEM. One-way ANOVA was performed on each sample, *P<0.05, indicating a significant difference between the two groups, **P<0.01, indicating that the difference between the two groups is highly significant. Results from the cultures were used for determining the optimal time periods for swainsonine production. The mass concentration peak area for SW was compared using linear regression. The colony diameters were measured by ruler. RT-qPCT data were analyzed using the 2 -△△CT method.