Primers for the PCR and Qpcr

Table information of transformed lines. Two types of promoter, i.e. the CaMV 35S promoter and PpCPS/KS promoter, were used to drive the heterologous transcription of sclareol biosynthetic genes. KOF1, KOF4, KOF6 and WTF2 were the CaMV 35S promoter type whereas WT-pBK3-14-1 and WT-pBK3-14-4 used PpCPS/KS promoter to drive sclareol biosynthetic genes.

Transformant / Construct / Targeting locus / Background
KOF1 / pUNI33-SsLPPS-2A-SsSS / illegitimate integration / PpCPS/KS KO
KOF4 / pUNI33-SsLPPS-2A-SsSS / illegitimate integration / PpCPS/KS KO
KOF6 / pUNI33-SsLPPS-2A-SsSS / illegitimate integration / PpCPS/KS KO
WTF2 / pUNI33-SsLPPS-2A-SsSS / illegitimate integration / WT
WT-pBK3-14-1 / pBK3-SsLPPS-2A-SsSS / PpCPS/KS locus (expected) / WT
WT-pBK3-14-4 / pBK3-SsLPPS-2A-SsSS / PpCPS/KS locus(expected) / WT

Primers for the PCR and qPCR

Target name: 1-Deoxy-D-xylulose 5-phosphate synthase

Abbreviation: DXS22

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s22_310V6.1

Primer (forward) and sequence: P70, GGAGTGAGGTTGTTGTTGTTGTTG

Primer (reverse): P71, CAAGGCAGCCATAGTGAGAAAC

Gel purified and sequenced: Yes

SNPs: No

Target name: 1-Deoxy-D-xylulose 5-phosphate synthase

Abbreviation: DXS139

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s139_98V6.1

Primer (forward) and sequence: P72, CGCTGTCCTTCTCTGTATTTGC

Primer (reverse): P73, GGAGCCGAGACGAACAATC

Gel purified and sequenced: Yes

SNPs: No

Target name: 1-Deoxy-D-xylulose 5-phosphate synthase

Abbreviation: DXS11

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s11_262V6.1

Primer (forward) and sequence: P74, CTTACCAACAGTCCAGTCTACCG

Primer (reverse): P75, ATGATGAGCATTCCACACAAGC

Gel purified and sequenced: Yes

SNPs: No

Target name: 1-Deoxy-D-xylulose 5-phosphate synthase

Abbreviation: DXS42

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s42_48V6.1

Primer (forward) and sequence: P76, GCACTCGCTTCACAGTCCTC

Primer (reverse): P77, TGAACTAAAGCCTGCTGACCA

Gel purified and sequenced: Yes

SNPs: No

Target name: 1-Deoxy-D-xylulose-5-phosphate reductoisomerase

Abbreviation: DXS62

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s62_157V6.4

Primer (forward) and sequence: P92, GGTCTCAGTTGCTTCCGAAATAC

Primer (reverse): P93, GAAAGAAAAGGAGAGCGTTGG

Gel purified and sequenced: Yes

SNPs: No

Target name: 1-Deoxy-D-xylulose-5-phosphate reductoisomerase

Abbreviation: DXR73

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s73_243V6.2

Primer (forward) and sequence: P94, ATGGATGGGCACGAGACTACAC

Primer (reverse): P95, CATTGTCTACCAGGAGTTCAGTTTC

Gel purified and sequenced: Yes

SNPs: No

Target name: 2-C-Methyl-d-erythritol 4-phosphate cytidylyltransferase

Abbreviation: CMS90

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s90_44V6.2

Primer (forward) and sequence: P159, GATGGTGTCTGAGGTAGTAGTAGGCA

Primer (reverse): P160, AGGCGTTTTGATACAATGGTGATA

Gel purified and sequenced: Yes

SNPs: No

Target name: 2-C-Methyl-d-erythritol 4-phosphate cytidylyltransferase

Abbreviation: CMS211

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s211_107V6.1

