Pentr topo

Full-length Arabidopsis MKK5, MPK3, MPK4, and MPK6 cDNAs were obtained using RT-PCR, cloned into the pENTR-TOPO cloning vector (Invitrogen) and sequenced. The Escherichia coli Strain BL-21 codon plus (Stratagene, LA Jolla, CA, USA) was transformed with the expression constructs, which was prepared by subcloning the genes into pGEX-6P-1 vector (Amersham Pharmacia Biotech). Growth of bacteria and isolation of recombinant GST fusion protein was as described in Matsuoka et al. (2002) (link).

The 700 bp msh CRM is described in Von Ohlen et al., 2009 [25] (link). All primers used in this study and the corresponding constructs generated can be found in Table S1. The various Drosophila msh-CRM constructs were subcloned in pCR-TOPO vectors (Invitrogen) and subsequently cloned into the [P]acman vector [28] (link) as NotI and KpnI restriction fragments. Site-directed mutagenesis PCR methods were adapted from [56] (link). The primers used to isolate the zebrafish msxB CRMs and the mouse msx1 CRM can be found in Table S1. Zebrafish constructs were cloned into pENTR-TOPO (Invitrogen), transferred to pTol2 by Gateway Recombination and injected in zebrafish embryos as previously described [30] (link).

All primers used in this work are listed in Table S2. The extensin signal peptide (SP) and the carbohydrate‐binding module (CBM) cassettes SP‐CBMs‐mCherry and SP‐CBM11‐Roo were constructed using overlapping PCR primers. After digestion with BamHI and SacI, the SP‐CBMs‐mCherry and SP‐CBM11‐Roo cassettes were cloned into pCAMBIA2300 (www.cambia.org/daisy/cambia/585). The SP‐CBM11‐IBP (blood iron‐binding peptide) cassette called CBM‐IBP was amplified from pCambia2300‐SP‐CBM11‐Roo using the primers CaccSP‐5′ and BIBP‐3′ and cloned into Gateway entry vector pENTR/TOPO (Invitrogen, www.invitrogen.com). The CBM‐IBPΔ cassette containing the mutated blood iron‐binding motif (BIBPΔ) was amplified from pENTR‐CBM‐IBP using the primers CaccSP‐5′ and BIBPΔ‐3′ and cloned into pENTR/TOPO. The constructs were analysed by PCR and restriction digests, and were sequenced using the M13 forward primer. The plasmids pENTR‐CBM11‐IBP/IBPΔ were cut with MluI, and the CBM11‐IBP/IBPΔ cassettes were transferred to pGWB14 (Nakagawa et al., 2007) (with C‐terminal HA fusion) and/or the oestrogen‐inducible vector pIST04 (modified from pEarleyGate101 with N‐terminal oestrogen‐inducible elements: FMV:NEV + 10x NIP promoter and C‐terminal Dendra2 fusion) (Jásik et al., 2013) using LR reactions (Invitrogen).

cDNA sequences of candidate genes predicted in the Ty-2 region were obtained from the Sol Genomics Network database. Primers were designed to amplify a 150- to 450-bp region from cDNA of the Wageningen Ty-2 line using Phusion DNA polymerase. Fragments targeting the candidate genes for silencing were amplified and cloned into pENTR-TOPO (Invitrogen), sequenced for confirmation and subsequently cloned into TRV2 vector (Liu et al. 2002 (link)) using the Gateway system. Plasmids were transformed into Agrobacterium tumefasciens strain GV3101. For sequence alignments, MEGA version 5 software was used.
Virus-induced gene silencing (VIGS) experiments were performed as described in Verlaan et al. (2013 (link)). Briefly, TRV infection was done through Agrobacterium-mediated infiltration on cotyledons of 10-day-old seedlings using syringes without needle. Two weeks after TRV inoculation, agro infiltration with TYLCV was performed.

