The GFP revolution!

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The GFP revolution!
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Green beings.
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Fluorescence A property of certain molecules
that absorb light of a specific wavelength, then
emit at another (longer) wavelength.
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Fluorescent variants of GFP.
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Plasmodesmata Communication channels in plants
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Viral movement protein trafficking.
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pSUC2 expression in root apex.
pSUC2GFP.ER
pSUC2GFP
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pAtML1-GFPGUS
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pAtML1-GFPKN1 pAtML1-GFPMP
L1 specific expression.
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Developing a Strategy to Determine the
Subcellular Localization of Unknown Genes in
Arabidopsis

• Charles Kopec
• Amitabh Mohanty

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Predicted Gene Function in Arabidopsis thaliana
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Goal

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

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YFP Fusion Constructs

• Internal YFP fusion
• 10 aa from C-terminus
• Flanked by 10x Ala and Gly linkers
• Designed to
• Not disrupt protein folding
• Not block C-terminal localization signals
• Contain flanking intergenic regions

FseI
SfiI
4kb Upstream Promoter
1.5 kb
Exons 10x Glycine linker YFP 10x Alanine linkers
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PCR Triple Template Method

• Does not require restriction enzymes to insert
  the YFP cassette.
• No cloning before bombardment.
• Design primers
• P1 upstream primer
• P2 points up, contains 5 portion of YFP cassette
• P3 points down, contains 3 portion of YFP
  cassette
• P4 downstream primer

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First round of PCR.
P1 / P2 P3 / P4 YFP Fse1 only Sfi1
only Double Undigested
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PCR Triple Template Method

• Second Amplification Overlap

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TTPCR Amplification
TTPCR for ?TIP
Unknown genes
1OO bp Ladder
P1-P2 PCR
kb 10 8 6 5 4
1 Kb Ladder
P2-P3 PCR
kb
TTPCR
YFP
10 8 6 5 4 3 2 1.5 1 0.5
TTPCR of reference protein genes and some of the
putative/hypothetical protein genes. ?TIP,
tonoplast intrinsic protein.
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attL1 L2
attL1 and P2
attL2 and P3
Gy2 (GSR_YFP)
VLY4 (VILLIN_YFP)
VLY4 (VILLIN_YFP)
VY4 (VIP_YFP)
VLC3 (VILLIN_CFP)
VC1 (VIP_CFP)
VLC3 (VILLIN_CFP)
GC3 (GSR_CFP)
VY4 (VIP_YFP)
TC5 (TIP_CFP)
VC1 (VIP_CFP)
Gy2 (GSR_YFP)
GC3 (GSR_CFP)
TC5 (TIP_CFP)
1 kb Ladder
TY2 (TIP_YFP)
TY2 (TIP_YFP)
pDONOR207
Gene P1-P2 size GSR 5.5 kb VIP1 3.9
kb TIP 3.7 kb VILLIN 5.0 kb
Clone confirmation by PCR for verification of
entry clones, colony PCR was performed with
primers from the vector (attL1 or L2) and either
P2 or P3 primer from the gene specific part of
the reference protein genes.
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CFP/Citrine YFP
Att B
Att B

(pDONOR 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 clonaseTM.
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Bombardment

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

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VIP1 bombarded into onion.
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3707 Bombarded Arabidopsis
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Reference Genes Gene Number Gene
Name Subcellular Location    Assigned to
Lab    Primers 1 SYP41 Trans-Golgi    NR(Cha
ry) 2 SYP42 Trans-Golgi    NR(Chary)    Primers
for SYP42 3 Mannosidase Golgi stacks    VC(Gouwei)
4 SYP21 Prevacuol    NR(Chary)    Primers for
SYP21 5 Delta TIP Vacuol Membrane   DJ(Amitabh) 6
SEC12 ER    NR(Chary)    Primers for
Sec12 7 SYP51 Late Secretory System   NR(Chary)   
Primers for SYP51 8 SYP61 Late Secretory
System   NR(Chary)    Primers for
SYP61 9 MAF1 Nuclear Membrane   DJ(Amitabh) 10 VIP
1 Intranuclear    DJ(Amitabh) 11 Toc75 Chloroplast
   VC(Brigitte) 12 AOX2 Mitochondria    VC(Brigit
te) 13 PIP2A Plasmamembrane    NR(Chary)    Primer
s for PIP2A 14 PME Cell Wall    VC(Gouwei) 15 PRP2
Cell Wall    NR(Chary)    Primers for
PRP2 16 TMV MP Plasmodesmata    VC(Brigitte) 17 TV
CV MP Plasmodrsmata    DJ(Amitabh) 18 VLN Microfil
ament arrays   DJ(Amitabh) 19 Fim1 Microfilament
arrays   VC(Gouwei) 20 TUA6 Microtubule
arrays   VC(Brigitte) 21 MFP2 Peroxisome    VC(Guo
wei) 22 PEX5 Peroxisome    VC(Guowei) 23 GSR1 Cyto
sol    NR(Chary) 24 GSK B Cytosol    VC(Brigitte)
25 AtELP Prevacuol    NR(Chary)    Primers for
AtELP --------------------------------------------
----------------------------general comments or
questions to curator_at_arabidopsis.org comments or
questions about seed stocks to
seedstock_at_arabidopsis.org comments or questions
about DNA stocks to dnastock_at_arabidopsis.org
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Collaborators Andy Maule Rick Nelson Wes
Bruce Mike Muszynski Hajime Sakai Ed
Buckler Sarah Hake Bob Schmidt Volker
Brendel Toby Kellogg Rob Martienssen Vitaly
Citovsky Natasha Raikhel Sue Rhee David
Erhardt Jim Haseloff Kathy Barton Alain Joliot
Dave Jackson Jae Yean Kim Michelle Cilia Namiko
Satoh Jing Wang Wim Deleu Zhuang Yuan
Anna Giulini Fumio Shiobara
Funding NSF, USDA, CSHL