Candidate OSC sequences from C. sinensis and A. indica were synthesized (Integrated DNA Technologies) in 2 fragments and recombined into the pYES2 vector (Thermo Fisher Scientific). MaOSC1 was cloned from M. azedarach into pYES2, and the cloned gene for AtLUP5, a previously characterized OSC (36 ), was sourced through TAIR (stock: U16880). Details of cloning methods of OSC candidates (SI Appendix, Table S2 ) are described in SI Appendix . Candidate OSCs were expressed in the yeast strain GIL77 (MATa/α gal2 hem3-6 erg7 ura3-176) (57 ) in 10-mL cultures. Triterpenes were extracted in hexane after saponification and analyzed by GC-MS (SI Appendix ). Purification of the AiOSC1 product is described in SI Appendix . In addition, candidate OSCs were cloned into pEAQ-HT-DEST1 (SI Appendix ) to enable agroinfiltration of N. benthamiana which was performed as previously described (44 (link)).
Pyes2 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PYES2 vector is a plasmid used in molecular biology for the expression of recombinant proteins in yeast. It contains a yeast 2-micron origin of replication, a yeast selectable marker, and multiple cloning sites for the insertion of a gene of interest.
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Lab products found in correlation
Hitrap q, by GE Healthcare (1 mentions)
Hitrap blue, by GE Healthcare (1 mentions)
Hitrap desalting, by GE Healthcare (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Yeast nitrogen base, by BD (1 mentions)
Uracil, by Takara Bio (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
Thermo start master mix, by Thermo Fisher Scientific (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Rneasy plant kit, by Qiagen (1 mentions)
Mint 2 cdna synthesis kit, by Evrogen (1 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (1 mentions)
Sanprep column dna gel extraction kit, by Sangon (1 mentions)
Pegfp plasmid, by Takara Bio (1 mentions)
Laser scanning confocal imaging system, by Olympus (1 mentions)
Optima 3000dv, by PerkinElmer (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
39 protocols using pyes2 vector
1
Heterologous Expression of Triterpenoid OSCs
2
Purification of Dna2 and SUMO Proteins
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Wild-type Dna2, Dna2 E675A, and Dna2 K1080E were expressed as fusions with N-terminal FLAG tag and C-terminal His tag. The protein was expressed from a galactose inducible promoter in yeast, and purified by affinity chromatography using Ni-NTA agarose (Qiagen) and M2 anti-FLAG affinity gel (Sigma)39 (link). Genes coding for the Dna2Δ405N variants (wt, E675A and K1080E), as well as for full-length Dna26K (six lysine residues replaced by alanines) were fused with N-terminal FLAG tag and a C-terminal His tag, cloned into a pYes2 vector (ThermoFisher) and expressed and purified as the full-length proteins. Yeast RPA was expressed in Escherichia coli and purified using HiTrap Blue, HiTrap Desalting and HiTrap Q chromatography columns (GE Healthcare)57 (link). The S. cerevisiae SUMO machinery proteins, including GST-Aos1/Uba2, His-Ubc9, His-FLAG-Smt3-KR (all lysines substituted by arginines, denoted as Smt3 in the text), His-Siz1 (1–465) and His-Siz2 were expressed in E. coli, and purified by affinity and ion exchange chromatography58 (link),59 (link). Yeast nuclear extracts were prepared by a standard procedure60 (link).
