Abstract
ABSTRACT Tripartite split-GFP complementation assay is a new protein-protein interaction technique improved from bimolecular fluorescence complementation (BiFC). Tripartite split GFP system is composed of a larger fragment, GFP1-9 (residues 1–193), and two shorter β-strands : GFP10 (residues 194–212) and GFP11 (residues 213–233). There are some disadvantages of BiFC because BiFC is based on bulky fragments that may increase the difficulty of protein folding and interfere with protein function. To test whether tripartite split-GFP is a useful tool in planta, we first chose two proteins involved in the phosphate starvation reponse: the phosphate starvation response 1 (PHR1) and the SPX domain-containing protein 1 (SPX1) proteins. The PHR1 and SPX1 sandwitch proteins (GFP10-PHR1-GFP11 and GFP10-SPX1-GFP11) were used for transient expression in tobacco leaves and localized in the nucleus. Next, we confirmed the previous results that PHR1 interacts with SPX1 using tripartite split-GFP system. This assay showed that PHR1 and SPX1 can form homo-dimer/oligomers itself. We used the Arabidopsis nitrogen limitation adaptation (NLA/BAH1) to verify the specificity of the interaction between PHR1 and SPX1. Our results showed that NLA interacts with both PHR1 and SPX1. In addition, we chose the other PHR1 family protein PHR1-like 3 (PHL3) to test the interaction of PHL3 with PHR1 and SPX1 respectively. The signal strength between PHR1, SPX1 and PHL3 are different. From the strongest to the weakest is PHR1 and PHL3, PHR1 dimer, SPX1 and PHR1, and then the signal of SPX1 and PHL3 was the strongest. Furthermore, we generated and obtained the Arabidopsis transgenic liens overexpessing GFP10-PHR1, SPX1-GFP11 and GFP1–9. Confonal analysis of these lines showed few signals in the root hair of seedlings, indicating that the tripartite spli-GFP system used in transgenic plants of Arabidopsis needs to be improved.