Function And Mechanism Analysis Of CLIPB8 And CLIPB10 In Melanization Pathway Of Bactrocera Dorsalis

Bactrocera dorsalis(Hendel)is a dangerously invasive insect pest with importantly economic significance,which has become a great threat to the healthy production of fruit and vegetable industry in China.Due to its strong fly ability and reproductive capacity,B.dorsalis distributes widely and spreads rapidly.B.dorsalis larvae feed inside fruit and cause fruits dacay and fall off,pupae live under the earth and develop to adult.Such special life history makes it difficult to control.Entomopathogens are natural resources for controlling pest population.Compared with insect natural predators,entomopathogens spread more rapidly and last for longer time in host’s community,making it be an ideal tool for pest management.However,entomopathogens application still faces many problems.One hurdle has been the challenge of insect immune system,which confers insects with powerful adaptability to environmental factors.As a consequence,to study the molecular mechanism of B.dorsalis immune system not only fills the gap in our understanding of how B.dorsalis adapt to dacaying environment,but also sets stage for applying entomopathogens in pest control.Insects do not possess adaptive immunity,mainly rely on humoral immunity and cellular immunity of innate immune system.Humoral immunity consists of antimicrobial peptide and melanization,which is mediated by Toll pathway and phenoloxidase activation,respectively.Clip serine proteases(cSPs)are a kind of arthropods and molluscs specific protease,play an important role in Toll pathway and phenoloxidase activation.In this study,taking the first step towards genome-wide gene identification,35 cSPs genes were obtained,and the relative expression level of 35 cSPs in different tissues and their expression pattern upon immune defense were analyzed.On this basis,CLIPB8 and CLIPB10 were selected as target genes for further study.With means of molecular biology,reverse genetics and biochemistry,this study uncovered the function and mechanism of CLIPB8 and CLIPB10 in melanization activation of B.dorsalis.The main results are as following:1.Genome-wide identification and phylogenetic analysis of cSPs genes in B.dorsalisBased on B.dorsalis genome data,35 cSPs were identified.Analyzing the properties of protein sequence,each protein consist 1 or more clip domain and 1 protease domain.Within these proteases,7 proteases lost its activity due to key amino acid residues mutation.Phylogenetic analysis showed that 35 cSPs were classified into 4 subfamilies,and subfamily A,B,C and D contain 7,17,6 and 5 genes,respectively.The evolutionary relationship of cSP between B.dorsalis and other insect species was analyzed.It was found that CLIPB6,CLIPB8,CLIPB10 and CLIPB11 were highly homologous with melanization-related genes,and CILPB3,CLIPB5 and CLIPC5 were homologous to Toll pathway-related genes.2.Expression pattern analysis of cSPs in B.dorsalisThe expression levels of cSPs in adult tissues were analyzed by RT-qPCR.Most cSPs were highly expressed in fat body.In addition,CLIPB3 and CLIPC5 were highly expressed in ovary and early embryo,and CLIPD5 was highly expressed in testis.The expression level changes of cSPs upon pathogen induction were also analyzed.The expression levels of CLIPB6,CLIPB8,CLIPB10 and CLIPB11 were significantly up-regulated under bacterial induction,but did not respond to fungal infection.The expression of CLIPB5,which was homologous to SPE of Drosophila melanogaster,increased rapidly after Beauveria bassiana infection.CLIPB7 and CLIPB13,which were homologous to Grass,showed different trends of expression changes under the bacteria induction and fungi infection.The expression of CLIPB7 was up-regulated under B.bassiana infection,while did not change significantly after Enterococcus faecalis induction.CLIPB13 responded to E.faecalis induction but did not respond to B.bassiana infection.3.Functional analysis of CLIPB8 and CLIPB10 via RNAi and CRISPR-Cas9 systemBy ds RNA injection,CLIPB10 were successfully silenced.RT-qPCR results showed that the expression of CLIPB10 was significantly down-regulated by 65.8% and 59.5% after injection of ds RNA for 24 h and 48 h,respectively.Through pathogens infection,we found that B.dorsalis were more susceptible to B.bassiana and E.faecalis when CILIP10 was silenced,while showed no defective immunity to P.aeruginosa.Based on the established CRISPR-Cas9 gene editing system in B.dorsalis,a homozygous mutant of CLIPB8 with 145 bp deletion was obtained.Pathogen infection experiments showed that knock out CLIPB8 alone did not affect the immune ability of B.dorsalis adults to pathogens B.bassiana and E.coli.4.Mechanism analysis of CLIPB8 and CILPB10 mediated melanization activation via heterologous expressionIn order to explore the function and mechanism of CLIPB8 and CLIPB10 in melanization activation,the interaction between CLIPB8,CLIPB10 and PPO was confirmed in vitro by heterologous expression system.Firstly,four PPO genes were identified based on the genomic information of B.dorsalis,named PPO1 a,PPO1b,PPO2 and PPO3,respectively.Then,four PPO recombinant proteins were successfully expressed and purified by prokaryotic expression.Enzyme activity assay results showed that PPO1 a,PPO2 and PPO3 could catalyze dopa to produce melanin,while PPO1 b showed no ability in dopa catalysis.The recombinant proteins CLIPB8 and CLIPB10 were also expressed and purified by insect baculovirus expression system.By co-incubation of CLIBP8 and CLIPB10 protease with PPO recombinant protein,we confirmed that CLIPB8 and CLIPB10 could cleave and activate PPO1 a and PPO2 in vitro,but showed no ability to activate PPO3.Based on the above experimental results,we concluded that two genes,CLIPB8 and CLIPB10,which were significantly up-regulated under pathogens infection,mediated the immune response of B.dorsalis to pathogens through directly cleaving and activating prophenoloxidase.