Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

No abstract

Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein-or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ;2 3 10 5 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

The objective of the present study was to test the hypothesis that fish gills can express more than one isoform of the Na+-K+-ATPase a subunit responsible for ion regulation in seawater and freshwater environments. Using rapid amplification of complementary DNA ends (RACE), we cloned and sequenced full-length cDNAs encoding Na+-K+-ATPase alpha 1 and alpha 3 subunits of tilapia (Oreochromis mossambicus). Clone TG33 is 3390 bp in length and encodes a polypeptide of 1023 amino acids, while clone TH3 is 3581 bp in length and encodes a protein of 1010 amino acids. Clones TG33 and TH3 showed 91% and 88% identities at the amino acid level with previously described animal Na+-K+-ATPase alpha 1 and alpha 3 subunits, respectively. Northern blot and reverse transcriptase polymerase chain reaction analyses indicated that the alpha 1 subunit is expressed predominantly in kidney and intestine, while the alpha 3 subunit is expressed mainly in brain and heart. However, lower levels of expression of both genes were detected in other tissues such as gill, spleen, and testis. The amounts of both alpha 1 and alpha 3 subunit messenger RNA in gill tissue increased with the level of environmental salinity. This provides direct evidence of enhanced transcription of N+-K+-ATPase alpha 1 and alpha 3 subunit genes upon salinity challenge.