Cloning & Constructs

All constructs generated in this study were synthesised using oligonucleotide primers (Appendix Table A1) and cloned either directly into their destination vector, or

indirectly via a sub-cloning vector. With the exception of two constructs, the restriction sites XbaI and SacI were exclusively used throughout this study due to their reliable cutting efficiency and the absence of these sites within the DNA coding sequence of RTN13, the myc-epitope and YFP. Unless otherwise stated, all constructs are driven by a 35S CaMV promoter. All vectors used are listed in table 2.1.

2.4.1 Sub-cloning

To facilitate the cloning process, constructs were regularly cloned first into the sub- cloning vectors pBluescript (Stratagene) and 35S-CaMV (Hellenset al., 2000). Both vectors are approximately 3kb (Appendix Figures A1-A2, respectively) and contain multiple cloning sites. The orientation in which the constructs were cloned in was irrelevant due to the digestion and re-cloning of the constructs once successfully inserted into the sub-cloning vector. Sequencing primers were designed to surround the multiple cloning sites.

2.4.2 Destination vectors

Following successful sub-cloning and sequencing, constructs were cloned into their final expression vectors. In the case of PEG-mediated protoplast transfections, constructs were cloned into the 35S-CaMV vector downstream of the 35S promoter (Hellens et al., 2000). The binary vectors pVKH18-En6 (Moore et al., 1998), pGreenII-0029 (Hellens et al., 2000) and pLH7000 (Hausmann & Töpfer, 1999) were used for Agrobacterium-mediated plant transformation. pVKH18-En6 (Appendix Figure A3) was the main vector used. It contains only two restriction sites, XbaI and SacI. It has hygromycin resistance in plants for use when making transgenic Arabidopsis. pGreenII-0029 (Appendix Figure A4) was only used in one instance, whereby the native promoter and genomic sequence of RTN13 was cloned upstream of YFP-OCS (octopine synthase terminator) for use in localising RTN13 expression in its native tissue. The wide range of restriction sites already available in pGreenII-0029 remained intact upstream of the YFP-OCS insertion. The binary vector pLH7000 (Appendix Figure A6) was used for expression exploiting the Bi- molecular Fluorescence Complementation (BiFC) system (kindly donated by Dr

Eugene Savenkov, Uppsala BioCenter SLU, Sweden). RTN13 and YFP fragment fusions were cloned to include a 35S promoter, and inserted upstream of a 35S terminator. All binary vectors were transformed into Agrobacterium tumefaciens

strain C58 harbouring the pSoup helper plasmid (Appendix Figure A5). These constructs were then co-expressed with constructs previously made (listed in table 2.2) either to exert an effect on cells, or to be used as fluorescent organelle markers.

Table 2.1 Vectors used in the generation of constructs

Vector Application Resistance Reference

pBluescript KS Sub-cloning Ampicillin (100μg/ml) Stratagene (Appendix Figure A1)

35S-CaMV cassette vector

Sub-cloning and PEG- mediated transfection Ampicillin (100μg/ml) Hellenset al., 2000 (Appendix Figure A2) pVKH18-En6 Agrobacterium- mediated plant transformation (binary vector) Kanamycin (50μg/ml – bacteria) Hygromycin (20μg/ml – Arabidopsis) Mooreet al., 1998 (Appendix Figure A3) pGreenII-0029 YFP- OCS Agrobacterium- mediated plant transformation (binary vector) Kanamycin (50μg/ml – bacteria) Kanamycin (50μg/ml – Arabidopsis) Hellenset al., 2000 (Appendix Figure A4)

pSoup Helper plasmid for Agrobacterium- mediated plant transformation Tetracycline (4μg/ml – bacteria) Hellenset al., 2000 (Appendix Figure A5) pLH7000 Agrobacterium- mediated plant transformation (binary vector) Streptomycin (100μg/ml – bacteria) Spectinomycin (100μg/ml – bacteria) Hausmann & Töpfer, 1999 (Appendix Figure A6)

Table 2.2 Previously generated 35S-driven constructs used in this study

Construct Application Reference

GFP-HDEL Luminal ER marker Batokoet al., 2000 RFP-HDEL Luminal ER marker Proprietary

ST-RFP Golgi marker Saint-Joreet al., 2002 GFP-calnexin ER-membrane marker Ironset al., 2003 αTIP-GFP Tonoplast marker Hunteret al., 2007

γTIP-GFP Tonoplast marker Hunteret al., 2007 SP:RFP:AFVY Vacuolar marker Hunteret al., 2007 YN Residues 1-154 of YFP expressed cytosolically. Zamyatninet al., 2006 YC Residues 155-239 of YFP expressed

cytosolically.

Zamyatninet al., 2006

YN-ER Residues 1-154 of YFP targeted to the ER by means of a signal peptide and HDEL retention motif.

Zamyatninet al., 2006

YC-ER Residues 155-239 of YFP targeted to the ER by means of a signal peptide and HDEL retention motif.

Zamyatninet al., 2006

Phaseolin Δ418 Truncated, secreted version of the vacuolar

storage protein phaseolin.

