10.2 　Cellular localization of a protein fused with a fluorescent protein (GFP, YFP, RFP)

Tomomichi Fujita

(A)　 Strategy

　

(1)　 cDNA-GFP fusion constructs are targeted to a certain platform locus and stably overexpressed and therein, localization of the fusion proteins are examined. We use the Pphb7, PpMADS2, or BS213 locus as a plat form.　

(2) cDNA-GFP fusion constructs are introduced into protoplasts and TRANSIENTLY expressed by a weak 35S promoter in the regenerated cells. Multi-copy cDNA-GFP constructs are usually introduced to a protoplast in our hands, which likely causes miss-localization of the fusion protein because of its excess amount. The transient assay is a much more convenient and rapid method than that with stable transformant in (1), and a method to introduce small number of plasmids should be established in future.

(caution) A rescue of knockout phenotype by cDNA-GFP proves the functionality of the fusion protein, although the rescue does not give a support for the localization of cDNA-GFP fusion protein.

(B)　 Available constructs using gateway system#

# original gateway constructs were kindly provided from Dr. Tsuyoshi Nakagawa (Shimane University, Japan)

(1) For stable transformation to BS213 targeting　 (Ref. for BS213 locus,　 Schaefer et al. 1997, Plant J.)

[C-mRFP fusion/35S promoter]

BS213 5'-35S-R1-Cm-ccdB-R2-mRFP-Tnos-Zeo selection cassette-BS213 3'　

[N-mRFP fusion/35S promoter]

BS213 5'-35S-mRFP-R1-Cm-ccdB-R2-Tnos-Zeo selection cassette-BS213 3'　

[C-mRFP fusion/7133 promoter]$

BS213 5'-7133-R1-Cm-ccdB-R2-mRFP-Tnos-Zeo selection cassette-BS213 3'　

[N-mRFP fusion/7133 promoter]

BS213 5'-7133-mRFP-R1-Cm-ccdB-R2-Tnos-Zeo selection cassette-BS213 3'　

(2) For transient overexpression　

35S-R1-Cm-ccdB-R2-sGFP-Tnos　　　　　 [C-GFP fusion]

35S-sGFP-R1-Cm-ccdB-R2-Tnos　　　　　 [N-GFP fusion]

35S-R1-Cm-ccdB-R2-mRFP-Tnos　　　　　 [C-RFP fusion]

35S-mRFP-R1-Cm-ccdB-R2-Tnos　　　　　 [N-RFP fusion]

35S-R1-Cm-ccdB-R2-YFP-Tnos [C-YFP fusion]

35S-YFP-R1-Cm-ccdB-R2-Tnos [N-YFP fusion]

7133 promoter acts stronger than 35S promoter in P. patens.　 Drs. I. Mitsuhara and Y. Ohashi kindly provided the promoter (Mitsuhara et al., Plant Cell Physiol.　 1996).

(Figure) A cDNA1-GFP fusion plasmid was transiently expressed into protoplasts and in a 3-cell stage, asymmetric protein localization in a basal cell was observed.