**3. Yeast two-hybrid screening for searching Hd3a-interacting proteins**

An important element in the characterization of Hd3a function is the identification of other proteins with which it interacts. To reveal the function of Hd3a in rice, yeast two-hybrid system was used as a tool for screening Hd3a-interacting proteins. This system consist of a fusion of protein interest to a DNA-binding domain (DBD-Hd3a) as a bait, and a fusion of cDNA library to a transcriptional activation domain (AD-cDNA library) as a prey (**Figure 5**). The DNA binding domain (DBD) recognizes a specific sequence in the DNA upstream of a promoter and the activation domain (AD) stimulates transcription by binding to RNA polymerase. If the two domains interact, they will activate transcription. In the two-hybrid system, both the DBD domain and the AD domain are fused to two other proteins. If the bait captures the prey, means that proteins are interacting, a complex will be formed and the reporter gene will be activated. The reporter gene is used to monitor for a successful interaction.

As a tool for searching Hd3a interactors, the construction of a bait and prey is important. cDNA library were constructed from leaf blades harvested 35 days after sowing at ZT 0, 2, and 4 when transition from vegetative to reproductive phase occurred and *Hd3a* was highly expressed [7]. The Hd3a full-length cDNA was constructed as a bait and expressed in yeast system (**Figure 6**).

Hd3a Florigen Recruits Different Proteins to Reveal Its Function in Plant Growth and Development http://dx.doi.org/10.5772/intechopen.70263 55

directly correlates with the start of emergence of the fourth leaf (counted downward from the flag leaf). At the time of flag leaf (small last leaf) emergence, the glumes flower primordial has already differentiated and pollen mother cells are being formed [28]. The flag leaf, contributes largely to the filling of grains because it supplies photosynthetic products mainly to the panicle. Flowering time in rice is indicated by the emergence of the flag leaf or the panicle (heading date). The panicle is initiated when the first bract primordium differentiates on the shoot apex, approximately 30 days before panicle emergence (heading). The start of differentiation of the glumes flower primordial follows after the rachis-branches differentiation has occurred (24 days before heading). The pistil and stamen differentiate 20 days before heading. Meiosis in the anther occurs 12 days before heading, and flower organ completion occurs 1–2 days before heading.

**Figure 4.** Rice plant during the developmental stages: vegetative phase, early reproductive phase (left panel) and during

seed maturation (right panel).

54 Plant Engineering

**3. Yeast two-hybrid screening for searching Hd3a-interacting proteins**

will be activated. The reporter gene is used to monitor for a successful interaction.

As a tool for searching Hd3a interactors, the construction of a bait and prey is important. cDNA library were constructed from leaf blades harvested 35 days after sowing at ZT 0, 2, and 4 when transition from vegetative to reproductive phase occurred and *Hd3a* was highly expressed [7]. The Hd3a full-length cDNA was constructed as a bait and expressed in yeast system (**Figure 6**).

An important element in the characterization of Hd3a function is the identification of other proteins with which it interacts. To reveal the function of Hd3a in rice, yeast two-hybrid system was used as a tool for screening Hd3a-interacting proteins. This system consist of a fusion of protein interest to a DNA-binding domain (DBD-Hd3a) as a bait, and a fusion of cDNA library to a transcriptional activation domain (AD-cDNA library) as a prey (**Figure 5**). The DNA binding domain (DBD) recognizes a specific sequence in the DNA upstream of a promoter and the activation domain (AD) stimulates transcription by binding to RNA polymerase. If the two domains interact, they will activate transcription. In the two-hybrid system, both the DBD domain and the AD domain are fused to two other proteins. If the bait captures the prey, means that proteins are interacting, a complex will be formed and the reporter gene

**Figure 5.** Yeast two-hybrid system. Positive interactions can be detected by selecting on plates lacking histidine, followed by screen for β-galactosidase expression.

For the large-scale yeast two-hybrid screen, a total of ~1.6 × 106 transformants were screened for activation of histidine synthase 3 (HIS3) onto selective medium plates without amino acids leucine (L), tryptophan (W) and histidine (SC-His). In the first screening on media SC-His, 354 colonies were obtained. The next screening on medium SC-His containing 3-AT, a total of 96 colonies' unique proteins were identified as positive clones. To eliminate false positive, we used 2.5 mM 3-AT, a competitive inhibitor of the *His3* gene product (histidine synthase),

**Figure 6.** The Hd3a protein expression (pBTM-Hd3a) in yeast strain L40. The protein was run on 10% SDS-PAGE and electrotransferred onto an immobilon-P membrane followed by detection of Hd3a-LexA fusion using the anti-LexA antibody. Molecular weight of Hd3a-Lex A and Lex A are 49 and 26 kDa, respectively.