Frigerioet al., 1998

Sec12 Guanine nucleotide exchange factor – over- expression inhibits initiation of transport of COPII vesicles from the ER and blocks secretion.

Phillipsonet al., 2001

YFP-RTN1 N-terminally tagged AtRTNLB1 Courtesy of Professor Chris Hawes, Oxford- Brookes University, UK RTN1-YFP C-terminally tagged AtRTNLB1

YFP-RTN2 N-terminally tagged AtRTNLB2 RTN2-YFP C-terminally tagged AtRTNLB2 YFP-RTN3 N-terminally tagged AtRTNLB3 RTN3-YFP C-terminally tagged AtRTNLB3 YFP-RTN4 N-terminally tagged AtRTNLB4 RTN4-YFP C-terminally tagged AtRTNLB4

2.4.3 Designing constructs

When designing the constructs used in this study (listed in table 2.3) consideration was taken to minimise the effect of the tag on the correct folding of proteins. In most cases, established tags, such as YFP and the myc-epitope, were fused at either the N-

or C-terminus of the protein to determine any effect the orientation may have. Where possible, an untagged form of the protein was also made. Constructs were usually made in two forms: one with a myc-tag for use in biochemical experiments, and one tagged with YFP for use with confocal microscopy, although both tags were used for both purposes. When truncations were implemented, conscious efforts were made to ensure that the truncated region was replaced, rather than left ‘free-standing’, usually with a myc-tag, which consists of a small, 10-residue stretch of amino acids.

Table 2.3 Constructs generated and used during the course of this study

Construct Vector Description

RTN untagged pVKH18-En6 The full length cDNA of RTN13 cloned in frame with a myc-epitope tag at either the N- or C-terminus or untagged (Fig. 3.6 onwards).

RTN-myc pVKH18-En6 35S-CaMV myc-RTN pVKH18-En6

35S-CaMV

RTN-myc-KKSE pVKH18-En6 As with RTN-myc, but the ER-retrieval motif has been transposed to the C-terminus of the myc-epitope tag. YFP-RTN pVKH18-En6 The full length cDNA of RTN13 cloned in frame with

YFP at either the N- or C-terminus. RTN-YFP pVKH18-En6

RTN-YFP-KKSE pVKH18-En6 As with RTN-YFP, but the ER-retrieval motif has been transposed to the C-terminus of YFP.

myc-RTN-ΔKKSE 35S-CaMV Myc-epitope tagged RTN13 mutant lacking the ER- retrieval motif, residues 203-206 (KKSE).

myc-RTN-ΔC 35S-CaMV Myc-epitope tagged RTN13 mutant lacking the C- terminus, residues 157-206.

ΔN-RTN-myc 35S-CaMV Myc-epitope tagged RTN13 mutant lacking the N- terminus, residues 1-22.

ΔN-RRKK-RTN-myc 35S-CaMV Myc-epitope tagged RTN13 mutant lacking the N- terminus, but retaining hypothetical stop-transfer signal, residues 23-26 (RRKK).

YFP-RTN-ΔKKSE pVKH18-En6 As with myc-RTN-ΔKKSE, but tagged with YFP. YFP-RTN-ΔC pVKH18-En6 As with myc-RTN-ΔC, but tagged with YFP.

ΔN-RTN-YFP pVKH18-En6 As with ΔN-RTN-myc, but tagged with YFP.

ΔN-RRKK-RTN-YFP pVKH18-En6 As with ΔN-RRKK-RTN-myc, but tagged with YFP. myc-RTN–ΔLoop 35S-CaMV Myc-epitope tagged RTN13 mutant - the loop (residues

75-107) have been replaced with 2x myc-epitope tags. YFP-RTN –ΔLoop pVKH18-En6 YFP-tagged RTN13 mutant - the loop (residues 75-107)

Table 2.3 (continued)

YFP-RTN –ΔTM1 pVKH18-En6 YFP-RTN13 mutant - the first trans-membrane domain has been shortened, residues 44-48.

YFP-RTN –ΔTM2 pVKH18-En6 YFP-RTN13 mutant - the first and second trans-

membrane domains have been shortened, residues 52-57. YFP-RTN –ΔTM3 pVKH18-En6 YFP-RTN13 mutant - the first, second and third trans-

membrane domains have been shortened, residues 125- 130.

YFP-RTN –ΔTM4 pVKH18-En6 YFP-RTN13 mutant - all four trans-membrane domains have been shortened, residues 134-139.

YN-RTN pLH7000 Residues 1-154 of YFP fused to the N-terminus of RTN13.

YC-RTN pLH7000 Residues 155-239 of YFP fused to the N-terminus of RTN13.

RTN-YN pLH7000 Residues 1-154 of YFP fused to the C-terminus of RTN13.

RTN-YC pLH7000 Residues 155-239 of YFP fused to the C-terminus of RTN13.

RTN –YC–RTN pLH7000 Residues 155-239 of YFP inserted in the loop region of RTN13 between residues 91-92.

Native-RTN-YFP pGreenII-0029 YFP-OCS

The genomic sequence of RTN13 cloned with the native promoter and in frame with YFP at the C-terminus.

2.5 Growth, maintenance, and manipulation ofArabidopsis thalianaand