| Plant parasitic nematodes have caused great losses to the global economy,and biological control has become a prevention method highly recommended by all countries in the world because it will not cause pollution to the environment.The biggest bottleneck of biological control of plant parasitic nematodes is its instability and low control efficiency in the field.The elucidation of the molecular mechanism of biocontrol strains infecting nematodes can provide a theoretical basis for enhancing the control effect of biocontrol bacteria.Perception is the first step during the process of bacterial infection of nematodes,which determines the success or failure of infection.Bacteria can sense signal molecules in the environment and regulate the expression of their own genes to adapt to the environment.In our previous studies,we have found that one bacterial strain Bacillus nematocida B16 isolated from Yunnan forest soil shows has high killing activities against many plant parasitic nematodes,which can sense the morpholine signal released by nematodes,thus forming spores in advance.Spores can protect B16 from chewing and phagocytosis,and then B16 successfully colonize in the nematodes’ intestines,destroy the intestines and kill nematodes.In contrast,B.subtilis 168 can not perceive the presence of nematodes when it meets nematodes and is eventually eaten as food.It can be seen that different species of Bacillus have different molecular mechanisms of response regulation to nematodes.Both B16 and 168 belong to Bacillus,why do B16 and 168 have opposite outcomes when they meet nematodes? How does B16 perceive nematodes?what are the differences between B16 and 168? Based on the problems above in this study,B16,which can perceive nematodes and form spores in advance,and B.subtilis 168,which cannot perceive nematodes and cannot form spores in advance,are the research objects.We compared the differences of transmembrane protein genes and cell membrane components between B16 and 168.Also,we compared the membrane protein differences of morpholine combined with two Bacillus species by adsorption affinity experiment to find the target membrane protein of morpholine acting on B16,and verified the target protein by molecular simulation docking experiment.Additionally,the transcriptome differences between the two Bacillus species before and after morpholine treatment were performed and analyzed to explore the related genes of Bacillus sense morpholine signaling molecules,and verify the potential sensing related genes obtained from the analysis via q PCR.Finally,it was found that B16 formed a biofilm after sensing morpholine,and further improved the signal pathway for B16 to sense nematode spore formation.The results reveal the differential perception of nematode morpholine signals between two Bacillus at the molecular level and provide reference for the biological control of plant parasitic nematodes.The main results of this study are as follows:1.Differences of membrane proteins between B16 and 168.By comparing B16 and 168 transmembrane protein genes,we found that compared to 168,B16 had 117 unique transmembrane proteins.Among them,15 proteins were located on the cell membrane.In addition,there were 5 proteins localized on non-membrane regions.The remaining 97 proteins did not detect subcellular localization,2 of which had no detected protein names,and the rest were tentatively designated disiganated as hypothetical proteins.The above results indicate that,the protein components of the two Bacillus species are quite different,especially the membrane protein genes.2.Differences of membrane components between B16 and 168.There are significant differences in the phospholipid composition of B16 and 168,and there are 28 components with a large difference in proportion.B16 has 20 unique ingredients.168 has 11 unique ingredients.There are significant differences in the fatty acid composition of B16 and 168.The type and quantity of each group of data in the two Bacillus are different,and there are four groups of data with multiple differences.Compared with 168,B16 has two kinds of multi-carbon chain fatty acids.There were three sets of data before and after B16 nematode induction all showed a significant increase in the proportion,while the type and amount of fatty acids before and after the induction of 168 nematode did not change.3.Differences of morpholine receptor proteins between B16 and 168.The results of adsorption affinity experiments showed that the binding degree of B16 to 2-aminoethylmorpholine was significantly higher than that of B.subtilis 168.After strong acid elution,SDS-PAGE experiments confirmed that there are 2 bands of morpholine receptor protein in B16 and 168,and the content of these two bands in B16 was significantly higher than 168.The two bands were further found to be peptide-binding protein S1 and oligopeptide ABC transporter substrate-binding protein Opp A S2 through mass spectrometry.Quantitative analysis results showed that the content of S1 and S2 protein of B16 was significantly higher than that of 168.The comparative analysis of the amino acid sequence of the two Bacillus species confirmed that the similarity of the amino acid sequence of protein S1 was 99.45%,and the similarity of S2 was 96.80%.The results of molecular simulation showed that the docking of S1 and S2 membrane protein of B16 with morpholine was closer to the adsorption affinity,and the adsorption number of morpholine was also more than 168.The affinity between morpholine with B16 S1、S2 protein was much higher than 168.4.Differential analysis of transcriptome after sensing morpholine signaling in two Bacillus species.There are significant differences in transcriptomics after B16 and 168 sense morpholine signaling molecules.There are 16 differential genes related to the regulation of cell membrane and transporter,63 differential genes associated with regulation of spor ulation,and 30 differential genes associated with phosphokinase,of which only 8 genes play the same regulatory effect in the same period of time when morpholine induces the two Bacillus.Among them,there are only two genes at 12h.In addition,the four genes tapA、tasA、epsI、epsF related to biofilm formation are all up-regulated 12h after morpholine induced B16,which promoted the formation of B16 biofilm.5.Differences in biofilm formation after sensing morpholine signals.After B16 senses morpholine,it not only regulates its own genes and forms spores in advance,but also forms a biofilm.The genes tapA、tasA、epsI、epsF associated with formation of biofilm were verified through q PCR.The relative expression of these four genes tas A,tap A,eps I,and eps F after 12h of B16 sensing morpholine signal compared with 168 are greatly up-regulated,which promotes the formation of biofilms.To sum up,although B16 and 168 are both Bacillus,there are great differences in cell membrane components.B16 can sense morpholine when facing the morpholine signal produced by nematodes,and interact with the target proteins S1 and S2 on the membrane.After being adsorbed,it enters the cell,so that B16 form biofilm ahead of time and forms spores at the same time,open its unique "Trojan horse mechanism ". |
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