The pkl1 ORF was amplified from testis cDNA using the following primer pairs: plk1-pentr-F1- 5’-CACCATGAGTGCTGCAATTGCAAAGCC-3’ and plk1-pentr-R1-5’- TTAGCGTGCTGAAGTAGCA GCTGTTGTGC-3’ and subsequently cloned into pENTR-TOPO (Invitrogen) to generate a middle entry clone, which was subsequently cloned into pDestTol2CG2 (contains a cardiac myosin light-chain GFP cassette) [51 (link)] along with a 5’ entry clone containing the beta-actin2 promoter [52 (link)] and a 3’ polyA entry clone using Gateway cloning technology (Invitrogen). 25pg of pDestTol2CG2B-actin-plk1 DNA along with 25 pg of Tol2 Transposase mRNA [51 (link)] was injected into the cytoplasm of one-cell stage embryos obtained from a p09ajug heterozygous female crossed to a 09ajug heterozygous male. Founder fish were identified by screening their corresponding progeny for cardiac GFP. A p09ajug (-/+) founder fish was subsequently in-crossed to p09aug to produce F1 p09ajug homozygous and heterozygous fish carrying the transgene, Tg(actb2:plk1). The transgene segregates in a Mendelian fashion through multiple generations, i.e. in an outcross, 50% of the progeny inherit the transgene, as indicated by green fluorescence in the heart and by genotyping for the transgene, indicating that it is a single transgene insertion.

Enhancer candidates were amplified from genomic DNA of Drosophila Kc167 cells (for primers see S3 Table). All candidates were subcloned to either pCR8/GW/TOPO (Invitrogen) or pENTR/TOPO (Invitrogen) and delivered into the firefly luciferase vector [45 ] using the Gateway LR Clonase II enzyme mix (Invitrogen). Kc cells (1x10⁵) were transfected using Fugene HD (Promega) with a total of 300 ng of various plasmid combinations (1:3 ratio of promoter reporter plasmid to Renilla). Luciferase activities were measured 48 h after transfection and after stimulation with either Wg ligand or CHIR99012 using the Dual-Luciferase Reporter Assay System (Promega). Every experiment was repeated at least twice with three replicates in each independent experiment. Enhancers’ sequences used are listed in S3 Table.

The CLB1 and SYT5 constructs used in this study were generated via PCR amplification using the reverse transcription–PCR (RT–PCR) product or genomic DNA as a template and gene-specific primers (Supplementarty Table S2 at JXB online), followed by cloning PCR products into pENTR/TOPO (Invitrogen, Carlsbad, CA, USA) or pDONR221. To generate the pUB10::CLB1-GFP construct, the CLB1 fragment was subcloned into the pB7m24GW,3 vector that contains a 615 bp UBQ10 promoter. To generate the p35S:SYT1-N-GFP, p35S:SYT1-C-GFP, p35S:SYT5-N-GFP, and p35S:SYT5-C-GFP constructs used in bimolecular fluorescence complementation (BiFC), combinations of pEN-L4-pro35S-R1 (Karimi et al., 2007 (link)), pEN-L4-proSYT5-R1, pEN-R2-N-GFP-L3 (Boruc et al., 2010 (link)), and pEN-R2-C-GFP-L3 (Boruc et al., 2010 (link)) were recombined with pEN-L1-SYT1genomic-L2 (Pérez-Sancho et al., 2015 (link)) and pEN-L1-SYT5genomic-L2 into pK7m34GW,0 (Karimi et al., 2005 (link)). To generate the pCLB1::CLB1-GFP construct, the CLB1 pENTR clone was recombined with the destination binary vector pGWB4. All resulting expression vectors were transformed in Arabidopsis via floral dip (Clough and Bent, 1998 (link)). The selection of transgenic lines was made on half-strength MS medium containing 25 μg ml^–1 hygromycin (pGWB4) or 15 μg ml^–1 glufosinate-ammonium (Sigma-Aldrich) (pB7m24GW,3).