Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in Pyricularia oryzae

Transcription

1 1 Supporting Information Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in Pyricularia oryzae Choong-Soo Yun, Takayuki Motoyama, and Hiroyuki Osada* Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama , Japan 7 8 *Correspondence: Tel: ; Fax: ; hisyo@riken.jp

2 26 Supplementary Methods DNA manipulation. Plasmids were purified with a QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer s instructions. DNA fragments were isolated from agarose gels with a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer s instructions. Polymerase chain reaction (PCR) was carried out with a DNA Engine Peltier Thermal Cycler (Bio-Rad). DNA amplification via PCR was conducted with KOD -Plus- Neo DNA polymerase (Toyobo). DNA fragment ligation and circularization for vector construction was conducted via infusion reaction using an In-Fusion HD cloning system (Clontech). DNA sequencing was carried out with a BigDye Terminator v3.1 Kit (Applied Biosystems). Sequencing products were run on an automated 3730xl capillary DNA Analyzer (Applied Biosystems). All other experiments were performed with standard methods (29). Gene overexpression. PoLAE1 gene overexpression strains were constructed as follows. Two kb of the upstream region of PoLAE1 and 2 kb of the downstream region including PoLAE1 were amplified from the genomic DNA of P. oryzae via PCR using the primers OELAE1_UP-F and OETAS1_UP-R (fragment 1), and OELAE1_DN-F and OELAE1_DN-R (fragment 2) respectively. The blasticidin S resistance gene expression unit (fragment 3) and the vector sequence of pbi121 between the right and left borders (fragment 4) were amplified using the same primers as used for the gene disruption. The TEF1 gene promoter region of A. oryzae was amplified from genomic DNA via PCR with primers OELAE_TEF1-F and OELAE_TEF1-R (fragment 5). All fragments were gel-purified and cloned with an In-Fusion HD Cloning Kit (Clontech) to yield pbi-. E. coli DH5α and A. tumefaciens transformations were conducted as described for gene disruption. PoLAE1 overexpression strains were selected via PCR with the primers OELAE1_CHK-F and OELAE1_CHK-R, which hybridize outside of the omitted 1 kb upstream region of the PoLAE1 gene. This primer set can amplify 1 kb of the 2

3 PoLAE1 ORF upstream region from wild-type strains, and the 2 kb hygromycin B resistance gene and TEF1 promoter expression unit from the MGG_07800 disruptants. The primers used are listed in Supplementary Table 1. Metabolite extraction and analysis. P. oryzae culture broth (100 µl) was extracted with 4 volumes of ethanol, evaporated with an N 2 stream, and dissolved in 200 µl methanol to analyze metabolites. SM analysis using UPLC MS was performed with an ACQUITY UPLC H-Class System (Waters Alliance) equipped with a mass spectrometer (API 3200, Applied Biosystems). The UPLC conditions were as follows: column, XTerra MS C18 (Waters), 5 µm ( mm); flow rate, 0.6 ml min -1 ; solvent A, water containing 0.05% formic acid; solvent B, acetonitrile. After the extracted sample was injected into a column equilibrated with 5% solvent B, the column was developed with a linear gradient from 5% to 100% solvent B over the course of 3 min and kept at 100% solvent B for another 2 min. Mass spectra were collected in the electrospray ionization-positive and electrospray ionization-negative modes. Quantitative PCR. For RNA extraction, each strain was cultured with 500 µl of YG media in a 24-well plate at 25 C without agitation for 5 d. The RNA was isolated with an RNeasy Plant Mini Kit (Qiagen) according to the manufacturer s instructions and treated with RQ1 DNase (Promega). The isolated RNA was reverse transcribed using a SuperScript VILO TM cdna Synthesis Kit (Invitrogen) according to the manufacturer s instructions, and quantitative PCR was carried out using a SsoFast TM EvaGreen Supermix Kit (Bio-Rad) on the CFX96, C1000 Thermal cycler (Bio-Rad). Thermal cycler conditions were as follows: 95 C 3 min (1 cycle); 95 C 10 s, 60 C 30 s (40 cycles). Amplified products were confirmed by melt curve analysis. The betatubulin gene (MGG_00604) was amplified as an endogenous control to normalize the amount of cdna sample added to the reaction mixture. Gene expression data were evaluated using Cqmethod. The primers used are listed in Supplementary Table

