Biomolecular fluorescence complementation (BiFC)

The method of choice to examine the in planta protein-protein association is Foster resonance energy transfer (FRET). However, efforts to detect the FRET between the ethylene receptor ETR1 and the CTRs were unsuccessful possibly due to the tendency of the ethylene receptor ETR1 to form aggregation bodies (data not shown). Therefore, biomolecular fluorescence complementation (BiFC) assay was used to investigate the receptor-CTR interaction.

The overall strategy of BiFC was outlined in Figure 3.46. In the BiFC assay, the tomato ethylene receptors were expressed in onion cells fused to the N-terminus of YFP (YFPN, aa 1-154) and the putative downstream kinases (CTRs) were expressed with the C-terminus of YFP fusion (YFPC, aa 155-238). Neither YFPN nor YFPC is fluorescent by itself, unless the ethylene receptor associates with CTR, in which case the N- and C-terminus of the YFP would be brought together and a functional YFP would be regenerated. Therefore, the fluorescent emission of the newly formed YFP would be an indication of the protein-protein association between the ethylene receptor and CTR.

As BiFC is a relatively novel technique and has not yet been used to study receptor-CTR interaction, a control experiment was carried out using the ethylene receptor NR. Because in this study, it has been shown that all the tomato CTRs except for CTR2 could co-localize with the ethylene receptor NR (Figure 3.44) and this is also

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in agreement with the previous yeast two-hybrid assay that LeCTR2 could not interact with NR (see section 3.8 for details). Therefore, it was reasoned that if the NR-LeCTR1 BiFC pair showed a positive result in the pilot experiment, whilst the NR-LeCTR2 pair could not, this BiFC method would be suitable to investigate the ethylene receptor-CTR interaction. The control BiFC experiment showed that NR has strong BiFC with CTR1 but not with CTR2 (Figure 3.47). Therefore, the interaction between the ethylene receptor LeETR1 and the four CTRs were subsequently examined. The results in Figure 3.48 show that only CTR1, 3 and 4 could have fluorescence complementation with LeETR1 in the BiFC assay. This suggests that LeCTR2 might not be able to interact with LeETR1, which is also in agreement with the ProQuest yeast two-hybrid assay results (see section 3.8).

However, it is not clear whether or not the recombinant LeCTR2 protein fused to the C-terminus of YFP (LeCTR2-YFPC) has been properly expressed; as the LeCTR2-YFPC protein by itself is not fluorescent and could not be detected by using the fluorescent microscope. For example, negative BiFC result would be obtained if the LeCTR2 has not been cloned in frame with the downstream YFPC or the expression of the recombinant protein was suppressed. To help resolve the issue, western blot using anti-GFP antibody could be performed to confirm the presence of the LeCTR2-YFPC protein. However, it is not practical to extract sufficient protein from onion epidermal cells for western blot analysis due to the low efficiency of the biolistic transformation.

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It has been reported that the N-terminus of YFP (YFPN) alone can show BiFC with any protein fused to the C-terminus of the YFP (YFPC) through self-assembling (Hu et al., 2000; Hu and Kerppola, 2003; Shyu et al., 2006). The YFPN protein would therefore act as a positive control to demonstrate that the protein fused to YFPC could be correctly expressed. In order to rule out the possibility that lack of a positive BiFC signal in the LeETR1-LeCTR2 experiment is due to the failure of LeCTR2-YFPC protein expression, a control experiment was performed to co-express YFPN with LeCTR2-YFPC. This combination generated a strong BiFC signal and confirmed that the LeCTR2-YFPC protein could be expressed (Figure 3.49). Therefore, the negative BiFC result of LeETR1-LeCTR2 in Figure 3.48 is unlikely to be resulted in experimental error such as lack of LeCTR2-YFPN protein expression.

Collectively, the BiFC analysis suggests that only LeCTR1, LeCTR3 and LeCTR4 could interact with the ethylene receptors LeETR1 when transiently expressed in the onion epidermal cells, while LeCTR2 might not take part in the protein-protein interaction with the ethylene receptor.

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Figure 3.46: Biomolecular fluorescence complementation (BiFC)

The ethylene receptors were fused to the N-terminus of YFP (N-YFP) and the CTRs were fused to the C-terminus of YFP (C-YFP). Neither the N- or C-terminal domain of the YFP would be expected to show any fluorescence by itself (left), unless CTR interacts with the receptor, in which case the two separated fragments of YFP would be brought together and the YFP emission would be observed (right).

In a BiFC assay, onion cells are co-transformed with three constructs that include: 1) The ethylene receptor fused to YFPN

2) CTR fused to the YFPC

3) A construct expressing the red fluorescent protein mRFP1, which act as the indicator for successful transformation.

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Figure 3.47: BiFC assay of the receptor NR with CTR1 and CTR2

The onion cell expressing NR-YFPN and CTR1-YFPC showed yellow fluorescence in the BiFC channel. This suggests that the LeCTR1 can associate with NR. The left images (yellow) were from the BiFC channel, in which the presence of YFP fluorescence indicates a positive protein-protein interaction result. The red images (right) were from the red fluorescent protein co-transformed with the BiFC constructs, which serves as the indicator of successful transformation. YFPN: N-terminus of YFP; YFPC: C-termini of YFP.

(A) An onion cell co-transformed with NR-YFPN, LeCTR1-YFPC and mRFP1. (B) An onion cell co-transformed with NR-YFPN, LeCTR2-YFPC and mRFP1.

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Figure 3.48: BiFC assay of the receptor ETR1 with 4 CTRs

Onion epidermal cells were co-transformed with three constructs: LeETR1-YFPN, CTR (1 to 4)-YFPC and mRFP1. The images of the BiFC channel (left) are shown in the colour yellow and the mRFP1 images (right) are indicator of successful transformation. YFPN/C: the N- and C-terminal fragment of YFP.

The tomato ethylene receptor ETR1 showed positive BiFC results with CTR1, CTR3 and CTR4, but not with LeCTR2 as indicated by the images of the BiFC channel (yellow).

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Figure 3.49: LeCTR2-YFPC showed positive BiFC with YFPN

An onion cell was co-transformed with three constructs: YFPN, CTR2-YFPC and mRFP1. The images of the BiFC channel (right) is shown in the colour yellow and the mRFP1 images (left) is the indicator of successful transformation.

The LeCTR2-YFPC construct previously showed no BiFC with the ethylene receptor NR and ETR1 (Figure 3.47 and 4.19). The BiFC between LeCTR2-YFPC and the positive control (YFPN) suggests that the LeCTR2 construct was functional and the LeCTR2-YFPC protein has been properly expressed.

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