As the material basis of life, protein play a critical role in all life activities (Liu et al., 2020). Proteins can operate alone or act as a roleplayer interacting with other proteins (s) (Alanis-Lobato, Andrade-Navarro, & Schaefer, 2017; Srihari, Yong, Patil, & Wong, 2015). More commonly, proteins may function by forming functional complexes (Wang et al., 2020) (Berggård, Linse, & James, 2007)to increase the variety of functional units present and enhance protein diversity (Miura, 2018). The interaction between proteins is essential for several cellular processes, encompassing signaling, molecular switching, ubiquitination, and so on (Kulandaisamy, Lathi, ViswaPoorani, Yugandhar, & Gromiha, 2017). The protein-protein complexes are also vital for effcientlycoordinating multiple enzymatic reactions into productive reaction pathways (Endutkin, Yudkina, Sidorenko, & Zharkov, 2019). Consequently, alterations in protein-protein interactions can yield valuable insight into the cell biology principles and reveal the mechanisms of numerous fundamental biological processes, such as transcription, translation, and cell cycle (Low et al., 2021). Therefore, understanding the interaction between proteins can help us better understand the life activity of cells and reveal the regulatory mechanism hidden beneath the surface.
Numerous approaches have been proposed to detect the interactions between proteins, i.e., pull-down assays (Tinnefeld, 2011), co-immunoprecipitation (Das, Ramachandran, vanWert, & Singal, 2004), bimolecular fluorescence complementation (BiFC) (Miller, Kim, Huh, & Park, 2015) and tandem affinity purification-mass spectrometry (TAP-MS) (Siva Sankar & Dengjel, 2021), and yeast two-hybrid (Y2H) (Paiano, Margiotta, De Luca, & Bucci, 2019). Among these various approaches, a two-hybrid assay is the most commonly used to identify the interacting proteins in vivo (Gonzalez & Kann, 2012). Y2H screening is a well-established genetic approach, which has the advantage of being the most inexpensive, time-saving, and straightforward method (Berggård et al., 2007; Paiano et al., 2019). The establishment of an assay rests on the observation that eukaryotic transcription factors are modular in structure, often consisting of two or more functionally independent domains, such as the DNA-binding domain (BD) and the activation domain (AD), which recruits the transcriptional machinery promoting transcription. These two domains still function separately when they are separated, but cannot activate transcription. Only when the separated two proteins are in close proximity the gene expression occurs. In the two-hybrid system, two plasmids are constructed. The former encodes the cDNA encoding the specific protein of interest fused with the BD domain, defined as "bait." The latter encodes the cDNA encoding the given protein fused with the AD domain, defined as "prey." The two-hybrid system has a unique advantage in detecting and analyzing protein-protein interactions. The method was simple to handle without the steps of recombinant production and subsequent purification of proteins and possessed high sensitivity. Most importantly, an in vivo assay more closely resembles cellular conditions. However, this approach has some limitations including the false positive and false negative results. Plus, Two general strategies have been employed to address these issues: remove the transcription activation domain or add the appropriate concentration of 3-AT (3-Aminotriazole) to the medium(Lathouwers, Wagemans, & Lavigne, 2018). However, these solutions have created new problems. For example, removing the transcription activation domain may affect the detection of protein interactions. Meanwhile, an elevated concentration of 3-AT will result in a false negative result.
The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is found in members of the ethylene-responsive element binding factor, such as ethylene-responsive element binding factors (ERF), Cys2/His2-type (C2H2) and auxin/indoleacetic acid (Aux/IAA) (Kazan, 2006). The EAR motif from these proteins has two distinct conserved amino acid sequences: L/FDLNL /F(x)P and LxLxL (where X can be any amino acid). Proteins containing these motifs are ubiquitously distributed across the plant kingdom and function as critical regulators by negatively regulating genes involved in various biological processes (Kagale, Links, & Rozwadowski, 2010). SRDX, of just 12 amino acids (LDLDLELRLGFA), is a common EAR motif in plant and suppress the expression of target genes, even in the presence of redundant transcription factors (Hiratsu, Matsui, Koyama, & Ohme-Takagi, 2003). Chimeric transcriptional repressors that included the strong repression domain SRDX could suppress the expression of their target genes and cause loss-of-function phenotypes (Cen et al., 2020; Heyl et al., 2008; Hwang, Susila, Nasim, Jung, & Ahn, 2019; Li, Xiong, & Li, 2020). As the SRDX could convert the transcriptional activator into a potent repressor, we attempted to suppress the autoactivation of the bait protein by fusing them with the SRDX in the Y2H system. In this study, we developed a novel method by fusing an SRDX at the C-terminal of target protein, yielding a dominant negative mutant capable of inhibiting the self-activation of the bait protein. Using this method, we chose five transcription factors, namely ApGNAT12, ApCPP5, ApVOZ1, ApMYB2, and ApWRKY41, which are involved in different physiologic process. Our findings demonstrated the efficacy of SRDX motif in reducing the “autoactivation” of bait protein. Our experiment has proved that this new approach, which offers numerous advantages including simplicity, reliability and efficiency, has broad applicability for solving the problem of the bait protein, may independently result in reporter gene activation.