The GFP revolution Green beings Fluorescence A property

The GFP revolution!

Green beings….

Fluorescence: A property of certain molecules that absorb light of a specific wavelength, then emit at another (longer) wavelength.

Fluorescent variants of GFP….

Plasmodesmata: Communication channels in plants

Viral movement protein trafficking.

p. SUC 2 expression in root apex. p. SUC 2: : GFP. ER p. SUC 2: : GFP

p. At. ML 1 -GFP~GUS

L 1 specific expression. p. At. ML 1 -GFP~KN 1 p. At. ML 1 -GFP~MP

Developing a Strategy to Determine the Subcellular Localization of Unknown Genes in Arabidopsis Charles Kopec Amitabh Mohanty

Predicted Gene Function in Arabidopsis thaliana

Goal • Determine the subcellular, tissue, and developmental localization of ~4000 genes of unknown function.

YFP Fusion Constructs • Internal YFP fusion – 10 aa from C-terminus • Flanked by 10 x Ala and Gly linkers • Designed to: – Not disrupt protein folding – Not block C-terminal localization signals • Contain flanking intergenic regions 4 kb Upstream Promoter Exons 10 x Glycine linker YFP 10 x Alanine linkers Fse. I Sfi. I 1. 5 kb

PCR: Triple Template Method • Does not require restriction enzymes to insert the YFP cassette. • No cloning before bombardment. • Design primers – – P 1 P 2 P 3 P 4 upstream primer points up, contains 5’ portion of YFP cassette points down, contains 3’ portion of YFP cassette downstream primer P 3 P 1 P 2 P 4

P 1 / P 2 P 3 / P 4 YFP Fse 1 only Sfi 1 only Double Undigested First round of PCR. P 1 / P 2 YFP P 3 / P 4

PCR: Triple Template Method • Second Amplification: Overlap P 1 / P 2 YFP P 3 / P 4 P 1 P 4

TTPCR Amplification 1 0. 5 1 kb ladder At 2 g 222170 At 2 g 27080 At 2 g 20830 At 2 g 16530 kb 10 8 6 5 4 At 2 g 215240 Unknown genes 1 kb ladder YFP 1 OO bp Ladder P 1 -P 2 PCR P 2 -P 3 PCR 10 8 6 5 4 3 2 1. 5 TTPCR kb 1 Kb Ladder TTPCR for TIP kb 12 7 6 5 4 3 2 1. 5 TTPCR of reference protein genes and some of the putative/hypothetical protein genes. TIP, tonoplast intrinsic protein.

att. L 1 & L 2 p. DONOR 207 VLY 4 (VILLIN_YFP) VLC 3 (VILLIN_CFP) TY 2 (TIP_YFP) TC 5 (TIP_CFP) VY 4 (VIP_YFP) VC 1 (VIP_CFP) Gy 2 (GSR_YFP) GC 3 (GSR_CFP) VLY 4 (VILLIN_YFP) att. L 2 and P 3 VLC 3 (VILLIN_CFP) TC 5 (TIP_CFP) TY 2 (TIP_YFP) VY 4 (VIP_YFP) VC 1 (VIP_CFP) Gy 2 (GSR_YFP) GC 3 (GSR_CFP) 1 kb Ladder att. L 1 and P 2 Gene GSR VIP 1 TIP VILLIN P 1 -P 2 size ~5. 5 kb ~3. 9 kb ~3. 7 kb ~5. 0 kb Clone confirmation by PCR for verification of entry clones, colony PCR was performed with primers from the vector (att. L 1 or L 2) and either P 2 or P 3 primer from the gene specific part of the reference protein genes.

Att B CFP/Citrine YFP Att B (p. DONOR 207) + BP Clonase + Insertion of PCR products into Gateway entry vector. The recombination reaction is catalyzed by BP clonase TM Transfer of the fragments into binary vectors Schematic representation of the LR recombination reaction. Transfer of entry clone to destination vector is catalyzed by an invitro recombination reaction by Gateway LR clonase. TM.

Bombardment • Coat 1 mm gold particles with constructs. • Bombard into Arabidopsis leaves and onion scales. • Determine subcellular localization using fluorescence dissecting, compound, and confocal microscopy.

VIP 1 bombarded into onion.

3707 Bombarded Arabidopsis

Reference Genes Gene Number Gene Name Subcellular Location Assigned to Lab Primers 1 SYP 41 Trans-Golgi NR(Chary) 2 SYP 42 Trans-Golgi NR(Chary) Primers for SYP 42 3 Mannosidase Golgi stacks VC(Gouwei) 4 SYP 21 Prevacuol NR(Chary) Primers for SYP 21 5 Delta TIP Vacuol Membrane DJ(Amitabh) 6 SEC 12 ER NR(Chary) Primers for Sec 12 7 SYP 51 Late Secretory System NR(Chary) Primers for SYP 51 8 SYP 61 Late Secretory System NR(Chary) Primers for SYP 61 9 MAF 1 Nuclear Membrane DJ(Amitabh) 10 VIP 1 Intranuclear DJ(Amitabh) 11 Toc 75 Chloroplast VC(Brigitte) 12 AOX 2 Mitochondria VC(Brigitte) 13 PIP 2 A Plasmamembrane NR(Chary) Primers for PIP 2 A 14 PME Cell Wall VC(Gouwei) 15 PRP 2 Cell Wall NR(Chary) Primers for PRP 2 16 TMV MP Plasmodesmata VC(Brigitte) 17 TVCV MP Plasmodrsmata DJ(Amitabh) 18 VLN Microfilament arrays DJ(Amitabh) 19 Fim 1 Microfilament arrays VC(Gouwei) 20 TUA 6 Microtubule arrays VC(Brigitte) 21 MFP 2 Peroxisome VC(Guowei) 22 PEX 5 Peroxisome VC(Guowei) 23 GSR 1 Cytosol NR(Chary) 24 GSK B Cytosol VC(Brigitte) 25 At. ELP Prevacuol NR(Chary) Primers for At. ELP ------------------------------------general comments or questions to: curator@arabidopsis. org comments or questions about seed stocks to: seedstock@arabidopsis. org comments or questions about DNA stocks to: dnastock@arabidopsis. org

Dave Jackson Jae Yean Kim Michelle Cilia Namiko Satoh Jing Wang Wim Deleu Collaborators: Andy Maule Rick Nelson Wes Bruce Mike Muszynski Hajime Sakai Ed Buckler Sarah Hake Bob Schmidt Volker Brendel Toby Kellogg Rob Martienssen Zhuang Yuan Anna Giulini Fumio Shiobara Vitaly Citovsky Natasha Raikhel Sue Rhee David Erhardt Jim Haseloff Kathy Barton Alain Joliot Funding: NSF, USDA, CSHL