Supplemental Materials and Methods S1.
ZeBaTA Vector Construction
Standard molecular manipulations (Sambrook and Russell, 2001) were performed to make the constructs. All plasmids derived from PCR products were verified by sequencing. All primers used for making constructs, insertions sequencing or RT-PCR are shown in Supplemental Table S1.
pGXT
A 721-bp DNA fragment containing a cassette of BamHI-XcmI-ccdB-XcmI-BamHI was amplified using plasmid pANDAmini (Miki and Shimamoto, 2004) as template with primers XT-F and XT-R. The fragment was inserted in the pGEM®-T easy vector (www.promega.com). The resulting plasmid was designated pGXT.
pXSN, pXSN-FLAG, pXSN-HA, and pXSN-Myc
Plasmid pA7-NYFP (Chen et al., 2006b) was digested with SpeI/SacI (blunted with T4 DNA polymerase) to delete the N-terminal YFP coding fragment. The re-circularized plasmid was digested with XhoI/SmaI (blunted with Klenow) to delete non-necessary sequences between the CaMV 35S promoter and the noparine synthase gene terminator (Tnos). The resulting plasmid was designated pA7-Nos.
A BamHI-digested fragment containing the XcmI-ccdB-XcmI cassette from plasmid pGXT was inserted into the BamHI site in between the CaMV 35S promoter and the noparine synthase gene terminator of plasmid pA7-Nos. The resulting plasmid was designated pXSN.
PCR fragments containing cassettes of BamHI-FLAG-XcmI-ccdB-XcmI-BamHI, BamHI-HA-XcmI-ccdB-XcmI-BamHI, or BamHI-Myc-XcmI-ccdB-XcmI-BamHI were amplified using plasmid pGXT as template with primer pairs XT-Flag/XT-R, XT-HaF/XT-R, or XT-MycF/XT-R, respectively. The fragments were digested with BamHI and then inserted into the BamHI site in between the CaMV 35S promoter and the noparine synthase gene terminator of plasmid pA7-Nos. The resulting plasmids were designated pXSN-FLAG, pXSN-HA, and pXSN-Myc, respectively.
pXUN, pXUN-FLAG, pXUN-HA, and pXUN-Myc
Plasmid pUbiGUS (Chen et al., 2006b) was digested with BamHI/SacI (blunted with T4 DNA polymerase) to delete the gus gene. The re-circularized plasmid was digested with XcmI, then blunted by deleting the single base of the XcmI-digested ends using T4 DNA polymerase. The deletion of the single base thus blocks the XcmI recognition site of maize ubiquitin-1 promoter. The resulting plasmid was designated pBUN.
A BamHI-digested fragment containing the XcmI-ccdB-XcmI cassette from plasmid pGXT was inserted into the BamHI site of plasmid pBUN. The resulting plasmid was designated pXUN.
PCR fragments containing cassettes of BamHI-FLAG-XcmI-ccdB-XcmI-BamHI, BamHI-HA-XcmI-ccdB-XcmI-BamHI, or BamHI-Myc-XcmI-ccdB-XcmI-BamHI were amplified using plasmid pGXT as template with primer pairs XT-Flag/XT-R, XT-HaF/XT-R, or XT-MycF/XT-R, respectively. The fragments were digested with BamHI and then inserted into the BamHI site of plasmid pBUN. The resulting plasmids were designated pXUN-FLAG, pXUN-HA, and pXUN-Myc, respectively.
pXUN-osaMIR528
Three PCR fragments were amplified using plasmid pNW55 (Warthmann et al., 2008) as template with primer pairs XT-ami-I-F/XT-ami-I-R, and XT-ami-III-F/XT-ami-III-R, and using plasmid pGXT as template with primer pair XT-ami-II-F/XT-ami-II-R, respectively. Over lapping PCR was performed using the three amplified fragments as template with primer pair XT-ami-I-F/XT-ami-III-R. The resultant fragment containing the cassette of 5ʹ Osa-MIR528 stemloop backbone-XcmI-ccdB-XcmI-3ʹ Osa-MIR528 stemloop backbone was digested with BamHI, and then inserted into the BamHI site of plasmid pBUN. The resulting plasmids were designated pXUN-osaMIR528.
pX-DG and pX-DR
A ScaI-digested fragment containing a cassette of CaMV 35S promoter-gfp-Tnos from plasmid pGDG (Goodin et al., 2002), and a ScaI-digested fragment containing a cassette of CaMV 35S promoter-DsRed-Tnos from plasmid pGDR (Goodin et al., 2002), were inserted into the PvuII site of plasmid pBluescript II KS (www.stratagene.com), respectively. The resulting plasmids were designated pBDG and pBDR, respectively.
A BamHI-digested fragment containing the XcmI-ccdB-XcmI cassette from plasmid pGXT was inserted into the BamHI site of plasmid pBDG and pBDR, respectively. The resulting plasmids were designated pXDG and pXDR, respectively.
pXGUS-P and pXGFP-P
PCR fragments of GUS and GFP coding sequences were amplified using pUbiGUS (Chen et al., 2006b), and pGDG (Goodin et al., 2002) as templates with primer pairs GusT-F/GusT-R and RSGFP-5/ RSGFP-3, respectively. The fragments were digested with BamHI and SacI, and then inserted into the BamHI/SacI site of plasmid pBI221 (Chen et al., 2003) to form intermediate vectors pBI221-GUST and pBI221-GFPT. The BamHI/EcoRI digested fragments containing the gus-Tnos or gfp-Tnos cassettes, respectively, from pBI221-GUST and pBI221-GFPT were inserted into the BamHI/EcoRI site of plasmid pBluescript II KS. The resulting plasmids were designated pBGUS-T and pBGFP-T, respectively.
A BamHI-digested fragment containing the XcmI-ccdB-XcmI cassette from plasmid pGXT was inserted into the BamHI site of plasmid pBGUS-T and pBGFP-T, respectively. The resulting plasmids were designated pXGUS-P and pXGFP-P, respectively.
pCXSN, pCXSN-FLAG, pCXSN-HA, and pCXSN-Myc
The binary plasmid pCAMBIA1300 (www.pcambia.org) was used as backbone to construct ZeBaTA vectors for Agrobacterium-mediated stable transformation of plants. PCR-based method was used to modified pCAMBIA1300 to block two XcmI recognition sites within the kanamycin resistance gene and the pVS1 Sta element by using primers with site-specific substitutions.
Two DNA fragments were amplified using plasmid pCAMBIA1300 as template with primer pairs KM-F1/KM-R1 and KM-F2/KM-R2, respectively. Overlapping PCR was performed using the two amplified fragments as template with primer pair KM-F1/KM-R2. The resultant fragment containing the modified kanamycin resistance gene was digested with SacII/NsiI, and then inserted into the SacII/NsiI site of pCAMBIA1300 to replace the fragment which contains the original kanamycin resistance gene. The resulting plasmid was digested with HindIII/NheI. The small HindIII/NheI-digested band was digested again with NdeI, and the HindIII/NdeI-fragment was recovered. To modify the pVS1 Sta element, a DNA fragment was amplified using plasmid CAMBIA1300 as template with primer pair STAM-F/STAM-R. The fragment was digested with NdeI/NheI, and a triple-fragment ligation was conducted by ligating the NdeI/NheI-digested PCR fragment, the big HindIII/NheI-digested band and the HindIII/NdeI-fragment. The resulting plasmid was digested with XcmI, then blunted by deleting the single base of the XcmI-digested ends using T4 DNA polymerase. The deletion of the single base thus blocks the last XcmI recognition site of the backbone of pCAMBIA1300. The resulting plasmid was designated pCAMBIA1300-XcmIΔ.
A HindIII/EcoRI digested fragment containing the CaMV 35S promoter and the noparine synthase gene terminator from plasmid pA7-Nos was inserted into the HindIII/EcoRI site of plasmid pCAMBIA1300-XcmIΔ. The resulting plasmid was designated pCSN.
The BamHI-digested fragments containing XcmI-ccdB-XcmI, FLAG-XcmI-ccdB-XcmI, HA-XcmI-ccdB-XcmI, or Myc-XcmI-ccdB-XcmI from plasmid pGXT, pXSN-FLAG, pXSN-HA, and pXSN-Myc were inserted respectively into the BamHI site in between the CaMV 35S promoter and the noparine synthase gene terminator of pCSN. The resulting plasmids were designated pCXSN, pCXSN-FLAG, pCXSN-HA, and pCXSN-Myc, respectively.
pCXUN, pCXUN-FLAG, pCXUN-HA, and pCXUN-Myc
A PvuII/HindIII-digested fragment containing the maize ubiquitin-1 promoter and the noparine synthase gene terminator from plasmid pBUN was inserted into the SmaI/HindIII site of plasmid pCAMBIA1300-XcmIΔ. The resulting plasmid was designated pCUN.
The BamHI-digested fragments containing XcmI-ccdB-XcmI, FLAG-XcmI-ccdB-XcmI, HA-XcmI-ccdB-XcmI, or Myc-XcmI-ccdB-XcmI from plasmid pGXT, pXSN-FLAG, pXSN-HA, and pXSN-Myc were inserted respectively into the BamHI site in between the maize ubiquitin-1 promoter and the noparine synthase gene terminator of pCUN. The resulting plasmids were designated pCXUN, pCXUN-FLAG, pCXUN-HA, and pCXUN-Myc, respectively.
pCXUN-osaMIR528
The overlapping PCR fragment containing the cassette of 5ʹ Osa-MIR528 stemloop backbone-XcmI-ccdB-XcmI-3ʹ Osa-MIR528 stemloop backbone was digested with BamHI, and then inserted into the BamHI site of plasmid pCUN. The resulting plasmids were designated pCXUN-osaMIR528.
pCX-DG and pCX-DR
A ScaI/SphI-digested fragment containing a cassette of CaMV 35S promoter-gfp-Tnos from plasmid pGDG (Goodin et al., 2002), and a ScaI/SphI-digested fragment containing a cassette of CaMV 35S promoter-DsRed-Tnos from plasmid pGDR (Goodin et al., 2002), were inserted into the SmaI/SphI site of plasmid pCAMBIA1300-XcmIΔ, respectively. The resulting plasmids were designated pCDG and pCDR, respectively.
A BamHI-digested fragment containing the XcmI-ccdB-XcmI cassette from plasmid pGXT was inserted into the BamHI site of plasmids pCDG and pCDR, respectively. The resulting plasmids were designated pCX-DG and pCX-DR, respectively.
pCXGUS-P and pCXGFP-P
The BamHI/EcoRI digested fragments containing the gus-Tnos or gfp-Tnos cassettes, respectively, from pBI221-GUST and pBI221-GFPT were inserted into the BamHI/EcoRI site of plasmid pCAMBIA1300-XcmIΔ, respectively. The resulting plasmids were designated pCGUS-T and pCGFP-T, respectively.
A BamHI-digested fragment containing the XcmI-ccdB-XcmI cassette from plasmid pGXT was inserted into the BamHI site of plasmid pCGUS-T and pCGFP-T, respectively. The resulting plasmids were designated pCXGUS-P and pCXGFP-P, respectively.
Construction of Plant Expression Vectors for Proof of Concept Testing
pXUN-GUS
A PCR fragment of gus gene was amplified using plasmid pUbiGUS as template with primers GusT-F and GusT-R. The fragment was ligated with XcmI-digested pXUN. The resulting plasmid was designated pXUN-GUS.
pXUN-HA-GFP
A PCR fragment of gfp gene was amplified using plasmid pGDG as template with primers HGFP-F and RSGFP-3'. The fragment was ligated with XcmI-digested pXUN-HA. The resulting plasmid was designated pXUN-HA-GFP.
pCXSN-atPDS-RNAi
A 217-bp DNA fragment of Arabidopsis thaliana PDS gene was amplified using genomic DNA of Arabidopsis thaliana Col-0 as template with primers AtPDSi-F and AtPDSi-R, and a 420-bp stuffer DNA fragment from gus gene was amplified using plasmid pANDAmini as template with primers GPLink-F and GPLink-R. Overlapping PCR was performed using the two amplified fragments as template with single primer AtPDSi-F. The resultant fragment was ligated with XcmI-digested pCXSN. The resulting plasmid was designated pCXSN-atPDS-RNAi.
pCXUN-amiPDS
Three PCR fragments were amplified using plasmid pNW55 as template with primer pairs NWB-F/amiPDS-II, amiPDS-I/amiPDS-III, and amiPDS-IV/NWB-R. Over lapping PCR was performed using the three amplified fragments as template with primer pair NWB-F/NWB-R. The resultant fragment was ligated with XcmI-digested pCXUN. The resulting plasmid was designated pCXUN-amiPDS.
pCXUN528-PDS
A PCR fragment was amplified using plasmid pNW55 as template with primers APDS-F and APDS-R. The fragment was ligated with XcmI-digested pCXUN-osaMIR528. The resulting plasmid was designated pCXUN528-PDS.
pXDG-Spin1 and pXDR-Spin1
A PCR fragment of spin1 gene was amplified using plasmid pA7spin1-NYFP (Chen et al., 2006b) as template with primers Spin-F and Spin-R. The fragment was ligated with XcmI-digested pXDG-Spin1 and pXDR-Spin1, respectively. The resulting plasmids were designated pXDG-Spin1 and pXDR-Spin1 respectively.
pCX-35S-GUS
A HindIII-digested fragment containing the CaMV 35S promoter from plasmid pA7spin1-NYFP was blunted with Klenow, and was tailed with A residues followed by the A-addition procedure (Technical manual of pGEM®-T and pGEM®-T easy vector systems, http://www.promega.com/tbs/tm042/tm042.pdf). The fragment was ligated with XcmI-digested pCXGUS-P. The resulting plasmid was designated pCX-35S-GUS.
LITERATURE CITED
Chen P, Wang C, Soong S, To K (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11: 287-293
Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang G-L (2006b) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7: 417-427
Goodin MM, Dietzgen RG, Schichnes D, Ruzin S, Jackson AO (2002) pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 31: 375-383
Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45: 490-495
Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual, Ed 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY
Warthmann N, Chen H, Ossowski S, Weigel D, Herve P (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS ONE 3: e1829
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