Primer (forward) and sequence: P157, CGACGAGCGTGAAGCAGG

Primer (reverse): P158, ACGACAAATGTGGTCCGAGGT

Gel purified and sequenced: Yes

SNPs: No

Target name: 4-Diphosphocytidyl-2-c-methyl-d-erythritol kinase

Abbreviation: CMK149

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s149_51V6.2

Primer (forward) and sequence: P163, TCGTCTCGGACACAATGTAGTATG

Primer (reverse): P164, GCCTTCAGAATTTTGATCCAGTG

Gel purified and sequenced: Yes

SNPs: No

Target name: 2-C-methyl-d-erythritol -cyclodiphosphate synthase

Abbreviation: MCS312

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s312_21V6.1

Primer (forward) and sequence: P161, TGCTTGTTGTTGATTTCATCGTAG

Primer (reverse): P162, GACCACACCTTAGACTCAAACCAC

Gel purified and sequenced: Yes

SNPs: No

Target name: Hydroxymethylbutenyl 4-diphosphate synthase

Abbreviation: HDS20

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s20_67V6.1

Primer (forward) and sequence: P149, AATGTGCCCTTGGTTTCTTCTTAG

Primer (reverse): P150, TATCGTCTCCACACAGCAACTCTAT

Gel purified and sequenced: Yes

SNPs: No

Target name: Hydroxymethylbutenyl 4-diphosphate synthase

Abbreviation: HDS71

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s71_230V6.1

Primer (forward) and sequence: P147, TTGGGAAGGATAGGAAACGCTC

Primer (reverse): P148, ACGGAAATGAAATGAATTGTTGTG

Gel purified and sequenced: Yes

SNPs: No

Target name: Hydroxymethylbutenyl 4-diphosphate synthase

Abbreviation: HDS85

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s85_12V6.1

Primer (forward) and sequence: P151, CGAATGCCGTGATGTCTGC

Primer (reverse): P152, GAACGCCAGCGATTAGCAC

Gel purified and sequenced: Yes

SNPs: No

Target name: 4-Hydroxy-3-methyl-but-2-enyl-diphosphate reductase

Abbreviation: HDR32

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s32_278V6.1

Primer (forward) and sequence: P106, CTCGTCATCATCTCACCACCATA

Primer (reverse): P107, AAGGGAGAGCGGAGGACAA

Gel purified and sequenced: Yes

SNPs: No

Target name: 4-Hydroxy-3-methyl-but-2-enyl-diphosphate reductase

Abbreviation: HDR210

Function: Terpenoids biosynthesis in the MEP pathway

Cosmoss accession no. : Pp1s210_40V6.1

Primer (forward) and sequence: P108, CTGCCCTCAGCCTATCTTCCA

Primer (reverse): P109, CCAACCAGGACTATCACCAGG

Gel purified and sequenced: Yes

SNPs: No

Target name: Isopentenyl diphosphate isomerase

Abbreviation: IDI28

Function: Terpenoids biosynthesis in the MEP and MVA pathway

Cosmoss accession no. : Pp1s28_399V6.1

Primer (forward) and sequence: P110, GGATAAATACCTTGGTGTCAGAAA

Primer (reverse): P111, GTAGTCCACTTCATGCTCGCC

Gel purified and sequenced: Yes

SNPs: No

Target name: 3-Hydroxy-3-methylglutaryl-coenzyme A synthase

Abbreviation: HMGS154

Function: Terpenoids biosynthesis in the MVA pathway

Cosmoss accession no. : Pp1s154_103V6.1

Primer (forward) and sequence: P153, AGATGAACAGACTGTAACAATGTGAAG

Primer (reverse): P154, TGAAGCAAGTCCACTACCATACGA

Gel purified and sequenced: Yes

SNPs: No

Target name: 3-Hydroxy-3-methylglutaryl-coenzyme A synthase

Abbreviation: HMGS180

Function: Terpenoids biosynthesis in the MVA pathway

Cosmoss accession no. : Pp1s180_129V6.1

Primer (forward) and sequence: P155, CAAACTGGATTCTCGCATAGCA

Primer (reverse): P156, GGGCGTAGAGTAGACTTGGAGC

Gel purified and sequenced: Yes

SNPs: No

Target name: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase

Abbreviation: HMGR1

Function: Terpenoids biosynthesis in the MVA pathway

Cosmoss accession no. : Pp1s1_155V6.1

Primer (forward) and sequence: P100, CCATTCGTCCAACATTTTGATGA

Primer (reverse): P101, GGGAGCAAGCAAGTTTAACACTG

Gel purified and sequenced: Yes

SNPs: No

Target name: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase

Abbreviation: HMGR61

Function: Terpenoids biosynthesis in the MVA pathway

Cosmoss accession no. : Pp1s61_10V6.1

Primer (forward) and sequence: P100, AGAGCACCTTAGACATCTTAGACCAA

Primer (reverse): P101, AGGAACATCGCCATTGCTAACC

Gel purified and sequenced: Yes

SNPs: No

Target name: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase

Abbreviation: HMGR139

Function: Terpenoids biosynthesis in the MVA pathway

Cosmoss accession no. : Pp1s139_2V6.1

Primer (forward) and sequence: P102, ATAGCCTCTAACAAGCAAACCTCG

Primer (reverse): P103, GTGCCTCGTGGAACACTGAAA

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranyl diphosphate synthase

Abbreviation: GPS8

Function: Terpenoids biosynthesis for the GPP biogenesis

Cosmoss accession no. : Pp1s8_131V6.2

Primer (forward) and sequence: P130, ATGGCAACCGACCTCCTCC

Primer (reverse): P131, AGCCGATTGGCAACAACCTG

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranyl diphosphate synthase

Abbreviation: GPS55

Function: Terpenoids biosynthesis for the GPP biogenesis

Cosmoss accession no. : Pp1s55_266V6.1

Primer (forward) and sequence: P132, TTGGAACACTTGGTCACAGGG

Primer (reverse): P133, CTAATACGGCAATGGATTTACAGC

Gel purified and sequenced: Yes

SNPs: No

Target name: Farnesyl diphosphate synthase

Abbreviation: FPS101

Function: Terpenoids biosynthesis for the FPP biogenesis

Cosmoss accession no. : Pp1s101_225V6.1

Primer (forward) and sequence: P134, CAGGACGACTATCTTGACTGTTACG

Primer (reverse): P135, TTCCTTCAATAACTGTTTCTGGG

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranylgeranyl diphosphate synthase

Abbreviation: GGPPS3

Function: Terpenoids biosynthesis for the GGPP biogenesis

Cosmoss accession no. : Pp1s3_343V6.1

Primer (forward) and sequence: P112, GCTTCTCTCCCTTCGTCTCTCATAG

Primer (reverse): P113, CCAGCACACCACACAACTCAAC

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranylgeranyl diphosphate synthase

Abbreviation: GGPPS167

Function: Terpenoids biosynthesis for the GGPP biogenesis

Cosmoss accession no. : Pp1s167_155V6.1

Primer (forward) and sequence: P116, AAGAGTTTGGAGTTTGATTTGGAC

Primer (reverse): P117, CTAACAAGGAATAACGCATAGCC

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranylgeranyl diphosphate reductase

Abbreviation: GGR20

Function: Chlorophyll biosynthesis

Cosmoss accession no. : Pp1s20_284V6.1

Primer (forward) and sequence: P120, AGGGTTCTTATCCGTTGATACTGC

Primer (reverse): P121, GATGTCCTGTTTTACAAGTGAATGAG

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranylgeranyl diphosphate reductase

Abbreviation: GGR100

Function: Chlorophyll biosynthesis

Cosmoss accession no. : Pp1s100_107V6.1

Primer (forward) and sequence: P118, CAACCAGTGCTGAGATGATTGTC

Primer (reverse): P119, CTTGAAGTAAGCCGATTGAAATG

Gel purified and sequenced: Yes

SNPs: No

Target name: Geranylgeranyl diphosphate reductase

Abbreviation: GGR425

Function: Chlorophyll biosynthesis

Cosmoss accession no. : Pp1s425_20V6.1

Primer (forward) and sequence: P122, GGATTAGGAATGCTAGATAATACTTTGC

Primer (reverse): P123, AATCAACGCTACTCAACAACTTCG

Gel purified and sequenced: Yes

SNPs: No

Target name: Phytoene synthase

Abbreviation: PSY134

Function: Carotenoids biosynthesis

Cosmoss accession no. : Pp1s134_62V6.1

Primer (forward) and sequence: P126, AGTCGTGAAGTTACCAAGCCTACC

Primer (reverse): P127, GTGGAGCTAAACAGGAGTGAATGA

Gel purified and sequenced: Yes

SNPs: No

Target name: Phytoene synthase

Abbreviation: PSY196

Function: Carotenoids biosynthesis

Cosmoss accession no. : Pp1s196_120V6.2

Primer (forward) and sequence: P124, TCTTACAATCTGCTGGGATGGTG

Primer (reverse): P125, CCTCAAGTGCCTGGACTGGTCA

Gel purified and sequenced: Yes

SNPs: No

Target name: Phytoene synthase

Abbreviation: PSY323

Function: Carotenoids biosynthesis

Cosmoss accession no. : Pp1s323_70V6.1

Primer (forward) and sequence: P128, CGGCACAATGAGAGTGTTACTTG

Primer (reverse): P129, ATGTGAGTGCGGTGTGATGTTAC

Gel purified and sequenced: Yes

SNPs: No

Target name: Copalyl pyrophosphate synthase/kaurene synthase

Abbreviation: PpCPS/KS

Function: Diterpene kaurene synthase in P. patens

Cosmoss accession no. : Pp1s130_5V6.1

Primer (forward) and sequence: P136, GCTTCCAGCACCTTGATACAGA

Primer (reverse): P137, CGACGCTTCTTTAGGCATTGA

Gel purified and sequenced: Yes

SNPs: No

Target name: 13-Labden-8,15-diol pyrophosphate synthase

Abbreviation: TPSsa3

Function: Diterpene 13-labden-8,15-diol pyrophosphate synthase in Salvia sclarea

Genbank accession no. : JN133923.1

Primer (forward) and sequence: P88, GGGAGCATAAAGGATAAGGAGC

Primer (reverse): P89, GTCGTGGTAGGCATCGTAGTAGAA

Gel purified and sequenced: Yes

SNPs: No

Target name: Sclareol synthase

Abbreviation: TPS1132

Function: Diterpene sclareol synthase in Salvia sclarea

Genbank accession no. : JN133922.1

Primer (forward) and sequence: P90, TGCCTACCACGACGATGAGAC

Primer (reverse): P91, AGCGACATAGGTCCTGGGTTAG

Gel purified and sequenced: Yes

SNPs: No

Target name: Polyubiquitin

Abbreviation: Ubi

Function: Polyubiquitin biosynthesis in P. patens

Cosmoss accession no. : Pp1s17_269V6.2

Primer (forward) and sequence: P80, CAAACTTGGCTGATTCATTCCG

Primer (reverse): P81, CGAACTACTGCGTCTAATGCTGA

Gel purified and sequenced: Yes

SNPs: No

Description of the vectors used:

Background: pCMABIA 1300

Background: pCMABIA 1300

Background: pCMABIA 1300, but largely changed. See construction steps in “Materials and Methods” part.

In-house starter pLIFE33 binary vector is derived from pCAMBIA1300, it has a USER (Uracil-Specific Excision Reagent) cassette (Geu-Flores, Nour-Eldin et al. 2007) that is driven by CaMV 35S promoter. USER cassette (GCTGAGGCTTAATTAAGGATCCTTAATTAAACCTCAGC) has two PacI and two BbvCI restriction site for cloning.

pCL755, which was used to knockout PpCPS/KS gene, was constructed as following steps:

1. Part of PpCPS/KS gene was amplified by nested PCR (the sequence shown in the supplementary materials);

2. The PCR product was cloned into pDONR201 and digested with XhoI;

3. The nptII cassette was obtained from pMBL6 by XhoI digestion and ligated

into the backbone vector from step 2. The resulting vector was referred to as pCL755