4 Supplementary Table 1. Strains, plasmids and primers used in this study Strains and plasmids Relevant characteristics Source or reference Strains Pyricularia oryzae Kita1 Pathogenic to rice plants Ref. 25 P. oryzae TAS2 (MGG_07800 ) gene disrupted P. oryzae This study P. oryzae Polae1 PoLAE1 (MGG_01233 ) gene disrupted P. oryzae This study P. oryzae OE::Polae1 Over-expressed PoLAE1 gene carried P. oryzae This study P. oryzae OSM1 (MGG_01822) gene disrupted P. oryzae Ref. 18 P. oryzae lae1 OSM1 and PoLAE1 gene disrupted P. oryzae This study P. oryzae OE::Polae1 TAS2 gene disrupted strain using over-expressed PoLAE1 gene carried P. oryzae This study E. coli DH5α F - ф80dlacz M15 (laczya-argf )U169 enda1 reca1 hsdr17 (r - K m + K ) Takara Plasmids pbi121 Binary vector, Km r, CaMV promoter, β-glucuronidase (GUS) Clontech pbi-m07800::hph pbi121 containing up & down stream of MGG_07800 and Hyg r unit between RB and LB This study pbi-polae1::hph pbi121 containing up & down stream of PoLAE1 and Hyg r unit between RB and LB This study pbi- pbi121 containing up stream of Lae1 including PoLAE1, BS r unit, TEF1 promoter between RB and LB This study pbi-osm1::hph pbi121 containing up & down stream of OSM1 and Hyg r unit between RB and LB Ref. 18 pbi-polae1::bs pbi121 containing up & down stream of PoLAE1 and BS r unit between RB and LB This study Primers Name Sequences Description pbi121-rb 5 -CAGATTGTCGTTTCCCGCCTTCAGTTT-3 For pbi vectors pbi121-lb 5 -CGTCCGCAATGTGTTATTAAGTTGTCTAAGC-3 For pbi vectors 5HPH 5 -AAGCTTATCGATACCGTCGACAGAAGATG-3 For pbi vectors 3HPH 5 -CGCGTTTTATTCTTGTTGACATGGAGC-3 For pbi vectors M07800_UP-F 5 -GGAAACGACAATCTGAAGGGCATAGAAGCGAATCGAGCCTGA-3 For pbi-m07800::hph M07800_UP-R 5 -CAAGAATAAAACGCGGCTGGTTCAAGAGCTCGGGTCCGAATG-3 For pbi-m07800::hph M07800_DN-F 5 -GGTATCGATAAGCTTCACAAACGTCAACTATCTTTAGCGTTG-3 For pbi-m07800::hph M07800_DN-R 5 -AACACATTGCGGACGGCTCTTCTACGTGTCAAAGTACGCCAT-3 For pbi-m07800::hph M07800_CHK_F 5 -TCTCATTCGGACCCGAGCTCTTGAACC-3 For pbi-m07800::hph M07800_CHK_R 5 -ATGTCGCGGCCAGGCATATATTGGAG-3 For pbi-m07800::hph LAE1_UP-F 5 -GGAAACGACAATCTGCAGCGGCCAGGGTGTCGTGATAGCTTC-3 For pbi-polae1::hph LAE1_UP-R 5 -CAAGAATAAAACGCGCAACTATATCGGGTTGGACAGAATAAGTTT-3 For pbi-polae1::hph LAE1_DN-F 5 -GGTATCGATAAGCTTACCCAAACATAGGCCAAAAAGACTCGT-3 