Whole Genome Sequencing And Expression Analysis Of Effector WalE1 Of PI-Wolbachia In Trichogramma Dendrolimi

Trichogramma dendrolimi(Hymenoptera: Trichogrammatidae)is an egg parasitoid of agricultural pests as a biological control agent.Parthenogenesis was observed in the wasps T.dendrolimi when they infected with Wolbachia.Compared with uninfected wasps,Wolbachiainfected strains show more potential usage as a biocontrol agent.Exploring the molecular mechanism of how Wolbachia regulates its host phenotypes can not only obtain a quality line,but provide some supports for Wolbachia-host interactions.This study focuses on Wal E1(Wolbachia actin-localizing effector 1),and analyzes its expression patterns.By heterologous expression,sublocalization in yeast and transcriptome analysis,we prove Wal E1 may interact with certain proteins of cytoskeleton.Combined quantitative assessment and Wolbachia titer,we speculate this interaction will contribute Wolbachia on cell-to-cell movement or reproduction.Our results provide a new light into the molecular mechanism of the interaction between obligate bacteria and host.Results of this study are as follow:1.Whole genome characterization and phylogenetic analysis of PI-Wolbachia parasitizing T.dendrolimi:By using alignment and removing host genes,we got a PI-Wolbachia complete genome,w Tde.w Tde genome size was 1108797 bp with 33.99% G+C%,1531 CDS genes,34 t RNA and 3 r RNA.This genome paralleled others PI-Wolbachia.In addition,we compared w Tde with other PIWolbachia(w Cle,w Pre,w Uni,w Fol)in NCBI public database and then removed the core genes(w Bm).The results showed these PI-Wolbachia shared 31 gene clusters and w Bm had no unique cluster.With this result it is possible to not only increase PI-Wolbachai genome informations but also explore the major Wolbachia factors which can induce PI2.Identify suitable reference genes for T.dendrolimi parasite,Wolbachia,during host development stages:Analysis of Wolbachia gene expression is an efficient approach in elucidating the molecular mechanism.In this study,we selected seven candidate genes,Wolbachia surface protein,wsp;cell division protein,ftsz;glyceraldehyde phosphate dehydrogenase,GAPDH;16s r RNA,recombination A,Rec A;elongation factors 4,EF4 and gyrase A,gyr A,and evaluated the stability of them under host different development stages.Of these genes,the expression of wsp,16 s,gyr A and EF4 had a highly expression,then they were subjected to detailed analysis.Normfinder,ge Norm,Bestkeeper and △ct were used to validate the candidates.The results demonstrated the most suitable gene was wsp.Our results lay foundation for future research into RT-q PCR normalization in T.dendrolimi development stages.3.Identification and bioinformatics analysis of the PI-Wolbachai effector Wal E1 and localization:Wal E1 is 1770 bp long and encodes a protein of 589 amino acids with an alpha-synuclein domain.Wal E1 homologs are evolutionarily conserved.The Wal E1 sequence was sub-cloned into a eukaryotic expression vectors p YES2.p YES2-Wal E1-EGFP was transformed into Saccharomyces cerevisiae W303-1A.Then for Wal E1 tracing,we used fluorescence of e GFP for signal detection and the cell cytoskeleton was stained with rhodamine-phalloidin.The results display Wal E1 is a soluble protein and in yeast cell,it is colocalization with the actin cytoskeleton.They further demonstrated that Wal E1 may interact with host cytoskeleton4.PI-Wolbachia Influences the expression patterns of cofilin and Wal E1 in T.dendrolimi development stages:RT-q PCR was performed to detect the expression patterns of cofilin and Wal E1 in Wolbachiainfected and Wolbachia-uninfected wasps during 5 development stages(egg,larva,prepupa,pupa and adult).For uninfected wasps,there were no Wal E1 expression,and the variation of the expression of cofilin was relatively stable.For infected wasps,Wal E1 expressed most abundantly in adult stage and cofilin in both pupal and adult stages.Compared with uninfected strain,cofilin was higher expressed in pupal and adult stages.Furthermore,we also analysis Wolbachia titer.As the wasp development increasing,the Wolbachia titer increases,and in prepupal,pupal or adult stages was significantly higher than other stages.All three expressions yielded similar results.This result indicates Wal E1 may influences Wolbachia titer through a cytoskeleton regulator cofilin and help Wolbachia in cell-to-cell movement or reproduction.