Introduction

3.1.1 Yeast Two-Hybrid System

The protein-protein interaction between the Arabidopsis ethylene receptor ETR1 and the downstream MAPKK kinase CTR1 was initially characterized by using the yeast two-hybrid assay (Clark et al., 1998). Yeast two-hybrid is a genetic tool to study protein interactions and to identify novel proteins that can associate with a known protein of interest without the need to know how and where the target protein acts (Bartel and Fields, 1997). The basis of the yeast two-hybrid system relies on the reconstitution of a functional transcription factor which has two separable domains: the DNA binding domain (DB) and the activation domain (AD). There are three types of commercially available yeast two-hybrid systems. The system I is based upon splitting the yeast transcription factor GAL4 into the GAL4-AD and GAL4-DB. Both the system II and III use the E. coli LexA as DB and differ in the choice of the activation domain. It is generally considered that the LexA-based systems are more sensitive than the GAL4 system and conversely the GAL4 system is more stringent and produces less false positive results (Criekinge and Beyaert, 1999).

The IntCR (Interacting with CTR2) clones were identified through screening a tomato fruit cDNA library (LexA-based yeast two-hybrid system) using the LeCTR2 N-terminus (aa 192-542) as bait (Lin and Grierson, unpublished data). The tomato fruit

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cDNA fragments were inserted into the prey vector pJG4-5, which fused the foreign DNA downstream of the activation domain. The bait cDNA (LeCTR2192-542) was cloned into the vector pEG202 in frame with the LexA DNA binding domain. The LexA-bait (DB-LeCTR2) fusion protein binds to the LexA operators upstream of the reporter gene. If the bait protein associates with an AD-prey protein, the interaction would activate the two reporter genes and the yeast cells would be able to grown in medium lacking leucine and convert the X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-glacto-pyranoside) substrate to a blue colour product (4-chloro-3-brom-indigo) (Figure 3.1).

Figure 3.1: The LexA-based yeast two-hybrid system

The bait protein was fused with the DNA binding domain (DB) of LexA, which can bind to the eight LexA binding sites in the reporter plasmid pSH18-34. The prey protein was fused to the activation domain (AD). If the association of the bait and prey occurs, a functional transcription factor is regenerated. This results in the activation of the reporter genes, and produces visible phenotypes. The activation of the Leu reporter gene allows the yeast to grow on synthetic dropout media lacking leucine and the gene product of the LacZ could change the colour of the yeast colony from white to blue.

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3.1.2 Fluorescent Proteins

The green fluorescent protein (GFP) was first discovered in the jellyfish Aequorea Victoria as a companion to the bioluminescent protein aequorin (Shimomura et al., 1962). Since then, GFP is widely used in various types of cells as a reporter gene due to its unique in vivo fluorescence ability and low toxicity to the host cell (Prasher et al., 1992). The use of GFP in higher plants was not feasible until a cryptic intron inside the coding sequence of the GFP was removed (Haseloff et al., 1997). In addition, extensive mutagenesis screens have been carried out and numerous GFP variants with distinct fluorescence characteristics have been generated (reviewed in Shaner et al., 2005). For instant, the S65T (Serine65changed to Threonine) GFP stabilized the fluorochrome in a permanently ionized form with a single absorbance peak at 489 nm, which became the backbone of the commercially available EGFP (Clontech). The identification of the spectra-shifted GFP variants, such as the blue-shifted cyan fluorescent protein (CFP) and the red-shifted yellow fluorescent protein (YFP) enables multiple proteins to be visualized simultaneously in the same cell (Heim et al., 1994).

Furthermore, it was realized that the Foster resonance energy transfer (FRET) from CFP to YFP could serve as an indicator of the distance between the two fluorescent proteins. FRET is the transfer of the electronic excitation energy between the donor (D) and the acceptor (A). The FRET energy transfer occurs without radiation emission (photons) and is results from the long-range interaction between the D and A dipoles. The efficiency of this process is dependent on the extent of the spectral overlap between the

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D emission spectrum and the A absorption spectrum, the quantum yield of A, the respective orientation of the proteins and most importantly, the distance between D and A (reviewed in Periasamy and Day, 2005).

In situations where two proteins are labelled with CFP and YFP, the only variable factor determining the FRET efficiency is the distance between the two fluorescent proteins. Therefore, by measuring the FRET efficiency, the distance of the two target proteins can be determined. However, it is difficult and impractical to obtain the orientation information (orientation factor 2

) for proteins in a biological system where the protein conformations change rapidly and result in a spread of possible 2

. Thus the average value of 2

=2/3 is generally used for the biological samples.

However, the early FRET measurements were hampered by the poor quantum yield and multiple excitation stages of CFP, plus the pH sensitivity and slow maturation rate of YFP. Therefore, three point mutations (S72A, Y145A and H148D) have been incorporated into CFP in order to stabilize its conformation and resulted in a new CFP variant, called “Cerulean”, which has increased quantum yield, higher excitation coefficient and a single exponential fluorescence life-time (Rizzo et al., 2004). On the other hand, the YFP variant Venus (F46L, F64L, M153T, V163A and S175G) has been generated with better pH sensitivity and folding efficiency when compared to EYFP (Nagai et al., 2002). It should be noted that a new pair of CFP/YFP variants CyPet-YPet had been specifically optimized for FRET experiment (Daugherty et al., 2005). There

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has not yet been any published literature on the applications of CyPet-YPet in plant cells and thus a comparison with the Cerulean-Venus pair is not feasible at present.

Quantification of FRET efficiency is generally achieved by measuring the change of the donor or the acceptor fluorescent intensity in acceptor photo-bleaching FRET or sensitized emission FRET. These methods either require complicated algorithms to remove the spectra bleedthrough or cause irreversible photo bleaching and require tissue fixation. The limitations of the intensity-based FRET could be overcome by using the fluorescence lifetime measurement FRET (FLIM-FRET), because the fluorescence lifetime is independent of the fluorophor concentration. However, FLIM-FRET is not widely used because of the requirement for a costly pulse laser source and fast photon-counting detectors.

An alternative technique known as biomolecular fluorescence complementation (BiFC) has been developed (Hu et al., 2002; Hu and Kerppola, 2003). BiFC is based on the ability of two non-fluorescent fragments of a fluorescent protein (e.g. YFP) to re-constitute a functional fluorescent complex. In this approach, neither fragment shows fluorescence by itself, unless the two fragments are brought together by interaction of the two target proteins fused to the fragments. Therefore, BiFC could serve as an indicator of target protein interaction. Because BiFC generates a strong fluorescence signal and direct read-out without the need of employing a complicated algorithm like FRET, it has become widely accepted for measuring in vivo protein-protein interaction.

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Moreover, multiple protein interactions could be visualized simultaneously within the same cell by using multi-colour BiFC (Hu and Kerppola, 2003; Shyu et al., 2006).