Supporting Information s19

Supporting information

Supplementary Methods

DNA manipulation

Escherichia coli DH5α was used for DNA manipulation. The polymerase chain reaction (PCR) was performed using PrimesSTAR HS DNA polymerase (TaKaRa, Otsu, Japan). The In-FusionÒ HD Cloning Kit (Clontech, Palo Alto, CA) was used for plasmid construction.

Media

Transformants were selected on Czapek-Dox (CD) medium (0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4•7H2O, 0.002% FeSO4•7H2O, and 2% dextrin, pH 5.5). CD medium supplemented with 0.5% uridine and 0.2% uracil was used for the growth of candidate transformants for pyrG mutants. Potato dextrose (PD) medium (Nissui, Tokyo, Japan) was used for the observation of conidial color. DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2PO4, 0.05% MgSO4•7H2O, pH 5.5) was used for the preparation of cell extracts.

Preparation of protein extracts and western blot analysis

A. oryzae mycelia grown in DPY liquid medium were frozen by liquid nitrogen and homogenized using multi-beads shocker (Yasui Kikai, Osaka, Japan). The homogenized mycelia were suspended in extraction buffer (50 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail (Sigma, St. Louis, MO)), and debris was removed by centrifugation at 1,000 × g for 10 min. Protein extracts were separated by electrophoresis on an 8% polyacrylamide gel containing sodium dodecyl sulfate, and then blotted onto ImmobilonÒ Transfer Membranes (Millipore, Bedford, MA). For Cas9 detection, the membrane was incubated with monoclonal ANTI-FLAG® M2 antibody (Sigma; 1:5,000 dilution) as a primary antibody and with 0.2 μg/mL peroxidase-labeled anti-mouse IgG (H+L) (Vector Laboratories, Burlingame, CA) as a secondary antibody. FLAG-tagged Cas9 was detected with a Western Lightning-ECL system (PerkinElmer, Waltham, MA) and the luminescent image analyzer LAS-4000miniEPUV (Fuji Photo Film, Tokyo, Japan).

Supplementary Material. Sequences of sgRNA, U6 promoter, U6 terminator, and codon-optimized cas9

Codon-optimized cas9

(Small letters and underlines indicate the FLAG-tag and SV40NLS encoding sequences, respectively.)

ATGgattacaaggatgacgacgataagATCATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACTCCATCGGTCTCGACATCGGTACCAACTCCGTCGGTTGGGCCGTCATCACCGACGAGTACAAGGTCCCCTCCAAGAAGTTCAAGGTCCTCGGTAACACCGACCGTCACTCCATCAAGAAGAACCTCATCGGTGCCCTCCTCTTCGACTCCGGTGAGACCGCCGAGGCCACCCGTCTCAAGCGTACCGCCCGTCGTCGTTACACCCGTCGTAAGAACCGTATCTGCTACCTCCAGGAGATCTTCTCCAACGAGATGGCTAAGGTCGATGACTCCTTCTTCCACCGTCTCGAGGAGTCCTTCCTCGTCGAGGAGGATAAGAAGCACGAGCGTCACCCCATCTTCGGTAACATCGTCGACGAGGTCGCCTACCACGAGAAGTACCCCACCATCTACCACCTCCGTAAGAAGCTTGTCGACTCTACTGATAAGGCTGACCTTCGTCTCATCTACCTCGCCCTCGCCCACATGATCAAGTTCCGTGGTCACTTCCTCATCGAGGGTGACCTCAACCCTGACAACTCCGATGTCGACAAGCTCTTCATCCAGCTCGTTCAGACTTACAACCAGCTCTTCGAAGAGAACCCCATCAACGCTTCCGGTGTTGACGCCAAGGCCATCCTCTCCGCTCGTCTCTCCAAGTCCCGTCGTCTCGAGAACCTCATCGCTCAGCTCCCTGGTGAGAAGAAGAACGGTCTCTTCGGTAACCTTATCGCCCTCTCTCTCGGTCTCACCCCCAACTTCAAGTCCAACTTCGACCTCGCCGAGGATGCCAAGCTTCAGCTCTCCAAGGACACCTACGATGACGATCTCGACAACCTCCTCGCCCAGATCGGTGACCAGTACGCTGACCTCTTCCTTGCCGCTAAGAACCTCTCCGACGCCATCCTCCTCTCCGATATCCTCCGTGTCAACACCGAGATCACCAAGGCTCCCCTCTCCGCTTCTATGATCAAGCGTTACGATGAGCACCACCAGGACCTCACTCTTCTCAAGGCCCTTGTCCGTCAGCAGCTCCCTGAGAAGTACAAGGAGATCTTCTTCGATCAGTCCAAGAACGGTTACGCCGGTTACATCGACGGCGGTGCTTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTCGAGAAGATGGACGGTACCGAGGAGCTCCTCGTCAAGCTCAACCGTGAGGATCTCCTCCGTAAGCAGCGTACTTTCGACAACGGTTCCATCCCCCACCAGATCCACCTTGGCGAACTCCACGCTATCCTCCGTCGTCAGGAGGACTTCTACCCCTTCCTCAAGGACAACCGTGAGAAGATCGAGAAGATCCTCACCTTCCGTATCCCCTACTACGTTGGCCCCCTCGCCCGTGGTAACTCCCGTTTCGCCTGGATGACTCGTAAGTCCGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTCGATAAGGGTGCCTCTGCCCAGTCCTTCATCGAGCGTATGACTAACTTCGACAAGAACCTCCCTAACGAGAAGGTCCTTCCTAAGCACTCTCTCCTTTACGAGTACTTCACTGTTTACAACGAGCTCACCAAGGTCAAGTACGTCACCGAAGGTATGCGTAAGCCCGCCTTCCTCTCTGGTGAGCAGAAGAAGGCTATCGTCGACCTCCTCTTCAAGACCAACCGTAAGGTTACCGTCAAGCAGCTCAAGGAAGACTACTTCAAGAAGATCGAGTGCTTCGACTCCGTTGAGATCTCCGGTGTCGAGGATCGTTTCAACGCTTCCCTCGGTACCTACCACGATCTCCTCAAGATCATCAAGGACAAGGACTTCCTCGACAACGAGGAGAACGAGGACATCCTTGAGGACATCGTCCTCACCCTTACCCTCTTCGAGGATCGTGAGATGATCGAAGAACGTCTCAAGACTTACGCTCACCTCTTCGACGACAAGGTCATGAAGCAGCTCAAGCGTCGTCGTTACACTGGTTGGGGCCGTCTCTCCCGTAAGCTGATCAACGGCATCCGTGACAAGCAGTCTGGTAAGACTATCCTCGACTTCCTTAAGTCCGATGGTTTCGCCAACCGTAACTTCATGCAGCTCATCCACGACGACTCTCTCACCTTCAAGGAGGACATCCAGAAGGCTCAGGTTTCCGGTCAGGGTGACTCCCTTCACGAGCACATCGCTAACCTCGCCGGTTCCCCCGCTATCAAGAAGGGTATCCTCCAGACCGTTAAGGTCGTCGATGAGCTCGTCAAGGTTATGGGTCGTCACAAGCCCGAGAACATCGTTATCGAGATGGCCCGTGAGAACCAGACTACCCAGAAGGGTCAGAAGAACTCTCGTGAACGTATGAAGCGTATCGAGGAGGGTATCAAGGAACTCGGCTCCCAGATCCTTAAGGAGCACCCCGTCGAGAACACCCAGCTTCAGAACGAGAAGCTCTACCTCTACTACCTCCAGAACGGCCGTGACATGTACGTTGATCAGGAGCTCGACATCAACCGTCTCTCCGACTACGACGTCGATCACATCGTCCCCCAGTCCTTCCTCAAGGATGACTCTATCGACAACAAGGTCCTCACCCGTTCCGATAAGAACCGTGGCAAGTCTGACAACGTCCCCTCCGAAGAGGTTGTCAAGAAGATGAAGAACTACTGGCGTCAGCTTCTCAACGCCAAGCTCATCACCCAGCGTAAGTTCGATAACCTCACTAAGGCTGAACGTGGTGGCCTCTCTGAGCTCGACAAGGCCGGCTTCATCAAGCGTCAGCTTGTTGAGACTCGTCAGATCACCAAGCACGTCGCCCAGATTCTCGACTCTCGCATGAACACCAAGTACGATGAGAACGACAAGCTCATCCGTGAGGTCAAGGTTATCACTCTCAAGTCTAAGCTCGTCTCCGACTTCCGTAAGGACTTCCAGTTCTACAAGGTCCGTGAGATCAACAACTACCACCACGCCCACGATGCCTACCTCAACGCTGTCGTTGGCACTGCTCTTATCAAGAAGTACCCCAAGCTTGAGTCCGAGTTCGTCTACGGTGACTACAAGGTCTACGATGTTCGTAAGATGATCGCTAAGTCCGAGCAGGAGATCGGTAAGGCCACCGCTAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCACCCTCGCCAACGGTGAGATCCGTAAGCGTCCCCTCATCGAGACCAACGGTGAGACTGGTGAGATCGTCTGGGACAAGGGTCGTGACTTCGCCACTGTCCGTAAGGTCCTCTCCATGCCCCAGGTCAACATCGTCAAGAAGACCGAGGTCCAGACCGGTGGCTTCTCCAAGGAATCTATCCTCCCCAAGCGTAACTCCGACAAGCTCATCGCTCGCAAGAAGGACTGGGACCCCAAGAAGTACGGCGGTTTCGACTCTCCCACTGTCGCTTACTCCGTCCTCGTTGTCGCCAAGGTCGAGAAGGGTAAGTCTAAGAAGCTCAAGTCCGTCAAGGAGCTCCTCGGCATCACCATCATGGAGCGTTCCTCCTTCGAGAAGAACCCCATCGACTTCCTCGAGGCCAAGGGTTACAAGGAGGTCAAGAAGGACCTCATCATCAAGCTTCCCAAGTACTCCCTCTTCGAGCTTGAGAACGGTCGTAAGCGTATGCTCGCTTCCGCCGGTGAGCTCCAGAAGGGTAACGAGCTCGCTCTCCCCTCCAAGTACGTCAACTTCCTCTACCTCGCCTCCCACTACGAGAAGCTCAAGGGTTCTCCCGAAGACAACGAGCAGAAGCAGCTCTTCGTCGAGCAGCACAAGCACTACCTTGATGAGATCATCGAGCAGATCTCCGAGTTCTCCAAGCGTGTCATCCTCGCCGACGCTAACCTCGATAAGGTCCTCTCCGCTTACAACAAGCACCGTGACAAGCCCATCCGTGAGCAGGCTGAGAACATCATCCACCTCTTCACTCTCACCAACCTTGGTGCCCCTGCTGCCTTCAAGTACTTCGACACCACCATCGACCGTAAGCGTTACACCTCTACCAAGGAGGTCCTCGACGCCACTCTCATCCACCAGTCCATTACCGGTCTCTACGAGACTCGTATCGACCTCTCTCAGCTCGGTGGTGACTCCCGTGCTGACCCCAAGAAGAAGCGTAAGGTCTGA

