Innate Immunity Responses Of Caenorhabditis Elegans Induced By Phytopathogen Xanthomonas Infection

Innate immunity is an important part of the immune response,it can initiate the acquired immunity,plays a decisive role in the body's immune response.Discussing the evolutionary origin of innate immunity will be useful for fully understanding the innate immune responses,provide a theoretical basis for selecting the strategies of regulating innate immunity,and further offer new ideas for the treatment of infectious diseases.The key issue of studying innate immunity is to establish the appropriate pathogen-animal infectious model.In this thesis,we take the following strategies to make model:on the pathogen selection,using plant pathogenic bacteria to exclude the innate immunity differences caused by animal pathogen species.Xanthomonas is a plant pathogenic bacterium.With the sequencing of the complete genome,Xanthomonas oryzae pv.Oryzae(Xoo)has become one of pathogen model for determing the induced immune responses in infected plants.On the host selection,Caenorhabditis elegans(C.elegans)only has innate immunity,and can both be infected by animal pathogens and plant pathogens.Therefore,we establish the Xanthomonas-C.elegans infection model to investigate the most conservative and basic innate immune responses in C.elegans,expect it can be an accumulated data for the evolution of innate immunity.In this study,five nthomonas are selected:PX086?PXO112?JXOV?Xpw andXpahpC,to infect the rice leaf then observe the withered degree,and feed the worms then analyse the survival rate of worms.Results indicate that similar virulence of Xanthomonas in rice could be ranked in C.elegans.Pathoginic extracellular metabolites are no-toxic to nematodes,and heat-killed bacteria have weakened effect on nematode death,showing Xanthomonas kills worm by live bacteria.SEM observation indicates pathogens proliferate in the worm intestine,and CFU increase along with the time extension.These evidences support that Xanthomonas can infect C.elegans successfully.Further,biofilm production is not directly related to their pathogenicity.The stronger infection ability of Xanthomonas,the lower pharyngeal pumping rate of C.elegans,and the worse selective in food choice plates.These illustrate Xanthomonas is not suit for C.elegans food source.Consequently,infection by Xanthomonas induced malnutrition and decreased survival.p38 MAPK and DAF-2/DAF-16 signal pathways control innate immune response to intestinal infection.PX099 and JXO? are more toxic in literature and are selected to study immune responses after worm is infected.Results show that PX099 and JXO? proliferate in the intestine of C.elegans,and make the intestinal region extend significantly.Infection inhibit the egg laying and leads to the brood size decreasing.These support that PX099 and JXO? can infect C.elegans like other five strains,increased death rate is because of infection and malnutrition.qRT-PCR is performed to determine the expression level of effectors associated the two pathways.Results indicate that sek-1,one component of the p38 MAPK pathway,is notably induced in wild type N2 and CF1295(daf-2;daf-16),C17H12.8 gene,an effector molecular downstream of sek-1,has significant increased expression level;however,the effectors mtl-1 and sod-3,downstream of DAF-2/DAF-16 pathway,can be triggered only when daf-2 is mutant.It suggests that p38 MAPK can always be activated,but DAF-2/DAF-16 can only be induced when daf-2 is mutant.daf-2 mutation can enhance the resistance of nematode,and expression level of downstream effectors can also be increased.Expression of sek-1 gene in CB1370(daf-2)mutant has no change,but it is significantly increased in the CF1295(daf-2;daf-16)double mutant,indicating that sek-1 can compensate the weakened resistance of worm because of daf-16 mutation in some degree.After infection by Xoo,DAF-16 does not tranlocate to the nuclear,however,phosphorylation of PMK-1 can be triggered during this process,and these also verify the RT-PCR results described above.Additionally,C.elegans also employ avoidance behavior to reduce infection.sek-1 and tol-1 mutation have no influence on worm avoidance behavior,suggesting that sek-1 and tol-1 are not involved in avoidance behavior.However,survival analysis show sek-1 and tol-1 participate the immune response on Xoo infection.sek-1 is involved in cell-autonomous innate immunity responses of worm intestine,while in the nervous system it regulate immunity in non-cell-autonomous manner.tol-1 and sek-1 genes have some overlap roles in C.elegans immune response to Xoo infection.p38 MAPK pathway can always be activated during the infection by Xoo,which indicate immune responses mediated by this pathway play a more general role,it may be more conserved in evolution.However,the traditional view is that DAF-2/DAF-16 pathway regulates the basal stress response of C.elegans,and p38 MAPK is a more specific immunity pathway.Our results suggest that p38 pathway may be a key for future research on innate immunity.