3
Investigating Ca2+ Sensitivity in Yeast
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The AtCCX4 cDNA was subcloned in the pYES2 vector (Thermo Fisher Scientific). The csg1, 2 mutants, reported to be sensitive to high Ca2+ (Beeler et al. 1994 (link)), were obtained through EUROSCARF (http://www.euroscarf.de ). The genotypes and the accession numbers of the strains are [MATa, his3, leu2, ura3, met15, ade2, YPL057c::kanMX4] and Y02771 (for csg1), and [MATa, his3, leu2, ura3, met15, ade2, YBR036c::kanMX4] and Y03173 (for csg2), respectively. Transformation using the csg2 strain was performed and the transformants, which grew on Ura-selective medium containing 0.67% yeast nitrogen base (w/v: BD science, USA), 2% sucrose (w/v), complete supplement dropout-URA (770 mg/L: FORMEDIUM, UK), and 2% agar (w/v) were selected as reported previously (Horie et al. 2001 (link)). Ca2+ hypersensitive assays were performed as described previously (Yadav et al. 2015 (link)). Briefly, transformants selected for uracil prototrophy on synthetic complete (SC) medium were grown at 30 °C for 7 d on the modified SC medium without (NH4)sSO4 (MP Biomedicals), supplemented with 2% (w/v) galactose, 5 g/L NH4Cl, and 1 or 100 mM CaCl2.
4
Mutagenesis of OLE1-FAH12 Gene
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Wild type amino acid residues were restored into the OLE1-FAH12 gene by mutagenesis using the QuikChange Site-directed Mutagenesis Kit (Stratagene) and following the manufacture's protocol, and the construct was then cloned into the pYES2 vector (ThermoFisher Scientific, Waltham, MA, USA; pYES2:HO-FAH12). The S49F and Q319H mutations were introduced using the primers S49FMutHOFAH12F/S49FMutHOFAH12R and Q319HMutHOFAH12F/Q319HMutHOFAH12R, respectively (Table 1), generating the constructs pYES2:Q319H-HO-FAH12 (m 1 OLE1), pYES2:S49F-HO-FAH12 (m 2 OLE1) and pYES2:S49F_Q319H-HO-FAH12 (m 3 OLE1).
5
Recombinant Expression of HBP2 Protein
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The gene sequence of HBP2 (Female-specific Histamine-Binding Protein 2, Rhipicephalus appendiculatus, Accesion number U96081) is deposited in the GenBank (NCBI, USA) with accession No. U96081, and it was obtained by requesting gene synthesis from Bioneer (Daejeon, Korea). The gene sequences of Cwp2p (Cell wall protein, the major mannoprotein of the yeast cell wall) is deposited in the GenBank with accession No. NM_001180025, and it was provided by Chungbuk National University (Lee et al. 2017 ).
The recombinant plasmid was constructed based on the pYES2 vector (Invitrogen) and was synthesized in the order of the Cwp2p signal peptide (60 bp), HA tag (27 bp) and histamine binding protein 2 (513 bp), Gly-Ser linker (30 bp) and Cwp2p gene (219 bp). The signal peptide and the stop codon of the histamine binding protein were excluded from the recombinant plasmid. The synthesized gene from HBP2 was provided in a pBHA vector containing the antibiotic Ampicillin resistance gene. After confirming the concentration of the vector, it was transformed into Escherichia coli DH5α to amplify the gene. E. coli DH5α cells were provided by the Real Biotech Corporation (Taipei, Taiwan).
The recombinant plasmid was constructed based on the pYES2 vector (Invitrogen) and was synthesized in the order of the Cwp2p signal peptide (60 bp), HA tag (27 bp) and histamine binding protein 2 (513 bp), Gly-Ser linker (30 bp) and Cwp2p gene (219 bp). The signal peptide and the stop codon of the histamine binding protein were excluded from the recombinant plasmid. The synthesized gene from HBP2 was provided in a pBHA vector containing the antibiotic Ampicillin resistance gene. After confirming the concentration of the vector, it was transformed into Escherichia coli DH5α to amplify the gene. E. coli DH5α cells were provided by the Real Biotech Corporation (Taipei, Taiwan).
6
Yeast-based azole resistance assay
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Full-length cDNA of FgSR was cloned into the pYES2 vector (Invitrogen Co., CA, USA), and transformed into the corresponding yeast mutant. The wild-type strain BY4741 and the mutant transformed with an empty pYES2 vector were used as controls. For complementation assays, the yeast transformants were grown at 30 °C on YPRG (1% yeast extract, 2% bactopeptone, 2% galactose) medium supplied with an azole compound. The experiments were independently repeated three times.
