Escherichia coli dh5α

Approximately 100 male heads and 100 female heads were pooled together from which total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The RNA was treated with DNase (Takara, Japan) and then reverse transcribed to cDNA using PrimeScript 1st strand cDNA synthesis kit (Takara, Japan).
Previously, one transcriptome dataset for L. striatellus was built by Zhang et al. (2010 (link)) and another one had been built in our laboratory (unpublished data). Based on the sequence data obtained from transcriptome searching using the basic local alignment search tool (BLAST), gene-specific primers were designed and used for cloning cry cDNA (Table S1). PCR were conducted using KOD FX DNA polymerase (Toyobo, Japan). The PCR conditions were determined empirically for the amplification of each gene. PCR products were verified by 1% agarose gel electrophoresis. Target bands were excised and purified using DNA gel extraction kit (Axygen, USA). The DNA fragments were TA ligated into pMD18-T vector (Takara, Japan) and transformed into Escherichia coli DH5α (TransGen, China). Positive clones were sequenced by SunnyBio (China).

The strains used in this study are listed in Table S1. Escherichia coli DH5α (Transgene, Beijing, China) was used for plasmid construction and cultured at 37 °C in Luria–Bertani broth containing ampicillin (100 μg/mL). The citric acid-producing strain A. niger D353.8 (kusA::hph, pyrG::hph, hyg^R) was stored in the lab [30 (link)]. A. niger strains were cultivated on defined minimal medium (MM), as reported previously [31 (link)], or on complete medium (CM) consisting of MM supplemented with 0.5% yeast extract and 0.1% casamino acids. Then, 1.5% agar was supplemented for plates. When necessary, 10 mM uracil was supplemented in the media for the pyrG mutants.

Total RNA was extracted using Trizol reagent (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. First strand cDNA was synthesized using M-MLV reverse transcriptase and modified oligo (dT), following the manufacturer’s instructions (TaKaRa Biotechnology, Dalian, China). Fragments of the rice heading date genes were amplified using the following PCR program: an initial denaturation cycle at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 35 s, and 72°C for 1 min, and then a final extension at 72°C for 10 min. The amplified products were separated on 1.0% agarose gel and target bands were purified using E.Z.N.A.® Gel Extraction Kit (Omega Bio-tek’s, Norcross, USA). Then the DNA fragments were cloned into a pGEM-T Easy Cloning Vector (Promega, Madison, WI, USA), transformed into Escherichia coli DH5α (TransGen Biotech Co., Ltd., Beijing, China), and sequenced following the techniques outlined by the Bio-Tech Research Center, Shandong Academy of Agricultural Science, Jinan, China.

Escherichia coli DH5α (Transgene, Beijing, China) was used for plasmid construction and cultured at 37 °C in Luria–Bertani broth containing ampicillin (100 μg/mL). The A. niger strains and plasmids used in this study are indicated in Additional file 1: Tables S1 and S2. A. niger G1 (amdS⁻, ∆glaA, and ∆pepA) was derived from A. niger NRRL3112 and presented by the Institute of Microbiology, CAS; this strain was used as the recipient strain for genome editing. A. niger strains were cultivated on minimal medium (MM) [18 (link)] containing 1% glucose, 70 mM NaNO₃, 110 mM KH₂PO₄, 70 mM KCl, 2 mM MgSO₄, and trace element solution or on complete medium (CM) consisting of MM supplemented with 0.5% yeast extract and 0.1% casamino acids. When using amdS as a selection marker, NaNO₃ in MM was replaced by 10 mM acetamide and 15 mM caesium chloride (MMSA). For growth on solid plates, 1.5% agar was supplemented. If necessary, 150 μg/mL of hygromycin was added.

All constructed strains in our study were generated from M. acridum CQMa102 which is conserved at China General Microbiological Culture Collection Center (WT, No. 0877), and cultured in 1/4 SDAY medium (one-quarter-strength Sabouraud dextrose agar medium which consists of 1% dextrose, 0.50% yeast extract, 0.25% mycological peptone and 2% agar, w/v) for 15 days at 28 °C. For testing the fungal cell wall integrity, fungal spot assays were conducted on 1/4 SDAY, 1/4 SDAY supplemented with 50 μg/mL CFW and 500 μg/mL CR at 28 °C for 5 days according to a previous method [34 (link)]. The diameters of the fungal colonies were measured to calculate the relative growth inhibition. For DNA manipulations and fungal transformations, the Escherichia coli DH5α (TransGen Biotech, Beijing, China) and Agrobacterium tumefaciens AGL-1 (TransGen Biotech, Beijing, China) growth on Luria–Bertani (LB) broth were selected at 37 °C and 28 °C, respectively.

Gordonia neofelifaecis (NRRL B-59395) was preserved in our laboratory (Ge et al. 2011 (link)) and cultivated in liquid LB medium (Luria–Bertani broth) at 37 °C. The strain had been originally isolated from the faeces of Neofelis nebulosa. Escherichia coli DH5α and E. coli BL21 (purchased from Transgen Biotech Co., Ltd, Beijing, China) were grown in LB broth or Super Optimal Broth (SOB, 2% peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄) (Hanahan 1983 (link)) at 37 °C/200 rpm. Kanamycin (25 μg/mL) was added to the growth medium when necessary. For growing on solid medium, 2% (w/v) agar was added.

Purified dsRNA was used as a template for cDNA cloning. The cDNA library was constructed using the methods described by Zhong (Zhong et al., 2016 (link)). Random primers (5’-GCCGGAGCTCTGCAGAATTCNNNNNN-3’) and specific primers (5’-GCCGGAGCTCTGCAGAATTC-3’) were used for reverse transcription and PCR amplification. The intermediate sequences were amplified with specific primers designed according to the obtained sequences, and the 5’ and 3’ terminal sequences were obtained using adaptor-ligated methods as previously reported (Xie et al., 2011 (link); Zhong et al., 2016 (link)). All amplicons were cloned into the pMD18-T vector (Takara, Dalian, China) and transformed into Escherichia coli DH5α (TransGen Biotech, Beijing, China). Positive clones were selected for Sanger sequencing, with each base pair sequenced at least three times.