Vector construction for functional validation and subcellular localization

Wild type and mutant versions of the albostrians gene were obtained by RT-PCR using cDNA of ‘Haisa’ and ‘M4205’ as template, respectively. Fragments amplified from ‘M4205’ carrying a 4 bp deletion at position 1123 - 1126 bp (count from adenine of start codon as +1) compared to the wild type. The insert was introduced into the HincII cloning site of pUbi-ABM (DNA Cloning Service, Hamburg, Germany) in a reaction volume of 20 µl containing 0.4 µl of digested pUbi-ABM (100 ng), 2 µl of RT-PCR product, 2 µl of 10x T4 DNA ligase buffer (100 mM MgCl2; 100 mM DTT; 5 mM ATP; 400 mM Tris-HCl; pH 7.8 at 25°C), 2 µl of 50% PEG 4000 solution, 1 µl of T4 DNA ligase (Fermentas GmbH, Schwerte, Germany) and 12.6 µl nuclease-free water. The reaction mix was incubated in a thermal cycler (Life Technologies GmbH, Darmstadt, Germany) with the program 22°C for 2 hours, followed by 70°C for 10 min to inactivate the enzyme. Thereafter, the ligated reaction mix was further digested at 37°C for 2 hours after adding 3 µl of 10x buffer 3.1 (New England Biolabs GmbH, Frankfurt am Main, Germany), 1 unit of HincII (New England Biolabs GmbH, Frankfurt am Main, Germany) and 6 µl nuclease-free water (HincII generates blunt end, this step aims to digest the religation vectors with no desired insert). Two microliter of the reaction mix was used for transformation of TOP10 competent cells (Life Technologies GmbH, Darmstadt, Germany) by the heat shock method described elsewhere (Froger and Hall, 2007). Positive clones were checked via restriction analysis and confirmed by Sanger sequencing. Subsequently, the

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expression cassette of the WT/MT albostrians gene was introduced into the SfiI cloning site of the binary vector p6d35S (Nagy et al., 2011). Binary vectors containing desired insert were transformed into A. tumefaciens strain AGL1 via electroporation (SAMBROOK et al., 1989). Agrobacterium-mediated transformation was performed by Dr. Götz Hensel in the research group of Plant Reproductive Biology (AG PRB) following the protocol described elsewhere (Himmelbach et al., 2007; Hensel et al., 2008). The derived constructs information is summarized in Table 2-2.

2.17.2 Inducing knock out (KO) mutant lines by a TALEN approach

TALEN (Transcription Activator-Like Effectors Nuclease) is an artificially engineered nuclease with the potential of genome editing as a result of causing double strand breaks (DSB), and its principle and application had been reviewed elsewhere (Joung and Sander, 2013; Puchta and Fauser, 2013; Voytas, 2013). The first report in barley using TALEN was to knock out the phytase gene (Wendt et al., 2013). Recently, expression of the functional GFP gene integrated in a transgenic barley line was successfully silenced through transformation with a GFP-specific TALEN pair (Gurushidze et al., 2014).

The module, array and last repeat TALEN plasmids ordered from Addgene (http://www.addgene.org/) were assembled in the research group of PRB as guided by the Golden Gate TALENs assembly protocol (Cermak et al., 2011). The assembled forward TALEN arm (pTAL_HvAs_F) and reverse TALEN arm (pTAL_HvAs_R) were each introduced into the AscI/BamHI and SpeI/ BamHI cloning sites of pSH60 and pSH34 (optimized backbone plasmids generated by Stefan Hiekel, AG PRB, IPK), respectively. The “self-cleaving” 2A peptides (T2A) were used to generate multiple proteins from a single promoter in many applications (de Felipe, 2004; Osborn et al., 2005; de Felipe et al., 2006). Hence, TALEN units of the two intermediate vectors were subsequently cloned into a 2A peptide-enabled dual expression vector pSH68 via AscI/BclI (BclI produces the same sticky overhangs as BamHI) and SpeI/BamHI digestion. The TALEN expression cassette (pSH68_TALEN_HvAs_F_T2A_TALEN_HvAs_R) was finally introduced into the SfiI cloning site of the binary vector p6id35STE9, which harbors hpt, a gene which confers hygromycin resistance, driven by the cauliflower mosaic virus double enhanced 35S (CaMVd35S) promoter. The binary vector containing both TALENs

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(p6id35STE9_TALEN_HvAs_F_T2A_TALEN_HvAs_R, with an alternative name pSH83 for convenient labeling) was then introduced into A. tumefaciens strain LBA4404pSB1 through electroporation (SAMBROOK et al., 1989). Aliquots of the liquid culture containing 15% glycerol were stored in -70°C for the downstream Agrobacterium-mediated transformation.

2.17.3 Subcellular localization of the WT and MT ALBOSTRIANS proteins

The coding sequence of the WT albostrians gene of barley cv. ‘Haisa’, as well as its two MT alternatives from M4205 and M2-TILLING mutant 6460-1 were fused to the N-terminus of the GFP reporter gene (Chiu et al., 1996), respectively, through ligation into the SpeI/HindIII cloning sites of the vector pSB179 (provided by Dr. Jochen Kumlehn), which contained an ampicillin resistance gene as selection marker. The digestion reactions were performed in a total volume of 10 µl containing 1 µg of plasmid, 5 µl of insert fragment, 1 µl of FastDigest SpeI (Fisher Scientific GmbH, Schwerte, Germany), 1 µl of FastDigest HindIII (Fisher Scientific GmbH, Schwerte, Germany), 1 µl of 10x FastDigest green buffer, and nuclease-free water. After incubation at 37°C for 20 min, the digested products were separated on a 2% (w/v) agarose gel and the expected fragments were recovered and purified using MiniElute Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany). Ligation reactions were carried out in a total volume of 20 µl after mixing 1 unit of T4 DNA ligase (Fermentas GmbH, Schwerte, Germany), 2 µl of 10x ligation buffer (100 mM MgCl2; 100 mM DTT; 5 mM ATP; 100 mM Tris-HCl, pH=7.8), 3 µl of purified PCR product, 1 µl of purified backbone fragment, and nuclease-free water. Reaction mix was incubated at 22°C for 2 hours. Then, the recombinant plasmid was transformed into TOP10 competent cell (Life Technologies GmbH, Darmstadt, Germany) via a heat shock method as described by Froger and Hall (2007). The obtained recombinant expression cassette was confirmed via Sanger sequencing and applied for the downstream bombardment experiment. The derived constructs information is summarized in Table 2-2.

32 Table 2-2: Summary of the constructed vectors.

Construct ID Source (Organism)^A Genotype Background^B Application

p6d35S-HvAs_WT Barley Haisa Complementation

p6d35S-HvAs_M4205 Barley Haisa Complementation

pSB179-HvAs_WT Barley Haisa Subcellular localization

pSB179-HvAs_M4205 Barley Haisa Subcellular localization

pSB179-HvAs_TILLING Barley Haisa Subcellular localization

pSH83 - - Inducing KO mutant lines

A_ The origin of the insertion fragment.

B_ The barley genotype used for amplification of the insertion fragment.

2.18 Biolistic transient expression assay for subcellular localization of the