Bimolecular Fluorescence Complementation (BiFC) in living
plant cells
To confirm protein-protein interaction in vivo, biochemical works using GST pull-down
and co-immunoprecipitation are generally used. Recently, however, some newexperimental techniques using fluorescent proteins for this purpose have beendeveloped, such as BiFC (bimolecular fluorescence complementation), andFRET/BRET (fluorescence/bioluminescence resonance energy transfer) [See review for
details: Bhat et al , Plant Methods, 2006]. Of these new methods, BiFC is the simplest
and easiest to introduce into the lab because a normal fluorescence microscope issufficient, whereas other methods require more specialized equipment. These new
approaches are valuable, as they allow researchers to observe what and whereinteractions occur at the subcellular level of living plant cells at the same time.

Vectors: Choose either vector set depending on which transformation method fortransient expression you will use.

Walter et al., Plant J., 40, 428-438 (2004)
A. pSPYNE-35S, pSPYCE-35S (plant expression binary vectors, for agro-infiltration)
B. pUC-SPYNE, pUC-SPYCE (pUC-based high copy plasmid vectors, for
PEG-mediated protoplast transformation)
Notes:- GatewayTM (Invitrogen) compatible destination vectors for all are also available.
- For Agro-infiltration, co-transformation of 35S::p19 with your constructs makes
enhanced expression. p19 is RNA silencing repressor protein from Tomato bushy
stunt virus (Voinnet et al., Plant J., 2003). 35S::p19 can be requested from Dr.

David baulcombe (John Innes center, UK).
- All vector plasmids can be requested directly from Dr. Klaus Harter (TubingenUniversity, Germany: klaus.harter@zmbp.uni-tuebingen.de).

Procedure:- Make BiFC constructs with your favorite genes.
- Co-transform plasmid set (SPYNE and SPYCE) by either transient expression assay
method A or B (see below).
- View the image of the living plant cells with YFP-derived fluorescence using
fluorescence microscope.

Transient expression assay methods:
A. Agro-infiltration for Nicotiana benthamiana (modified protocol of David
Baulcombe lab)
1) Inoculate your agro into 5 ml LB to make pre-culture (or directly inoculate into 50 ml
LB) by using glycerol stock or picking up the colony from the petri-dish. Grow it at 28oC,O/N. Use:-5 ml (or 50 ml) L-broth + antibiotics (Kanamycin, Rifampicin, etc…)
If you’d like to use pre-culture for 50ml inoculation, use 1ml of pre-culture. For 50 ml
culture, add the following ingredients:-10 µI 100mM Acetosyringone (20 µM final): Stock must be made into DMSO
-500µI 1M MES-KOH, pH5.7 (10 mM final) [optional, filter-sterilized]
2) Precipitate the bacteria by centrifuge (5000g, 15-20 min, RT) and resuspend thepellet in 5ml of Agro-infiltration buffer containing:-10 mM MgCI2-10 mM MES-KOH, pH 5.7-150 -200µM Acetosyringone (freshly adding just before using)3) Check OD600 by making 1:10 dilution into another eppendorf tube and adjust agro’s
OD to 0.5 (1 agro/injection), 1.0 (2 agros/injection), 1.5 (3 agros/injection) using
Agro-infiltration buffer.
4) Leave it on the bench for 2 to 4 hours before agro-infiltration (or overnight).
5) Mix them up (final OD of each agro is 0.5) and perform the infiltration with a 1-2 mlsyringe. Simply press the syringe (no needle) on the underside of the leaf, and exert acounter-pressure with your finger on the other side. Infiltrate the maximum number ofleaves, avoiding cotyledons. Use top leaves if you expect high expression of transgene.

6) 2-5 days after infiltration (5 days is best), harvest the samples or perform yourexperiment.
B. PEG-mediated transformation for Arabidopsis mesophyll protoplasts
We strongly recommend using Jen Sheen’s lab’s protocol for this work because they
established this experiment very well. You can download the protocol file from their website (registration required). They also have an excellent movie featuring the details of allprocedures.

References:Bhat, R.A., Lahaye, T. and Panstruga, R. (2006) The visible touch: in planta
visualization of protein-protein interactions by fluorophore-based methods, Plant
Methods, 2, 12Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic,D., Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004)
Visualization of protein interactions in living plant cells using bimolecular fluorescencecomplementation, Plant J., 40, 428-438
Jen Shhen lab’s web site: http://genetics.mgh.harvard.edu/sheenweb/main_page.html
David Baulcombe lab’s web site:
http://www.ayeaye.tsl.ac.uk/index.php?option=com_content&task=view&id=42&Itemid=
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