Screening and Bioinformatics Analysis of Proteins Interacting with StMAPKK1 in Potato by Yeast Two-Hybrid System

Mitogen-activated protein kinase (MAPK) cascade reaction is one of important and complex signal networks involved in plant growth and development, hormone and stress response. As one of its main members, mitogen-activated protein kinase kinase (MAPKK) is located in the middle of the cascade reaction and plays a key role in signal collection and divergence. There were shown that potato ( Solanum tuberosum L.) StMAPKK1 (PGSC0003DMT400000744) gene responded to drought stress. Therefore, StMAPKK1 gene was firstly selected to screen its interacting protein. In this study, the bait vector pGBKT7-StMAPKK1 was constructed by homologous recombination and used to screen potato cDNA library by yeast ( Saccharomyces cerevisiae ) two-hybrid system. Five StMAPKK1-interacting proteins, hydrolase (hydrolyzing O-glycosyl compound), RING-H2 subgroup RHE protein, cyanate hydratase, ARF GTPase activator, and a C2 domain-containing protein, were obtained through this screening and were identificated by bioinformatics analysis, and the interaction was verified by small-scale hybridization verification. The results provided theoretical basis for further study on the signal pathway and biological function of potato StMAPKK1.

There are three main numbers of MAPK cascade reaction, mitogen activated protein kinase kinase kinase (MAPKKK), mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK). Phosphorylation at specific motifs acts on the upstream and downstream of certain members to form cascade signaling pathways (Iftikhar et al, 2017). The number of MAPKKs members is the least, and it can be divided into four subfamilies A, B, C, D. MAPKKs is a double kinase, it can be activated by upstream MAPKKKs phosphorylation and activate downsteam MAPKs through phosphorylation. MAPKK responsed to drought stress in plants, AtMAPKKK18-AtMKK3-AtMPK1/2 involved in drought resistance regulation with ABA induction dependence (Li et al., 2017). Overexpression of ZmMKK1 or ZmMKK4 in Arabidopsis enhanced drought resistance (Cai et al., 2014;Kong et al., 2011). GhMKK3 in cotton enhanced drought resistance through regulating stomatal size and root growth (Wang et al., 2016). Also, studies have found that OsMKK1 were involved in salt stress signaling in rice  and OsMKK6 played a role in low temperature and salt stress (Xie et al., 2012).

Construction of yeast two-hybrid bait vector pGBKT7-StMAPKK1
The full length transcription sequence of StMAPKK1 gene (PGSC0003DMT400000744) is 1 666 bp. The inserted fragment designed according to the design principle of homologous recombination primers would have homologous sequence 21 bp at the end of insertion fragment 5' and 3' respectively, so the product size is about 1 708 bp, which is consistent with the results of electrophoresis ( Figure 1A). pGBKT7 plasmid size is 7 303 bp. The linear plasmid was obtained from pGBKT7 plasmid digestion by PstⅠand NdeⅠ. The size of the linear plasmid is about 7 270 bp, and the electrophoresis detection was in accordance with the expected size ( Figure 1B).
The recombinant product was transformed into E.coli DH5α competent cell and coated on LB solid plate (50 mg/L Kan). Six single colonies were identified by PCR, and there were obvious bands at about 1 708 bp ( Figure  1C). StMAPKK1 gene had enzyme cutting sites of NdeⅠ at 572 bp and 582 bp, so the recombinant plasmid pGBKT7-StMAPKK1 digestion by PstⅠand NdeⅠ should have three fragments about 7 270 bp, 1 126 bp and 572 bp, respectively. The results of electrophoresis ( Figure 1D) were in good agreement with the expected results. Mainwhile, the results of sequencing showed that StMAPKK1 gene had been successfully inserted into pGBKT7 vector ( Figure 2). 1.2 Detection of toxicity and self-activating activity of yeast two-hybrid bait vector Y187 yeast containing pGBKT7 empty vector or pGBKT7-StMAPKK1 bait vector was coated on single deficient medium SD/-Trp/X-α-gal. The growth number of the two strains was about the same (Figure 3), which indicated that the bait vector has no toxic to yeast cells. At the same time, the two strains did not grow on SD/-Trp/-His/ X-α-gal or SD/-Trp/-Ade/X-α-gal, indicating that the strain had no self-activating activity and could not activate downstream reporter genes alone.

