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