Screening And Preliminary Functional Analysis Of Tomato SlMPK1 Interaction Protein

Mitogen activated protein kinase (MAPK) cascade is considered to be one of the main ways to transfer extracellular stimuli into intracellular response in plant. MAPK cascade plays an important role in regulating the physiological state and gene expression of plant responsing to various abiotic stress. The cascade consists of MAPKKK-MAPKK-MAPK. With the development of high-throughput screening techniques such as yeast two-hybrid system and phosphorylated proteomics technology, more and more interactive proteins of MAPK can be identified, especially the MAPK downstream target proteins. Therefore, identifying interactive proteins of MAPK and to investigating their functions are essential to plant biological science researches.Our previous studies indicated that SlMPK1 plays an important role in the process of response to heat stress, but the mechanism is still unclear. In this study, we used yeast two hybrid system (Y2H) to screen for SlMPK1 interacting proteins from the cDNA library of high-temperature-induced tomato, and found many possible interactive proteins of SlMPK1. Some possible SlMPK1 protein interactions were verified in yeast and were confirmed by using of bimolecular fluorescence complementation (BiFC) technology. The subcellular localization of some proteins were also identified successfully. In addition, qRT-PCR was used for analyzing the expression patterns of coding gene of interacting proteins in different tomato organs and in tomato leaves under heat stress. The main findings are as follows:1. Screening SlMPK1-interactive proteins by Y2H.The Y2H bait vector pGBKT7-SlMPK1 was constructed, and was confirmed to be no toxic effect and self-activation in Y2HGold. Bait plasmids were co-transformed with pGADT7/EV into yeast and the recombinant yeast strain was confirmed to be no self-activation on SD/-Trp/-Leu/-His/-Ade/+AbA+X-α-gal+20 mM 3-AT medium plate. We transformed cDNA library plasmids with yeast containing bait vector pGBKT7-SlMPK1 and screened recombinant yeast strains using SD/-Trp/-Leu/-His/-Ade/+AbA+X-a-gal+20 mM 3-AT. Finally, a number of proteins interacting with SlMPK1 were screened from the cDNA library.2. Validation of screening results of SlMPK1 interacting proteins in yeast cells.In order to verify the screening results of SlMPK1 interacting proteins preliminarily, we cloned the full-length sequence of coding genes of interacting proteins and constructed Y2H vectors:pGADT7/04330, pGADT7/MVQ1, pGADT7/SKI, pGADT7/SIMPKK9, pGBKT7/04330, pGBKT7/SKI, pGBKT7/MVQI and pGBKT7/SlMPKK9. Then, we co-transformed them to Y2HGold with pGBKT7/SlMPK1 and pGADT7/SlMPK1 separately and screened recombinant yeast strains by SD medium plates. The results showed that SlMPK1 interacted with 04330、SKI、MVQ1 and SlMPKK9.3. Validation of screening result of SlMPK1 interacting proteins by using BiFC.In order to further verify screening results of SlMPK1 interacting proteins, the co-expressions of SlMPK1-cYFP with 04330-nYFP, SKI-nYFP, MVQ1-nYFP, SlMPKK2-nYFP and SlMPKK9-nYFP were conducted in tobacco leaves. The results showed that SlMPK1 interacted with 04330, SKI and MVQ1 in plant living cells; Interaction between SlMPK1 and 04330 was mainly localized in cytoplasm and nucleus; Interaction between SlMPK1 and SKI was localized in nucleus; Interaction between SlMPK1 and MVQ1 might be localized in cytoplasm and nuclear membrane.4. Sub-cellular localization of S1MPK1 interacting proteins.In order to define the sub-cellular localization of SlMPK1 interacting proteins, we constructed GFP fusion expression vectors:pEGAD/SlMPK1, pEGAD/04330, pEGAD/SKI, pEGAD/MVQ1 and pEGAD/SlMPKK9, and transformed them to agrobacterium (EHA105). Then,the fusion expression vectors were transformated into tobacco leaf. The results showed that SlMPK1,04330 and SlMPKK9 were mainly localized in the cell nucleus and cytoplasm; SKI was localized in the nucleus; MVQ1 might be localized in the cytoplasm and nuclear membrane.5. Analyzing expression characteristics of coding gene of SlMPK1 interacting proteins.We analyzed SlMPK1,04330, SKI, MVQ1 and SIMPKK9 expression characteristics in different organs of tomato and in tomato leaf under heat stress by qRT-PCR. The results showed that:SlMPK1,04330, SKI, MVQ1 and SIMPKK9 were all expressed in roots, stems, leaf, flower, green fruit and red fruit; The relative gene expression level of SIMPK1,04330 and SKI increased under heat stress; The relative gene expression levels of MVQ1 and SIMPKK9 reduced. The results suggested that SlMPK1,04330, SKI, MVQ1 and SlMPKK9 genes are involved in response to heat stress.