For pbi-polae1::hph LAE1_DN-R 5 -AACACATTGCGGACGGCGACAGTTACCCCATGCTGCCTCTTT-3 For pbi-polae1::hph LAE1_CHK_F 5 -AAACTTATTCTGTCCAACCCGATATAGTTG-3 For pbi-polae1::hph LAE1_CHK_R 5 -ACGAGTCTTTTTGGCCTATGTTTGGGT-3 For pbi-polae1::hph OELAE1_UP_F 5 -GGAAACGACAATCTGGAACCGTTTAGAAGAAGAAGCATTTTTGCC-3 For pbi- OELAE1_UP_R 5 -CAAGAATAAAACGCGGTACAAAACCCAGGTGCGGCAGGCAAG-3 For pbi- OELAE1_DN_F 5 -CTACAAAGTTCGCACATGTCAAAGTATGCCCCCTGAATTTATCC-3 For pbi- OELAE1_DN_R 5 -AACACATTGCGGACGTTTTATCCCAGTAGGTGCTGTGAAGAAGGT-3 For pbi- OELAE_TEF1_F 5 -GGTATCGATAAGCTTGACCAGACAGGCGCCACTCGGCCGGGC-3 For pbi- OELAE_TEF1_R 5 -GTGCGAACTTTGTAGTTCTTTGTAAGA-3 For pbi- OELAE1_CHK_F 5 -CTTGCCTGCCGCACCTGGGTTTTGTAC-3 For pbi- OELAE1_CHK_R 5 -GGATAAATTCAGGGGGCATACTTTGACAT-3 For pbi- QPoTAS_F 5 -GACGCCGAAAGGAACCTGTA-3 For qpcr QPoTAS_R 5 -CGGGGCAAAAAGTGGTGATG-3 For qpcr QPoTub_F 5 -TCTTCATGGTTGGCTTCGCT-3 For qpcr QPoTub_R 5 -CCTGAAGTCAGAAGCAGCCA-3 For qpcr QPoLAE_F 5 -CGGACGAGATGGTTCAACATGCGCC-3 For qpcr QPoLAE_R 5 -GAACTGCCAGTCGATCTCAAATTGTTC-3 For qpcr QPoTAS2_F 5 -CCACCTGGTCGACAGCCGCGGCTGCG-3 For qpcr QPoTAS2_R 5 -CGCGGATGGACTGTGTAGGTGCCATC-3 For qpcr QPoOSM_F 5 -ATGGCGGAATTCGTGCGGGCCCAGATC-3 For qpcr QPoOSM_R 5 -CATAATCTTCTTGATGGCGACATTCTG-3 For qpcr

5 0.25 TAS1 a) b) PoLAE / + + / c) TAS2 d) OSM / + + / Supplementary Figure 1. Transcriptional levels of TAS1, TAS2, PoLAE1 and OSM1 in P. oryzae Kita1 () and its mutants used in this study. Quantitative PCR was conducted with RNAs isolated from cultures grown for 5d in YG medium. The mrna levels were normalized to beta-tublin gene expression levels. All samples were assayed in triplicate and averaged with standard deviation.

6 PoLAE1 min. Supplementary Figure 2. UPLC analysis of metabolites extracted from P. oryzae Kita 1 (), and PoLAE1 deletion and overexpression strains. Each strain was staticcultured for 5 days at 25 C in YG medium. Arrow indicates the TeA peak.

7 SAHA Aza 2,000 µm 2,000 µm 200 µm 200 µm Absorbance 20 µm Absorbance 20 µm 0 µm 0 µm min min. Supplementary Figure 3. UPLC analysis of metabolites extracted from P. oryzae Kita1 cultured with various dosage of suberoylanilide hydroxamic acid (SAHA) or 5-azacytidine (Aza). Each strain was static-cultured for 5 days at 25 C in YG medium. Dotted red line indicates TeA retention time.