Development Of A Novel System For Co-expressing Multiple Chimeric Fluorescent Proteins In Plants And Its Application In Protein Polar Exocytosis During Plant Cell Wall Formation

The knowledge of the subcellular localization of a protein is crucial for understanding its physiological functions in cells.Chimeric fluorescent fusion with proteins that are interested in is a widely-used method to study their subcellular localizations and dynamics.In particlular,co-expression of the target protein with reportor proteins of organelles and secretion vesicles in the cell is useful to elucidate the biogenesis and spation-temperal interactions of the proteins.For conventional protein co-expression approaches,multiple independent expression plasmids for expressing individual fusion proteins are mixed and used for the co-expression of chimeric fluorescent fusion proteins in plants.There are several drawbacks of the conventional methods,including low co-expression efficiency,expression-level variations,and time-consuming in genetic crossing and screening.In this study,we firstly set up a robust and novel system for co-expression of multiple chimeric fluorescent proteins in plants.Secondly,we applied our newly developed system to study the roles of protein polar transportation and targeting in plant cell wall formation and cell polarilization.We have set up a robust system that contains multiple semi-independent expressing cassettes for co-expression of multiple chimeric fluorescent fusion proteins can by a single vector.As a validation of this system,we co-expressed fluorescently fused vacuolar sorting receptor and secretory carrier membrane protein via either transient expression or stable genetic transformation in plant cells.The results showed that their perspective subcellular localizations were consistent with previous studies.Furthermore,each protein expression cassette contains its own functional protein expression elements,and therefore,it can be flexibly adjusted to meet diverse expression demand.Also,it is easy and convenient to perform the assembly and manipulation of DNA fragments by using an optimized one-step reaction without additional digestion and ligation steps.In addition,it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications such as FRET and BiFC.Thereafter,we applied this novel and new approach to study polar protein transportation and targeting for the plant cell formation and redigity.Plant cell wall mainly consists of cellulose,callose and pectin as well as a small portion of other polysaccharides and proteins.The synthesis of these cell wall materials requires the participation of cellulose synthase,pectin methylesterase and callose synthase.Pectin methylesterase NtPPME1 are synthesized in ER and secreted to the PM or extracellular space via secretion vesicles derived from Golgi apperaus.Cellulose synthase complex and callose synthase complexes are assembled in endoplasmic reticulum and secreted to plasma membrane via vesicle.However,the detail underlying mechanisms regulating the polar secretion of cellulose synthase,pectin methylesterase and callose synthase still remain unknown.In this study,we use polarized and rapid growing pollen tubes as the model cell system for studying the regulatory mechanisms for protein polar exocytosis during plant cell wall formation.We have cloned the key proteins from protein exocyts complex which is critical for mediating exocytic fusion of the secretion vesicles with the PM.Furthermore,we have also cloned the reporter proteins for different lipid components to elucidate the roles of lipid in vesicle exocytosis.By a combination of our newly developed convenient and robust chimeric fluorescent fusion protein co-expression system,we aim to explore the underpinning mechanisms of protein polar exocytosis in regulating plant cell wall formation.