The Impact Of Phosphatidylcholine On Translation And Translocation Of T3SS Component Proteins In Pseudomonas Syringae

Pseudomonas syringae pv.syringae Vane Hall 1336 is important plant pathogens which has a wide range of hosts,and causes a variety of disease on branches,leaves,flowers,fruit of woody plants and herbs.In our previous studies,we found that Pseudomonas syringae pv.syringae Vane Hall 1336 only uses phosphatidylcholine synthase(Pcs)pathway to synthesize phosphatidylcholine(PC).The deletion of the pcs gene by knockout of phosphatidylcholine synthase gene made the pcs~-mutant unable to synthesize phosphatidylcholine.When phosphatidylcholine is absent in its membranes,Pseudomonas syringae pv.syringae Vane Hall 1336 almost loses bacterial virulence,and its virulence is mainly associated with those effectors secreted via type?secretion system(T3SS).It has been proposed that the probable mechanisms for the impact of PC on bacterial toxicity are phosphatidylcholine may affect the expression of T3SS-associated genes,the protein transport from cytoplasm to the cell membrane or the protein assembly in bacterial membranes or the topological structure of T3SS-associated proteins in cell membranes.In order to investigate the molecular mechanism of the impact of phosphatidylcholine on the function of type?secretion system,It is necessary to compare the difference of T3SS-associated proteins between the wild type and 1336 pcs~-mutant at transcription level,translation level and transportion level.In our provious study,we found that the absence of phosphatidylcholine did not affect the expression of T3SS-associated genes at transcription level.Hence,in this study,we mainly focus on gene cloning,protein expression,protein pufication,properation of polyclonal antibodies of all T3SS component proteins of P.syringae pv.syringae van Hall 1336,and then analysis by western blotting to assese the difference of T3SSS component proteins in cytoplasm and cell membranes between the wild type and the1336 pcs~-mutant.In this study,4 gene of the T3SS apparatus were successfully cloned,7 proteins of the T3SS apparatus were successfully expressed,purified,and obtained their polyclonal antibodies,and 13 proteins of the T3SS apparatus were successfully analysis by western blotting.Analysis of western blotting showed that 12 proteins in cytoplasmic fractions did not show significant difference between the wild type and the 1336 pcs~-mutant.Only HrpR protein displayed a noticeable accumulation in the cytoplasm of the 1336 pcs~-mutant compared to the wild type.By contrast,4 proteins obviously reduced their contents in the membrane extract of the 1336 pcs~-mutant as compared with those for the wild type.Less HrpB,HrpE,HrpJ and HrpR in the membranes of the 1336 pcs~-mutant indicated that the absence of phosphatidylcholine might affect protein transport form bacterial cytoplasm to membranes.