The Analysis Of Interaction Of Exocyst Subunit SEC6 And PME17 And Their Roles In Arabidopsis Thaliana

Proper vesicle tethering and membrane fusion are essential for forming cell plate. The exocyst is an octameric vesicle tethering complex that functions upstream of SNARE mediated exocytotic vesicle fusion with the plasma membrane. In Arabidopsis, Previous studies showed that the SEC6 localizes to the cell plate during cytokinesis. To learn about more the function of SEC6 protein in Arabidopsis, By screening the arabidopsis cDNA library in yeast-two hybrid system, we obtained an interacting protein PME17 （Pectin Methylesterase 17）, which is one kind of Pectin Methylesterasein protein. We confirmed the interaction of SEC6 and PME17 by a number of approaches, and identified the function during the cell plate formation through the phenotype.U SEC6 interacts with PME17 in vitro and in vivoIn order to verify the interaction between SEC6 and PME17 in vivo, the full length PME17 and three truncations:PME17-1 （25-511aa）, PME17-C （172-521aa） and PME17-N （27-206aa） were constructed, the physical interaction between SEC6 and different truncates were assayed by yeast two-hybird. The C （172-521aa） part of PME17 protein is necessary for the interaction between PME17 and SEC6; We verified the interaction between SEC6 and PME17 in vitro by GST-pull down assay, and then confirmed the physical interaction between SEC6 and PME17-C; SEC6-PME17 interaction was further confirmed by the bimolecular fluorescence complementation （BiFC） assay in onion epidermal cells and N. benthamiana leaves. These results confirmed SEC6 and PME17 is a pair of interacting protein.2, The location of PME17 and the homozygous plants of pmel7In the previous study, fluorescence microscopy showed SEC6-GFP fluorescent signals were concentrated on the cell plate of dividing cells until the end of cytokinesis. In order to study the location of AtPME17, we produced the transgenic tobacco BY-2 cell and Arabidopsis transgenic plants of p35S:PME17-GFP, We also found that PME17 was involved in the entire process of cell plate formation.The homozygous plants of pme17 （Salk₀59908） were obtained by PCR assay and PME17 mRNA expression analysis. In the previous study we have been obtain the pollen rescued complementation lines PRsec6, the PRsec6 mutant plants showed the phenotype in primary root development and embryo morphogenesis, and pollen tube growth and maturity rates of seed. These results suggested that SEC6 might be working in primary root development. As the microarray information and expression profiling indicated, pme17 was constitutive expression and expressed high in root, that indicated pme17 might play some role in root.Referring to the microarray information, we identified PMEl 7 might be involved in abiotic or biotic stress response in Arabidopsis thaliana.There are two novel facets in our study. On one hand, the interaction of Exocyst subunit SEC6 and PME17 was firstly reported in plant. Futher study firstly showed that PME17 located in the cell plate. On the other hand, PME17 might be interact with the exocyst complex and participate in the cell plate formation through the vesicle trafficking.