The Study Of Cytoplasmic Incompatibility Mechanism Induced By Wolbachia Based On MicroRNA In Drosophila Melanogaster

Wolbachia is an endosymbiotic bacterium that induces a wide range of effects in its insect hosts including cytoplasmic incompatibility(CI),which is expressed as embryonic lethality when Wolbachia-infected males mate with uninfected females,or with different strains females.In recent years,CI phenomenon has been a research hotspot.However,little is known of the molecular mechanisms underlying CI,which is to further study.Evidences have revealed that bacteria can regulate many cellular processes in their hosts using small non-coding RNAs.Therefore,we investigate whether Wolbachia can induce cytoplasmic incompatibility(CI)in Drosophila host by the pathways of miRNAs.In this study,small RNA(sRNA)libraries were first compared between Wolbachia-infected and uninfected fly testis.there are 18 novel miRNAs are identified.Of which novel miR-1,3,4,5 are appeared in both Dmel wMel and Dmel T testes,while miR-2 is expressed only in Dmel T testes,13 novel miRNAs(from miR-6 to miR-18)are specifically appeared in Dmel wMel.In addition,10 miRNAs appeared significantly changes in expression.When comparing Wolbachia-infected flies to uninfected flies,4 miRNAs(nov-miR-12,dme-miR-986-3p,dme-miR-283-3p,dme-miR-306-3p)were up-regulated and 6 miRNAs(dme-miR-992-5p,dme-miR-1009-3p,dme-miR-972-5p,dme-miR-137-3p,dme-miR-34-3p,dme-miR-2a-2-5p)were down-regulated.By testing the qRT-PCR of the above 10 differentially expressed miRNAs,analysis results are consistent with the results of sRNA sequencing,Of these up-regulated miRNAs,nov-miR-12 exhibited the highest upregulation in the testes of D.melanogaster when infected by Wolbachia.To further identify the biological functions of the differentially expressed miRNAs,miRanda and Mireap were used to predict the target genes of miRNAs including expressed specifically in Dmel T(nov-miR-2)and expressed specifically in Dmel wMel(nov-miR-6?nov-miR-18)as well as 10 DE miRNAs,which in total resulted in the identification of 10738 target gene.The biological functions of the predicted target genes were then analyzed using the gene ontology(GO)annotations of the D.melanogaster genes.The putative target genes cover a wide range of biological process mainly including cellular proccess,organism process,biological regulation,metabolic process,developmental process,reproduction.Our previous studies have found that Wolbachia infection in the Drosophila testis occurred in most genes associated with sperm expression,which is down-regulation expressed.Other studies have shown that Wolbachia can change the expression level of host miRNA and regulate the expression of its target genes,and then create a sufficient condition for the survival of the symbiotic bacteria.The nov-miR-12 with the highest expression difference is selected as our research object.Then a target gene(pipsqueak,psq)of nov-miR-12 was identified to be most complementary in its 3'UTR.The expression of nov-miR-12 in the testis of infected Wolbachia was significantly increased and the expression of psq gene was significantly decreased by qRT-PCR.It was presumed that nov-miR-12 could negatively control psq Transcription level.To further determine whether psq mRNA is repressed by nov-miR-12,we performed two independent experiments.There are two vitro cell experiments in this study.On the one hand,Wolbachia-free Drosophila S2 cells were transfected with a specific synthetic mimic or inhibitor(complementary sequence)of nov-miR-12.After 48 h,we observed significantly lower transcript level of the psq in cells transfected with the miRNA mimic than that in cells transfected with control.By contrast,the psq gene expression exhibited significant up-regulation after nov-miR-12 inhibitor was transfected.On the other hand,it constructed three dual luciferase reporter.The seed region of psq containing null UTR,wild-type and 4 bp mutation were respectively cloned into the psiCHECK2 dual luciferase reporter plasmid.Luciferase reporters were cotransfected with either nov-miR-12 mimic or miRNA NC into S2 cells.co-transfection of nov-miR-12 mimic with the psq 3'UTR resulted in an extremely significant decrease in luciferase activity compared to other groups.No decrease in luciferase activity was observed when nov-miR-12 mimic was transfected together with the mutant reporter or null plasmid.These indicate that the predicted site is the direct target of nov-miR-12 and it is solely responsible for nov-miR-12 targeting of the psq 3'UTR.In order to further study the function of psq in Drosophila,Specifically knock down of psq in Wolbachia-free fly gland and detecet the hatching rate of the embryos of the offspring in knock down the male fly.After knock down of psq,the hatching rate of the offspring was significantly decreased.With the further development of embryos,nuclear adhesions were appeared forming into clusters irregularly.While in the embryos compared with the controls,the nuclear division was synchronous and the nuclei were completely segregated.These results suggest that Wolbachia may induce CI in insect hosts by altering host gene expression through miRNA-target pathway.Our findings provide new insights for understanding the complex interactions between endosymbionts and their insect hosts and in particular the underlying mechanisms of Wolbachia-induced CI.