7
Functional Characterization of Aoerg19 Mutant
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The erg19 (Y41208) mutant was purchased from EUROSCARF1 and the yeast strain BY4741 was the wild-type control. Plasmid pYES2-Aoerg19 contains Aoerg19 full-length coding sequence (CDS) under the control of the GAL1 promoter. To construct pYES2-Aoerg19, the CDS of Aoerg19 was amplified using PCR (the primer sequences are listed in Supplementary Table S2 ) and HindIII and EcoRI restriction sites were added to facilitate cloning of the fragments into the pYES2 vector (Invitrogen, Shanghai, China). The recombinant plasmid was sequenced. Then, the pYES2-Aoerg19 construct was transformed into the yeast mutant. The control and transformants were grown on YPD (1% yeast extract, 2% Bacto-Peptone, 2% glucose), and YPG (1% yeast extract, 2% Bacto-Peptone, 2% galactose) to assess the phenotypes.
8
Purification of DNA Repair Proteins
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Wild-type yDna2 as well yDna2 E675A and yDna2 K1080E variants were expressed and purified as described previously (Levikova et al. 2013 (link)). The construct for the expression of the yDna2 ΔN variant was prepared by cloning the yDNA2 sequence lacking the first 1215 residues (corresponding to 405 amino acids) between the 5′-terminal Flag tag and the 3′-terminal 6His tag into BamHI and EcoRI sites in a pYes2 vector (Invitrogen). The truncated protein was expressed and purified as the full-length protein. Wild-type hDNA2, hDNA2 K654E, and hDNA2 K654R were prepared as described previously (Pinto et al. 2016 (link)). No difference was observed between the hDNA2 K654E and hDNA2 K654R variants in the experiments presented in this report (data not shown). Wild-type BLM, WRN, BLM K695A, and WRN K577M were expressed and purified as described (Pinto et al. 2016 (link)). Sgs1, Top3–Rmi1, and Mre11–Rad50–Xrs2 were expressed and purified as described previously (Cejka and Kowalczykowski 2010 (link); Cejka et al. 2010 (link); Cannavo and Cejka 2014 (link)). The yRPA and hRPA proteins were expressed and purified as described (Henricksen et al. 1994 (link); Kantake et al. 2003 (link)).
9
Complementation of Yeast Mtg2 Deletion Mutant
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The yeast wild type strain BY4741 and the MTG2 deletion mutant BY4741ΔMTG2 was provided by EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/ ). The full-length BcMtg2 cDNA was amplified from total RNA of B05.10 using the primer pair P35/P36 (Table S1 ). The PCR product was digested with BamHI and XbaI and cloned into the pYES2 vector (Invitrogen), and transformed into the yeast mutant BY4741ΔMTG2. Yeast transformants were selected on synthetic medium lacking uracil (Clontech). Additionally, the wild-type strain BY4741 and BY4741ΔMTG2 mutant transformed with empty pYES2 vector were used as controls. In the complementation assays, the yeast transformants were grown at 30 °C for 4 days on YPRG medium (1% yeast extract, 2% peptone, 1% galactose, 1% raffinose, 2% agar) modified with various stress agents including Congo red, NaCl, and H2O2 at concentrations shown in figure legends. The experiments were repeated three times independently.
10
Functional Complementation of Scper1 Mutant
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The full length of MoPER1 cDNA were amplified using primers FL21/FL22 (Additional file 3 : Table S1). The PCR product was disgested with HindIII/XhoI and cloned into the pYES2 vector (Invitrogen, Shanghai, China), and transformed into the ∆Scper1 mutant. Colonies were selected on SD medium without uracil. As a control, the ∆Scper1 mutant were transformed with the empty pYES2. Yeast cells were incubated on liquid YPD medium (2% glucose, 2% peptone, and 1% yeast extract), and aliquots (5 μl) of 10-fold serial dilution were grown in SD (galactose) and SD-CFW (galactose+ 20 μg/ml CFW) plates at 30 °C for 4 days and photographed.
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