| Bacterial type Ⅵ secretory system（Type Ⅵ secretion system,T6SS）has been widely studied as a survival mechanism evolved by bacteria in adverse environment.It was initially considered as a typical virulence factor.Later,it was found that it has many functions,such as antifungal,mediating the uptake of metal ions,regulating the formation of biofilm and so on.The biological function of T6SS mainly depends on the secretion of effector proteins into host cells.In recent years,many studies have found that T6SS mediates the interaction between bacteria and plants,but there is no evidence that T6SS effector proteins can be directly injected into plant cells to play a role,so it is particularly important to find T6SS effector proteins and explore the mechanism of T6SS in mediating bacteria-plant interactions.In the previous study,we found a new T6SS effector protein TsePl in Pseudomonas aeruginosa,and it was found that it can mediate the virulence of Pseudomonas aeruginosa to plants by toxicity test.This suggests that T6SS effector proteins may mediate the interaction between Pseudomonas and plant hosts.Therefore,in this study,T6SS was taken as the main research object,Pseudomonas syringae pv.tomato DC3000 and Pseudomonas aeruginosa PAO1 were taken as the starting strains,and the function of T6SS effector protein in the interaction between pathogenic Pseudomonas and plant host was explored by homology comparison,bacterial two-hybrid experiment,GST-Pull down,Western blot and infection experiment.The specific results are as follows:（1）On the basis of previous experiments,the virulence of Pseudomonas aeruginosa to mung bean mediated by effector protein TseP1 was confirmed by further mung bean infection model and translocation experiment.（2）The results of bioinformatics analysis and NCBI-BLAST homology search showed that there was a homologous protein PSPTO5011（hereinafter referred to as Pst5011）of TseP l in the plant pathogen Pseudomonas syringae DC3000.（3）The results of bacterial two-hybrid experiment showed that there was no interaction between Pst5011 and the key structure VgrG of type Ⅵ secretion system HSI-Ⅱ in Pseudomonas syringae,but directly interacted with another key structural protein Hcp2.The results of GST-Pull down test were consistent with those of bacterial two-hybrid test.（4）The results of secretion experiment showed that the secretion of effector protein Pst5011 was dependent on HSI-Ⅱ in Pseudomonas syringae,but not on HSI-Ⅰ,indicating that Pst5011 was the secretory substrate of HSI-Ⅱ of Pseudomonas syringae.（5）The results of tomato infection experiment showed that the deletion of Pst5011 and HSI-Ⅱ secretory structure gene clpV2 reduced the virulence of Pseudomonas syringae to tomato,and the virulence returned to the wild level after genetic complementary deletion.The results showed that Pst5011 could mediate the virulence of Pseudomonas syringae to tomato and has similar biological functions to TseP1.（6）The results of GST Pull-down experiment showed that there were prey proteins in tomato that might interact with effector protein Pst5011,indicating that there may be target proteins interacting with T6SS effector protein Pst5011 in plant host.Based on the above results,we proposed a mechanism model of T6SS effector protein promoting the infection of pathogenic Pseudomonas into plant host:Pseudomonas effector protein Pst5011/TsePl inhibits the defense response of plant host by anchoring to secretory structural proteins such as VgrG or Hcp and secreting them into the plant host,and interacting with target proteins in the plant host.Finally,it promotes the virulence of pathogenic Pseudomonas to the plant host. |
