pCLV3:HEC1-linker-GR, pCUC2:HEC1-linker-GR and p16:HEC1-linker-GR constructs were generated by ligation of HEC1 coding sequence (CDS) with a 33 aa Serine-Glycin linker and GR tag into pDONOR221 vector and recombined in pGreenIIs constructs (Schuster et al., 2014 (link)). pKNOLLE:fast-mFluorescentTimer-NLS was generated from fast m-FluorescentTimer CDS fused to N7 NLS and introduced by subsequent BP and LR reactions (Thermo Fischer Scientist, Waltham, Massachusetts, USA) in a pGreenIIs vector containing 2.1 kb of genomic sequence upstream of the KNOLLE start codon. The CUC2 promoter used correspond to the 3.2 kb genomic sequence upstream the ATG. pCLV3:HEC1-linker-GFP, pCUC2:HEC1-linker-GFP, pCUC2:3xGFP-NLS and p35S:HEC1-linker-GFP were cloned using the Green Gate system (Lampropoulos et al., 2013 (link)). N7-NLS was used as NLS tag (Daum et al., 2014 (link)).
For Yeast-two-Hybrid and Bi-Fluorescence complementation (BiFC) assay, HEC1, HEC2, HEC3, MP, BDL and PEP CDS were PCR amplified using Phusion Taq-polymerase (New England Biolabs, Inc., Massachusetts, USA) and subsequently cloned by Gibson assembly in pGILDA/pB42AD (Yeast-two-Hybrid) (Gibson, 2011 (link)) or ligated in pGreenII0179 (SPYCE constructs) or pGreenII0229 (SPYNE cassettes) via NotI (BiFC complementation) (RodrÃguez-Cazorla et al., 2015 (link); Waadt et al., 2008 (link))
For Yeast-two-Hybrid and Bi-Fluorescence complementation (BiFC) assay, HEC1, HEC2, HEC3, MP, BDL and PEP CDS were PCR amplified using Phusion Taq-polymerase (New England Biolabs, Inc., Massachusetts, USA) and subsequently cloned by Gibson assembly in pGILDA/pB42AD (Yeast-two-Hybrid) (Gibson, 2011 (link)) or ligated in pGreenII0179 (SPYCE constructs) or pGreenII0229 (SPYNE cassettes) via NotI (BiFC complementation) (RodrÃguez-Cazorla et al., 2015 (link); Waadt et al., 2008 (link))
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