Identification And Functional Study Of T6SS Effector Protein TseP1 Of Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a non-fermenting Gram-negative bacteria,widely distributed in various environments due to its strong environmental adaptability,such as air,water,soil,plants,mammalian skin,mucous membranes and so on.At the same time,P.aeruginosa is also a pathogen with a very wide range of infection,and its infection targets mainly include plants,insects and vertebrates.Studies have shown that the Type VI secretion system(T6SS)of P.aeruginosa plays an important role in its adaptation to various environments and pathogenesis.P.aeruginosa encodes three independent sets of T6 SS,namely H1-,H2-and H3-T6 SS,which have various biological functions,such as mediating metal ion uptake,sterilization,mediates the interaction between bacteria and eukaryotic cells and so on.The various biological functions of T6 SS in bacteria are mainly realized through its secreted effector proteins,and specific effector proteins have been identified for the above functions of T6 SS,and its mechanism of action has been well analyzed.However,although some studies have reported that T6 SS plays an important role in the pathogenesis of some pathogens to plants,its mechanism of action is still unclear,mainly because the T6 SS effector proteins that mediate the interaction between bacteria and plants have not been identified.In this study,using P.aeruginosa as the model bacteria and T6 SS as the main research object,we discovered a T6 SS effector protein PA0115 that can act on plants.We named it TseP1 and analyzed its biological function.The specific research results are as follows:1.Using the Hcp bait protein,five proteins that can interact with Hcp1 were screened from P.aeruginosa,which are three T6 SS structural proteins VgrG1 a,VgrG2 b and VgrG3 and two unknown functional proteins TseP1 and PA1969.The interaction of full-length TseP1,PA1969,VgrG1 a,VgrG2 b and VgrG3 with Hcp1 was detected by bacterial two-hybridization.The results showed that Hcp1 interacted with VgrG1 a,VgrG2 b,VgrG3 and TseP1,but not with PA1969.In addition,we also studied the interaction between TseP1 and three VgrG proteins of H1-T6 SS,VgrG1 a,VgrG1 b and VgrG1 c,and found that TseP1 can directly interact with VgrG1 a,but not VgrG1 b and VgrG1 c.2.The results of GST-Pulldown experiment showed that TseP1 could interact with VgrG1 a and Hcp1,but not with VgrG1 b,VgrG1 c and Hcp3,which was completely consistent with the results of bacterial two-hybridization.Therefore,we determined the pairwise interaction among the three proteins TseP1,VgrG1 a,and Hcp1.The results of protein secretion assay showed that the secretion of TseP1 was dependent on H1-T6 SS,but not on H2-T6 SS and H3-T6 SS.3.Through Escherichia coli toxicity experiment,Galleria mellonella competitive infection experiment and G.mellonella toxicity experiment,it was found that TseP1 does not have toxic effects on E.coli;TseP1 neither affects the survival of P.aeruginosa in G.mellonella larvae,nor does it affect the G.mellonella larvae have toxic effects.In order to further study the biological function of TseP1,the protein structure of TseP1 and its homologous proteins were analyzed in this study,and the results showed that: TseP1 is a protein containing an acetyltransferase domain,and the homologous proteins are widely distributed in bacteria,and most of these bacteria are plant-associated bacteria.4.By comparing the ability of P.aeruginosa wild-type strain PAO1 and ?tseP1 strain to infect mung bean and jujube and their virulence to mung bean,we found that TseP1 can enhance the ability of P.aeruginosa to infect plants.The ability of transfection or survival in plants is stronger,and TseP1 also makes P.aeruginosa more toxicto plants.Based on the above research results,this study proposed a mechanism model of Tsep1 acting on plants: when P.aeruginosa infects plants,Tsep1 is translocated to the plant host by H1-T6 SS,binds to the target protein and acetylates it.This modification inhibits the activity of the plant's immune receptor R protein,resulting in the inhibition of the effector-triggered immunity triggered by the effector protein,thus promoting the infection of P.aeruginosa to plants.