Plasmids for transient expression of genes in N. benthamiana

Starting plasmid for transient expression of genes in N. benthamiana. This plasmid is based on pGreen0000 (Hellens et al., 2000) and conveys Kan resistance.

pGreen- See1 (w/o SP)

Plasmid was used for the transient expression of See1 in N. benthamiana. See1 (um02239) was amplified without its secretion signal by using the primers OAR45 /46 and cloned via restriction sites XbaI and SacI in pGreen-CP1A (Mueller et al.2013) by taking out CP1A with the same restriction sites.

pGreen- See1 -mCherry

Plasmid was used for the transient expression of See1 in N. benthamiana. It contains See1 (um02239), with and without its secretion signal which was fused to mCherry to obtain a fusion protein. This PCR product was amplified by the primers OAR48 and OAR49 for with signal peptide construct and OAR50/49 for without signal peptide construct. Both the PCR products were cloned via XbaI and SacI in pGreen-CP1A (Mueller et al.2013) by exicing out CP1A with the same restriction site.The plasmid was used for the localization of See1 upon transient expression in N. benthamiana via Agrobacterium transformation.

pGreen- See1 –Myc (Schilling, 2014)

This plasmid was used for the transient expression of See1 in N. benthamiana for the in planta co-immunoprecipitation. It contains See1 (um02239), without its secretion signal and was fused to Myc epitope to obtain a fusion protein. This PCR product was amplified by the primers OLS147 and OLS148 and cloned via XbaI and SacI in pGreen vector.

pGreen- SGT1 –HA (Schilling, 2014)

Plasmid was used for the transient expression of ZmSGT1 in N. benthamiana for the in planta co-immunoprecipitation. It contains ZmSGT1 which was fused to HA epitope to obtain a fusion protein. This PCR product was amplified by the primers OLS149 and OLS152 and cloned via XbaI and BamHI in pGreen vector.

pGreen- SPYNE-mCherry (Hemetsberger et al., 2012; Schilling, 2014)

eBiFC expression vector for expression of candidate genes N. benthamiana under the control of the 35S promoter. For the amplification of mCherry from p123-mCherry the primer pair OCFH11 / OCFH10 was used so that an N-terminal RSIATA linker sequence was inserted. The PCR product was cloned via the restriction sites XhoI and XmaI in pUC-SPYCE-35S. The open reading frame was then ligated with HindIII and EcoRI restriction digests in the vector pGreen0029. To remove unnecessary restriction sites in two multiple cloning sites two inverse PCR reactions with the primer pairs OCFH48 / OCFH49 and OCFH50 / OCFH51 were performed.

pGreen- SPYCE-CFP (Hemetsberger et al., 2012; Schilling, 2014)

eBiFC expression vector for expression of candidate genes N. benthamiana under the control of the 35S promoter. For the amplification of p123-CFP, CFP from the primer pair OCFH9 / OCFH10 was used so that an N-terminal RSIATA linker sequence was inserted.

The PCR product was cloned via the restriction sites XhoI and XmaI in pUC-SPYCE-35S.

The open reading frame was then ligated to a restriction digestion with HindIII and EcoRI in the vector pGreen0029. To remove unnecessary restriction sites in two multiple cloning sites two inverse PCR reactions with the primer pairs OCFH48 / OCFH49 and OCFH50 / OCFH51 were performed.

pGreen- SPYCE-CFP _ZmSGT1 (Schilling, 2014)

eBiFC expression vector for expression of candidate genes ZmSGT1 in N. benthamiana under the control of the 35S promoter. For the amplification of ZmSGT1 the primer pair OLS120 and OLS121 was used and the product was cloned with the restriction sites BamHI and XhoI in the already generated SPYCE-CFP vector by Hemetsberger et al., 2012.

pGreen- SPYNE-mCherry _Umsee1 (Schilling, 2014)

eBiFC expression vector for expression of candidate genes Umsee1 in N. benthamiana under the control of the 35S promoter. For the amplification of Umsee1 without its secretion signal the primer pair OLS119 and OLS120 was used and the product was cloned with the restriction sites BamHI and XhoI in the already generated SPYNE-mCherry vector by Hemetsberger et al., 2012.

pGreen- PIP –YFP ( obtained from Armin Djamei)

This plasmid was used as the control for the transient expression assay of See1 in Z.

mays via ballistic bombardment. The fusion protein PIP-YFP specifically localizes to the plant nucleus and hence easily distinguishes the transformed cell along with the See1-mCherry vector which has a nucleo-cytoplasmic localization

pENTR-D-TOPO (Invitrogen, Karlsruhe)

This is a directional entry TOPO vector for the Gateway technology which does not use any restriction enzymes and is dependent on principle of homologous recombination. The intergration of the gene depends on the recognition sequence CACC at the N terminal of the of the entry site.

pENTR- D-TOPO-Umsee1

Entry clone for the generation of the destination vectors for transient plant expression of See1 under an inducible promoter destination vector. The Umsee1 was amplified by using the primers OAR86/88 for fragment with secretion signal and with the primers OAR89/88 for product without the secretion signal. Both the products generated were cloned as per the manufacturer’s instructions.

pENTR -D-TOPO-ZmSGT1

Entry clone for the generation of the destination vector for transient plant expression of ZmSGT1. The gene was amplified by using the primers OAR133/134 for amplification without stop codon for a C terminal fusion and by OAR133/135 with stop codon for a N terminal fusion destination vector. Product generated was cloned as per the manufacturer’s instructions.

pTA7001 (Procured from Prof. Nam Hai Chua, USA)

A DEX inducible plant expression vector which is used for the stable expression in A.

thaliana or transient expression in N. benthamiana for in planta assays.

pTA7001- See1-HA

The vector was used for the induction of Umsee1 under DEX promoter for the in planta phosphorylation assay. The Umsee1 without its secretion signal was amplified along with the primers OAR92 and OAR93 which was integrated with a HA tag to formed the fusion protein with See1-HA and cloned with the restriction sites SpeI and XhoI.

pGWBSH- HA-ccDB-Nos (procured from IBB, Poland)

This plasmid is Gateway destination empty vector with an N terminal HA tag for the purification of the protein.

pGWBSH -ccDB-Strep-His-Nos (procured from IBB, Poland)

This plasmid is Gateway destination empty vector with a C terminal Strep and His tag for the purification of the protein.

pGWBSH -ZmSGT1-Strep-His (generated from IBB, Poland)

This plasmid is Gateway destination empty vector with a C terminal Strep and His tag for the purification of the protein. This vector was used for the cloning and expression of ZmSGT1 in planta phosphorylation assay. The ZmSGT1 was cloned via recombination from the pENTR-D-TOPO-ZmSGT1 without the stop codon.

pROK2-NbSGT1-6X HIS.Ala-Ala-Strep II (Hoser et al., 2013)

This is a destination vector with the SGT1 from N. benthamiana and fused to a 6X HIS Strep II tag. The construct was used as a control in the in planta phosphorylation assay and was previously generated by Rafal Hoser from IBB, Poland.

pROK2-SIPK (Hoser et al., 2013)

This is a destination vector with the SIPK from N. benthamiana. The construct was used for the transient in planta phosphorylation assay and was previously generated by Rafal Hoser from IBB, Poland.

pTA7002-NtMEK2^DD/NtMEK2^KR (Hoser et al., 2013)

The vector was used for the induction of the constitutive active kinase MEK2^DD and the inactive kinase MEK2^KR from N. benthamiana which are under DEX promoter and used for the in planta phosphorylation assay. These constructs were previously generated by Rafal Hoser from IBB, Poland.