U6 promoter

TGGTTCACTTCTCTTTAGAAATCAACTGTGGGTTTTGCTTTTTGCTTCATTCTCTTTGTCTTCTCCATCTTTGATCAAATCCTGGACTTTCTCAATCCCCAGCTAATTCAATCATAGTCAGTTTTCTATTTTTATTATTTCTTTTTCTTTTGAAATGTGATTAACAACCAGTCCGTTATATATCTTGTACCCAGATTACGCCCAACTCGTGCTCCTCAGCCACAAAGATACTCAATTGATAGCCAAGATACATACATACCACAAAGTAAGGACTCCATGCATTGAGTATTACTCATCGTATTCTAGACTACTCCAAAACTCAGCACATAGACAAACAATACGAACCTCGTCTAGGGGTGATTCAGAGGCGGCAAAGCGGGGTTTTCGCATTTGATGTTCCTGGCACTTATGTAAGCCCACGCTTCCCGCTCAACTAAACCATCAGCCAATCAGACTGCTCAGATTTATCTTTTGAAGGGTAAATAAATCATTGTAAAGAAGAACAAGT

U6 terminator

TTTTTTTTTGAGCATTTATCAGCTTGATATAGAGGTAGGAATGTATGGAGGTGCAGAATGGCTATTTTGTTATTGGAGCGGGTTCGAAACGGAGGGCAGGAGACTTTTTCTAAATACGTCACGTGATATAGAGCTGCT

sgRNA

(protospacer sequence)-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC

Supplementary Table 1. Primers used in this study

Primer name Primer name Sequence 5' to 3'

Primers to amplify codon-optimized cas9

CAS9-in-fusion-6R CTACTACAGATCCCCTCAGACCTTACGCTTCTTCTTGGGGTC

FLAG-NLS-cas9-F TGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACTCCATCGGTC

IF5-FLAG-NLS-F TCGAGCTCGGTACCCATGGATTACAAGGATGACGACGATAAGATCATGGCCCCAAAGAAGAAGCGG

Primers to amplify the U6 promoter and terminator and sgRNA sequence

pUNA+U6p-F ATAGGAAAGGATCCTTAATGCCGGCTCATTCAAACGGAAATACGAGGGAC

gRNA+U6t-F1 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG

gRNA+U6t-R1 ACATTCCTACCTCTATATCAAGCTGATAAATGCTCAAAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGA

gRNA+U6t-F2 TCAGCTTGATATAGAGGTAGGAATGTATGGAGGTGCAGAATGGCTATTTTGTTATTGGAGCGGGTTCGAAACGGA

gRNA+U6t-R2 AGCAGCTCTATATCACGTGACGTATTTAGAAAAAGTCTCCTGCCCTCCGTTTCGAACCCGCTCCAATAACAAAAT

pUNA+U6t-R3 GCAGGTCGACTCTAGAGCAGCTCTATATCACGTGACGTATTTAGA

Primers with protospacer sequences to amplify the U6 promoter

gwA1-PU6-R GTGACGCGCCAGTAGATCCACACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG

gwA2-PU6-R GCTTATGGCTCTTAGACAGCCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG

gwA3-PU6-R ATAAGCTGCGAGGCATCTTTCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG

gwA4-PU6-R GTGCGCGGGTCTTCTTAGTTCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG

gyA-PU6-R TTAGTGAGAATCATTTGGCGCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG

gpG-PU6-R CTCGGAGCCGTAGGGGAAGTCCACTTGTTCTTCTTTACAATGATTTATTTACCCTTCAAAAG

Primers with protospacer sequences to amplify the U6 terminator

gwA1-TU6-F GTGGATCTACTGGCGCGTCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

gwA2-TU6-F GGCTGTCTAAGAGCCATAAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

gwA3-TU6-F GAAAGATGCCTCGCAGCTTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

gwA4-TU6-F GAACTAAGAAGACCCGCGCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

gyA-TU6-F GCGCCAAATGATTCTCACTAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

gpG-TU6-F GACTTCCCCTACGGCTCCGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC