All the primers used in this study are listed in S1 Table . The PCR-amplified regions and ligation junctions were confirmed by sequence analysis for all vectors.
Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine. Sulfonylurea herbicides, e.g. CS, bind reversibly to the ALS-FAD-thiamine pyrophosphate-Mg2+-decarboxylated pyruvate complex and also compete for the second pyruvate binding site in ALS [29 (link)]. Mutagenesis at these sites in ALS confers tolerance to sulfonylurea herbicides. For example, mALSs containing mutations at P197H, R198S, and W574L conferred resistance to CS in Arabidopsis [30 (link)]. To generate a CS-resistance marker gene for use in M. polymorpha, its ALS sequence was mutagenized to contain corresponding mutations (P207S/R208S/W582L) to those described above, as follows. First, M. polymorpha ALS cDNA was amplified by RT-PCR with the primer set ALS-P1 and ALS-P2 using first-strand cDNA synthesized from a 10-day-old thallus. The resultant ALS cDNA was cloned into the pENTR/D-TOPO vector (Life Technologies, Gaithersburg, MD, USA). A point mutation at W582L in the ALS sequence was introduced by PCR-based site-directed mutagenesis with the primer pair ALS-W582L-F and ALS-W582L-R, using the ALS cDNA plasmid as the template. The PCR product was digested with DpnI, and the digested PCR product was transformed into Escherichia coli competent cells (DH5α). The mutation in ALS was confirmed by sequencing the resultant plasmid. The mALS was prepared by repeating the same procedure with the primer set ALS-P207S-R208S-F and ALS-P207S-R208S-F using the W582L-mutated ALS as a template. The mALS cDNA was cloned into pCAMBIA1300 to replace the hpt gene, generating the plasmid pCAMBIA1300-mALS.
The marker cassettes pro35S×2:hpt:ter35S, pro35S×2:aacC1:ter35S, pro35S×2:mALS:ter35S, and pro35S×2:nptII:ter35S were prepared by PCR with the primer set Marker_LinF and Marker_RinF using pCAMBIA1300, pPZP221, pCAMBIA1300-mALS, and pCAMBIA2301, respectively, as the template. The marker cassettes were cloned into EcoRI-digested pGWB400 [31 (link)] using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA) to replace the proNOS:nptII:terNOS cassette, generating pMpGWB100, pMpGWB200, pMpGWB300, and pMpGWB400, respectively. The E. coli DH5α cells harboring these plasmids were selected on LB medium containing 100 mg/l spectinomycin.
The Gateway-compatible binary vectors pMpGWBs were constructed using the same strategy as that used by Nakagawa et al. to construct pGWBs [31 (link),32 (link)]. Detailed procedures of pMpGWBs construction are described inS1 Text . The E. coli (strain DB3.1) cells harboring pMpGWBs were selected on LB media containing 100 mg/l spectinomycin and 30 mg/l chloramphenicol.
The vector for expression of TagRFP-LTI6b was constructed as follows: the GFP-LTI6b coding sequence was PCR-amplified using the primers GFP-LTI6b_GW_L1 and GFP-LTI6b_GW_R1 and cloned into pENTR/D-TOPO. The GFP coding sequence flanked by two NotI sites in this plasmid was replaced with the TagRFP-coding sequence with two similarly flanking NotI sites, which was PCR-amplified using the primers pENTRD_NotI_TagRFP_F and TagRFP_NotI_R. This plasmid was used for LR recombination with pMpGWB103 to generate pMpGWB103-TagRFP-LTI6b.
To construct the vector for expression of SP-GFP-HDEL, the SP-GFP-HDEL-coding sequence [33 ] was PCR-amplified using the primers SP_Lc and HDEL_R and cloned into pENTR/D-TOPO. The resulting vector was used for LR recombination with pMpGWB303 to generate pMpGWB303-SP-GFP-HDEL.
To construct the vectors for expression of tdTomato-NLS and GUS under the endogenous ELONGATION FACTOR1α promoter (proEF), the 1,729-bp promoter sequence of MpEF1α [34 (link)] was amplified by PCR using the primers MpEF-P_L1 and MpEF-P_R1 and cloned into pENTR/D-TOPO. The resulting vector was used for LR recombination with pMpGWB216 and pMpGWB404 to generate pMpGWB216-proEF and pMpGWB404-proEF, respectively.
Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine. Sulfonylurea herbicides, e.g. CS, bind reversibly to the ALS-FAD-thiamine pyrophosphate-Mg2+-decarboxylated pyruvate complex and also compete for the second pyruvate binding site in ALS [29 (link)]. Mutagenesis at these sites in ALS confers tolerance to sulfonylurea herbicides. For example, mALSs containing mutations at P197H, R198S, and W574L conferred resistance to CS in Arabidopsis [30 (link)]. To generate a CS-resistance marker gene for use in M. polymorpha, its ALS sequence was mutagenized to contain corresponding mutations (P207S/R208S/W582L) to those described above, as follows. First, M. polymorpha ALS cDNA was amplified by RT-PCR with the primer set ALS-P1 and ALS-P2 using first-strand cDNA synthesized from a 10-day-old thallus. The resultant ALS cDNA was cloned into the pENTR/D-TOPO vector (Life Technologies, Gaithersburg, MD, USA). A point mutation at W582L in the ALS sequence was introduced by PCR-based site-directed mutagenesis with the primer pair ALS-W582L-F and ALS-W582L-R, using the ALS cDNA plasmid as the template. The PCR product was digested with DpnI, and the digested PCR product was transformed into Escherichia coli competent cells (DH5α). The mutation in ALS was confirmed by sequencing the resultant plasmid. The mALS was prepared by repeating the same procedure with the primer set ALS-P207S-R208S-F and ALS-P207S-R208S-F using the W582L-mutated ALS as a template. The mALS cDNA was cloned into pCAMBIA1300 to replace the hpt gene, generating the plasmid pCAMBIA1300-mALS.
The marker cassettes pro35S×2:hpt:ter35S, pro35S×2:aacC1:ter35S, pro35S×2:mALS:ter35S, and pro35S×2:nptII:ter35S were prepared by PCR with the primer set Marker_LinF and Marker_RinF using pCAMBIA1300, pPZP221, pCAMBIA1300-mALS, and pCAMBIA2301, respectively, as the template. The marker cassettes were cloned into EcoRI-digested pGWB400 [31 (link)] using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA) to replace the proNOS:nptII:terNOS cassette, generating pMpGWB100, pMpGWB200, pMpGWB300, and pMpGWB400, respectively. The E. coli DH5α cells harboring these plasmids were selected on LB medium containing 100 mg/l spectinomycin.
The Gateway-compatible binary vectors pMpGWBs were constructed using the same strategy as that used by Nakagawa et al. to construct pGWBs [31 (link),32 (link)]. Detailed procedures of pMpGWBs construction are described in
The vector for expression of TagRFP-LTI6b was constructed as follows: the GFP-LTI6b coding sequence was PCR-amplified using the primers GFP-LTI6b_GW_L1 and GFP-LTI6b_GW_R1 and cloned into pENTR/D-TOPO. The GFP coding sequence flanked by two NotI sites in this plasmid was replaced with the TagRFP-coding sequence with two similarly flanking NotI sites, which was PCR-amplified using the primers pENTRD_NotI_TagRFP_F and TagRFP_NotI_R. This plasmid was used for LR recombination with pMpGWB103 to generate pMpGWB103-TagRFP-LTI6b.
To construct the vector for expression of SP-GFP-HDEL, the SP-GFP-HDEL-coding sequence [33 ] was PCR-amplified using the primers SP_Lc and HDEL_R and cloned into pENTR/D-TOPO. The resulting vector was used for LR recombination with pMpGWB303 to generate pMpGWB303-SP-GFP-HDEL.
To construct the vectors for expression of tdTomato-NLS and GUS under the endogenous ELONGATION FACTOR1α promoter (proEF), the 1,729-bp promoter sequence of MpEF1α [34 (link)] was amplified by PCR using the primers MpEF-P_L1 and MpEF-P_R1 and cloned into pENTR/D-TOPO. The resulting vector was used for LR recombination with pMpGWB216 and pMpGWB404 to generate pMpGWB216-proEF and pMpGWB404-proEF, respectively.
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