Small-scale hybridization verification of positive clone
The hybrid solution of bait protein and library was coated on 50 quadruple dropout media QDO. A total of 406 single colonies with large diameter were selected for identification and screening by X-α-Gal staining. 210 Molecular Plant Breeding 2020, Vol.11, No.3, 1-8 http://genbreedpublisher.com/index.php/mpb 4 colonies which grew within 48 hours and displayed as blue were regarded as positive clones. The positive rate was 51.72%. Five different StMAPKK1 interacting proteins named C1 to C5, respectively, were identified by electrophoresis and sequencing. The proportion was 87%, 2.9%, 4.8%, 2.9% and 2.9%, respectively.
Five StMAPKK1 interacting protein gene plasmids were randomly selected and transferred back into AH109 yeast. The AH109 with plasmid was crossbred with Y187 containing bait vector and coated on QDO/X-α-Gal medium to culture. After two days of culture, the colonies with different blue depths grew at all five points ( Figure  4). The results showed that all the five positive cloned genes could be verified by small-scale cross rotation, and the interaction between them was true and credible.

Identification of positive clones
By sequencing and analysis of interacting protein genes, five StMAPKK1 interacting proteins C1-C5 were obtained, and then the basic physical and chemical properties of these genes were obtained (Table 1). The names of these five genes from NCBI were hydrolase, (hydrolyzing O-glycosyl compounds), RING-H2 subgroup RHE protein, cyanate hydratase, ARF GTPase activator and C2 domain-containing protein.

Discussion
After sequencing the positive clones, A total of five StMAPKK1 interacting proteins were obtained: Hydrolase (hydrolyzing O-glycosyl compounds), RING-H2 subgroup RHE protein, cyanate hydratase, ARF GTPase activator and C2 domain-containing protein. Among them, the interacting protein C1 is hydrolase (hydrolyzing O-glycosyl compounds), and it can be also noted as galactitol-sucrose galactosyltransferase. It belongs to the GH36C subfamily of 11 subfamilies (GH36A to GH36K) in the glycoside hydrolase family 36. The members of the family can also be called raffinose synthase (RS) or seed imbibition protein I (Sip I). Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway, it plays an important role in gaining drought resistance and prolonging the life of plant seeds (Peterbauer et al., 2002). In germinating maize seeds, some studies have shown that raffinose synthases are exclusively responsible for raffinose family oligosaccharides (RFO) breakdown (Andreas et al., 2008). CsRS expression was induced by low temperature and exogenous plant hormone abscisic acid (ABA) in cucumber leaves and fruits, RS activity and the content of raffinose increased gradually (Sui et al., 2012). Also in beet (Beta vulgaris L.), two kinds of raffinose synthase identified in the study were proved to be involved in cold stress and salt stress responses (Kito et al., 2018). The interacting protein C2 was RING-H2 subgroup RHE protein, it is a cyclic H2 protein with E3 ubiquitin ligase activity and belongs to ATL (Arabidopsis toxicos para levadura) gene family (MartínezGarcía et al., 1996). It was proved that PtaRHE1 was involved in the development of secondary phloem fibers in poplar hybrid varieties (Baldacci-Cresp et al., 2015). There are also some studies shown that the ATL gene family may be involved in early defense responses to pathogen attack (Salinas-Mondragón et al., 1999). The interacting protein C3 cyanate hydratase also known as cyanoate lyase (EC:4.2.1.104), is responsible for the hydrolysis of cyanate ester and exists in bacteria and plants, so that the organism with this kind of enzyme can overcome the toxicity of environmental cyanate ester (Sung and Fuchs, 1988). The interacting protein C4 ARF (ADP-ribosylation factor) GTPase activating factor is often involved in vesicle transport, especially in coatomer-coated vesicle transport between Golgi cisternae (Rothman and Wieland, 1996). In domain analysis, this activating factor contains C2 domain, which is involved in regulating membrane transport (Thomas and Rizo, 1996). At the same time, the interacting protein C5, a C2 domain-containing protein, the C2 domain could exhibit a distinct characteristic of binding to a variety of different ligands and substrates, including Ca 2+ , phospholipids, phosphoinositides, and intracellular proteins (Cho and Stahelin, 2006). A protein, OsPBP1, containing C2 domain in rice has been shown to regulate pollen fertility through Ca 2+ and phospholipid signaling pathways (Yang et al., 2008).
In this experiment, the StMAPKK1 interacting proteins screened by yeast two-hybrid technique were different from the expected target results, other types of kinases in the MAPKs cascade reaction, such as MAPKKKs and MAPKs, were not obtained. There were some studies showed that there are other cascade pathways in addition to the typical MAPK pathway MAPKKK-MAPKK-MAPK, which may be cross-transmitted with other signal pathways.