which is the reporter gene for the interaction in the yeast two-hybrid system. The 96 positive clones with the size ranged between 600-2000 bp then were checked to confirm the presence of "in frame" cDNA-AD fusion. Some clones were found in full-length of cDNA, but the other clones were not. They lack of N-terminal coding region. The clones were then retransformed into yeast containing Hd3a bait to confirm their interaction on selective medium SC-His plus 3-AT (**Figure 7**). The flowchart of yeast two hybrid screens is presented in (**Figure 8**). For the result, a diverse range of interactor proteins can be identified in this yeast two hybrid system (**Table 2**).

The main group of proteins identified in this yeast two-hybrid screen belongs to the class of signal transduction pathway components (57%). Others are classified as proteins that are involved in carbohydrate metabolism, protein/RNA/DNA synthesis, and proteins with unknown function. Interestingly, our yeast two-hybrid screening identified a diverse range of proteins that are mainly involved in signaling. In this chapter, we focus on three candidates: (i) GF14c (G box factor 14c), a 14-3-3 protein identified as a protein that is involved in signaling pathway. The recent results revealed that Ha3a, 14-3-3 and FD form a hexameric florigen activation complex (FAC) and in shoot apical meristem cells, 14-3-3 protein act as intracellular receptor for florigen [29]; (ii) BRI1-kinase domain (KD)-interacting protein 116b, and (iii) a novel myb transcription factor-like protein, namely OsKANADI1.

**Figure 7.** Interaction of Hd3a with its partners in yeast two-hybrid system. The growth of yeast colonies on the plate (LWH) lacking leucine (L), tryptophan (W), and histidine and with 2.5 mM 3-AT (3-aminotriazole) indicates a positive interaction between Hd3a and the particular Hd3a-interacting proteins. 3-AT is a competitive inhibitor of the *HIS3* gene product (histidine synthase), which is the reporter gene for the interaction in the yeast two-hybrid system. Each clone of Hd3a interacting protein (HIP) was spotted onto selective plate.

Hd3a Florigen Recruits Different Proteins to Reveal Its Function in Plant Growth and Development http://dx.doi.org/10.5772/intechopen.70263 57

which is the reporter gene for the interaction in the yeast two-hybrid system. The 96 positive clones with the size ranged between 600-2000 bp then were checked to confirm the presence of "in frame" cDNA-AD fusion. Some clones were found in full-length of cDNA, but the other clones were not. They lack of N-terminal coding region. The clones were then retransformed into yeast containing Hd3a bait to confirm their interaction on selective medium SC-His plus 3-AT (**Figure 7**). The flowchart of yeast two hybrid screens is presented in (**Figure 8**). For the result, a diverse range of interactor proteins can be identified in this yeast two hybrid system

The main group of proteins identified in this yeast two-hybrid screen belongs to the class of signal transduction pathway components (57%). Others are classified as proteins that are involved in carbohydrate metabolism, protein/RNA/DNA synthesis, and proteins with unknown function. Interestingly, our yeast two-hybrid screening identified a diverse range of proteins that are mainly involved in signaling. In this chapter, we focus on three candidates: (i) GF14c (G box factor 14c), a 14-3-3 protein identified as a protein that is involved in signaling pathway. The recent results revealed that Ha3a, 14-3-3 and FD form a hexameric florigen activation complex (FAC) and in shoot apical meristem cells, 14-3-3 protein act as intracellular receptor for florigen [29]; (ii) BRI1-kinase domain (KD)-interacting protein 116b, and (iii) a

**Figure 7.** Interaction of Hd3a with its partners in yeast two-hybrid system. The growth of yeast colonies on the plate (LWH) lacking leucine (L), tryptophan (W), and histidine and with 2.5 mM 3-AT (3-aminotriazole) indicates a positive interaction between Hd3a and the particular Hd3a-interacting proteins. 3-AT is a competitive inhibitor of the *HIS3* gene product (histidine synthase), which is the reporter gene for the interaction in the yeast two-hybrid system. Each clone of

Hd3a interacting protein (HIP) was spotted onto selective plate.

novel myb transcription factor-like protein, namely OsKANADI1.

(**Table 2**).

56 Plant Engineering

**Figure 8.** Flowchart of yeast two-hybrid screening. (A) Interactor hunt using L40 yeast strain; (B) Retrieving putative interactors.



\*\* Length in amino acids of protein.

**Table 2.** Proteins identified by yeast two-hybrid screening.
