Gateway lr clonase 2 reaction

Transient translations of eYFP reporter constructs were generated based on pGWB540 vectors^{72 (link)}. The promoter region (~1 kb) and the first exon until the ATG start codon of MPK20 (pMPK20) and ACT8 (pACT8) were amplified from Arabidopsis wild-type genomic DNA by PCR. The PCR products were ligated to the NotI-HF and AscI digested pENTR-D-Topo vector through isothermal assembly to yield entry clones. Short promoter-proximal RNA (sppRNAs) deletion constructs were generated from entry clones. The 58 bp sppRNA region in the first exon of the MPK20 5′-UTR was deleted (pMPK20-ΔsppRNA), and the 64 bp sppRNA region was deleted in the ACT8 5′-UTRs (pACT8-ΔsppRNA). Entry vectors were used in Invitrogen, Gateway LR Clonase II reaction according to manufacturer conditions with pGWB540 (enhanced YFP) to generate expression vectors: pMPK20-eYFP, pΔMPK20-eYFP, pACT8-eYFP and pΔACT8-eYFP. The expression vectors were transformed into Agrobacterium tumefaciens strain GV3850 by electroporation under 2.5 kV, 400 Ω and 25 μF. Agrobacteria containing expression vectors were respectively co-infiltrated with the p19 suppressor of silencing into Nicotiana benthamiana leaves. The eYFP signal was assayed on the third day after infiltration.

Mouse Map1b FL cDNA was provided by Beat M. Riederer (Department of Cell Biology and Morphology, University of Lausanne, Switzerland). Sub-cloning into a modified pENTR1A, containing 5’ GFP and 3’ mCherry was conducted using Map1b specific primers (FP: CGCGGGACGCGT-ATGGCGACCGTGGTGGTGGA, RP: CGCGGGGCGGCCGCCC-TACAGTTCTATCTTGCA). cDNA of human tubulin alpha 1a (TUB1A) and human actin alpha 1 (ACTA1) was part of our common lab stock. They were swap cloned into a modified version of pENTR1A with 5’ GFP or mCherry using the restriction enyzmes mluI and notI (New England Biolabs, Germany) Sub-cloning into pLenti6/V5-Dest (Invitrogen) was done according to Invitrogen’s Gateway LR Clonase II reaction protocol. All constructs were sequenced to confirm desired constructs.