Role Of Mi-FAR-1 Protein In Bacillus Cereus BCM2-mediated Defense Against Nematodes In Tomato

Meloidogyne incognita is one of the most widely distributed and harmful pathogenic nematodes in the world.It can infect almost all cultivated crops and bring huge losses to the world economy every year.Biological control is considered to be an important means to control root knot nematodes because of its safety,efficiency and environmental friendliness.Therefore,it is of great significance to find the microorganisms that can effectively control plant pathogenic nematodes and study the mechanism of controlling nematodes.The previous experiments showed that the Bacillus cereus BCM2 strain isolated from plants could effectively control M.incognita,and the crude proteins extracted from the oesophageal glands of M.incognita can enhance the activity of BCM2-mediated phenylalanine ammonia lyase(Phenylalanine ammonia lyase,PAL)in tomato,and significantly induce the colonization of BCM2 in tomato to enhance the defense against M.incognita.Further analysis of the crude proteins obtained showed that M.incognita fatty acid-and retinol-binding protein(M.incognita fatty acid-and retinol-binding protein,Mi-FAR-1)were the main components of the crude proteins and this protein was selected for study because of its function of changing host defense.It provides a theoretical basis for biocontrol bacteria BCM2 to better control M.incognita.Since the Mi-FAR-1 protein secreted by M.incognita is very few,the protein was obtained by using the eukaryotic expression system for further study.Mi-FAR-1 gene was cloned from M.incognita.Mi-FAR-1 protein was successfully obtained from Pichia pastoris.The expression of Mi-FAR-1 protein was confirmed to have the activity of binding fatty acids and retinol in vitro by fluorescence-based ligand analysis(Fluorescence-based ligand analysis).The Mi-FAR-1 protein and the second stage juveniles(Second stage juveniles,J2s)of M.incognita were added to BCM2-colonized tomatoes.Tomatoes without BCM2 colonization were used as the control group.Then,M.incognita was added.The expression levels of PAL,lipoxygenase(Lipoxygenase,Lox D),Proteinase inhibitor II(Proteinase inhibitor ?,Pin II)and ?-thionin(?-thionin)in tomato roots were detected by quantitative real-time PCR(Quantitative real-time PCR,q RT-PCR).The activities of peroxidase(Peroxidase,POD)and superoxide dismutase(Superoxide dismutase,SOD)were detected by spectrophotometry and hydroxylamine method.The number of root galls and egg masses in tomato roots were investigated.The results were as follows.The first result showed that the expression of PAL in tomato roots of BCM2 colonized tomato was significantly higher than that of the treatment group without BCM2 when Mi-FAR-1 protein was added to the tomatoes colonized by BCM2 for 6 h and the protein was added again for 6 h(54 h).It indicated that Mi-FAR-1 protein extracted from the oesophageal glands of M.incognita was one of the factors that induced tomato colonized by BCM2 to enhance defense against M.incognita.The second result showed that Mi-FAR-1 protein could significantly enhance the expression of Lox D,Pin II and ?-thionin genes of jasmonic acid pathway(Jasmonic acid,JA)in tomato colonized by BCM2.The expression of Lox D,Pin II and ?-thionin genes decreased after 48 h.The expression of three defense genes in tomato colonized by BCM2 could also significantly increase when the protein is added again,indicating that Mi-FAR-1could induce defense enhancement in tomato system colonized by BCM2 than that in tomato without BCM2 colonization.The third result showed that adding Mi-FAR-1 protein to tomatoes colonized by BCM2 could promote tomato growth and significantly inhibit the formation of root galls and egg masses when compared with the tomatoes uncolonized BCM2.The number of root galls and egg masses were decreased by 71% and 68%.In summary,the analysis of the above results showed that the interaction between BCM2 and Mi-FAR-1 protein of M.incognita in tomato could increase the expression of defense genes in the jasmonic acid pathway in tomato and enhance the antioxidant enzyme activity of tomato,so as to effectively enhance the control effect of BCM2 on M.incognita.The results of this study showed that the Mi-FAR-1 protein secreted from the oesophageal glands of M.incognita could enhance the defense effect of tomato colonized by BCM2 against M.incognita.These studies provide a theoretical basis for biocontrol bacteria BCM2 to better control M.incognita.