| Crop pathogenic nematodes are seriously harmful.It's difficult to control plant parasitic nematodes because of the rapid population growth.Due to the limitations of traditional control methods and the enhancement of people's awareness of environmental protection,people are more focused in efficient,green and environmentally friendly biological control.People pay more attention to bacteria as the biocontrol agents against plant-parasite nematodes because of their fast growth,easy cultivation and good killing effects.Bacillus nematocida B16 showed significant killing activities against a variety of plant parasitic nematodes,and its successful colonization in the nematode gut is the key factor to complete the infection and obtain the maximum biocontrol efficiency.In the previous study,an endophyte SCO41(Phytobacter diazotrophicus)was isolated from the free-living nematode C.elegans intestine,and it was found that the strain SCO41 can inhibit the growth of B.nematicide B16 in vitro and the colonization of B.nematicide B16 in vivo.The main inhibitory factor was found to be the flagellin P1.However,the molecular mechanism of the effect of the protein P1 on B.nematicide B16 is still unclear.It has been found that bacterial flagellin can specifically bind to Toll-like receptor 5(TLR5),thereby triggering the host's innate immune response.This reaction is common in eukaryotes and has not been reported in prokaryotes.So is there a similar binding between P1 and B.nematicide B16? It's still not clear.In this study,B.nematicide B16 and P.diazotrophicus SCO41 were selected as the research objects.The mechanism of the protein P1 secreted by P.diazotrophicus SCO41 on B.nematicide B16 were explored via the experiments of the activity assay,FITC fluorescent label,proteomic analysis and molecular biology methods.The results provide a new perspective on the interaction between the pathogenic bacteria,the gut symbiotic bacteria and the nematode hosts.Modifing B.nematicide B16 to enhance its adaptability under natural conditions,and defend the resistance to endophytes by means of genetic engineering.Finally make B.nematicide B16 achieve the purpose of fully controlling nematodes.The main results of this study are as follows:1.Activity test and localization analysis of P1 on B.nematocida B16.The biological activity test of the protein P1 includes the in vitro antibacterial activity test and morphological change observation of B.nematocida B16.The antibacterial activity test showed that the recombinant protein P1 had obvious inhibitory effect on B.nematocida B16.The observation of morphological changes showed that the B.nematocida B16 cells treated with protein buffer had normal shape,and the B.nematocida B16 cells treated with purified protein P1 and recombinant protein P1 became thinner and longer,and some of them became bent.The localization experiments found that green fluorescence appeared on the cytoplasm and the cell membrane of B.nematocida B16,and the fluorescence intensity at the cell division was significantly enhanced.It indicated that the protein P1 was localized in both the cytoplasm and the cell membrane of B16 strain,in the latter especially at the position of cell division.The results suggest that the protein P1 may inhibit B.nematocida B16 colonization in the nematode gut by affecting the normal division of B.nematocida B16.2.Proteomic analysis.The total protein before and after the recombinant protein P1 treatment of B.nematocida B16 was extracted,and the iTRAQ analysis of differential protein expressions in the control and the experimental group of B.nematocida B16 was performed.It was found that 203 and 506 proteins were up-regulated in B.nematocida B16 after 4 h and24 h treatment with recombinant protein P1,and 124 proteins were simultaneously up-regulated at both time points.The up-regulated proteins can be divided into ribosomal proteins,dehydrogenases,transferases,elongation factors,etc.There were 16 and 541down-regulated proteins treated with recombinant protein P1 for 4 h and 24 h,respectively.The down-regulated proteins can be divided into spore formation-related proteins,transferases,dehydrogenases,peptidases,binding proteins,etc.After B.nematocida B16 was treated with recombinant protein P1 for 24 h,the expression of elongation factor protein was increased significantly,and the expression of stress proteins including spore germination,sporulation,and oxidative stress-related stress proteins decreased significantly.It showed that the proteins P1 were mainly related to biological processes such as metabolism and stress response.KEGG pathway analysis revealed that the differential proteins were involved in glycolysis,infection,HIF-1 signaling pathway and two-component system process.The results indicate that the protein P1 may inhibit the activity of B.nematocida B16 by reducing its ability to adapt to external stresses,thereby achieving the purpose of inhibiting nematode colonization.3.Changes in differential protein expression were verified by qPCR.The real-time fluorescence quantitative PCR experiments were performed on the differentially expressed proteins,and it was showed that the relative expression of elongation factor-related genes fus A,tuf,tsf,gre A and efp was significantly up-regulated,and the relative expression of spore formation-related gene spo0 A was significantly down-regulated after recombinant protein P1 treated B.nematocida B16.The spore count experiments and spore staining experiments showed that B.nematocida B16 had a reduced number of spores after treated with recombinant protein P1 for 24 h.It indicated that the protein P1 inhibited the formation of B.nematocida B16 spores.The results show that the protein P1 may affect the corresponding life activities of B.nematocida B16 by inhibiting B.nematocida B16 from forming spores,thereby inhibiting the colonization of B.nematocida B16 in the intestine of nematodes.4.Molecular docking analysis.According to the FITC fluorescent label,it could be assumed that the protein P1 acts on the cytoplasm and the cell membrane of B.nematocida B16.Therefore,we preferentially screened the membrane proteins and kinases in iTRAQ and compared them with the whole genome of B.nematocida B16 to obtain their protein sequences.Retrieving the three-dimensional structures from the Protein Data Bank and using ZDOCK for the protein-to-protein docking,it found three potential receptor proteins that the protein P1 acts on B.nematocida B16.The output results of ZDOCK analysis indicated that the scores of serine/threonine protein kinase,the membrane protein insertase YidC,and membrane protein CydB with the protein P1 are 1668.462,1945.839,and 1994.676,respectively.The results indicate that the protein P1 may combine with kinases and membrane proteins in B.nematocida B16 and affect bacterial growth,division and cell function,thereby inhibiting the colonization of B.nematocida B16 nematodes in the intestine.In conclusion,the protein P1 may inhibit B.nematocida B16 colonization in the nematode intestinal tract by affecting the normal division of B.nematocida B16;it may also inhibit the activity of B.nematocida B16 by reducing its ability to adapt to external stresses,thereby achieving the purpose of inhibiting nematode colonization.The protein P1 acts on the cytoplasm and the cell membrane of B.nematocida B16.It was found that the protein P1 can bind to potential receptor proteins of B.nematocida B16,which are serine/threonine protein kinase,the membrane protein insertase YidC,and membrane protein CydB,respectively.The protein P1 could interact with the amino acid residues at the binding interface of the three potential receptors proteins of B.nematocida B16,respectively,which will lay the foundation for the subsequent study of the molecular mechanism of the protein P1 at specific sites in B.nematocida B16. |
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