Studies On The Function Of Whitefly Horizontally Transferred Genes In Biotin And Pantothenate Synthesis

The whitefly Bemisia tabaci is a complex of more than 36 cryptic species.Within the B.tabaci species complex,B.tabaci MEAM1 is a globally important and invasive agricultural pest.In China,B.tabaci MEAM1 bears Portiera and Hamiltonella in the same bacteriocyte and in the whole body cavity.The genome of Portiera is highly reduced.Previously,we found some horizontally transferred genes(HTGs)were encoded in the genome of the whitefly B.tabaci MEAM1 and highly expressed in bacteriocytes.These genes are presumably involved in synthesis of biotin and pantothenate.However,the manner in which HTGs contribute to whitefly-symbiont interactions and coevolution is unclear.In this study,the function of the horizontally transferred bioA,bioD,bioB,and panBC in the B.tabaci MEAM1 symbiosis system was studied.The main results are as follows.1.A microbiological assay for biotin determination in insects was establishedWe firstly investigated how experimental conditions influence the results of biotin assay.Our findings reveal that using bacteria strains at the third-fifth generations,incubating cultures for at least 19 h and at OD630,we obtained good linear relationships between absorbance and biotin concentration with high correlation coefficients.Then an efficient microbiological assay to measure biotin in insects was developed using the whitefly B.tabaci MEAM1,Trialeurodes vaporariorum,and Nilaparvata lugens.Our approach,which shows high sensitivity,rapidity and accuracy,will be very useful for biotin assay in other small insects.2.The effects of symbionts on the synthesis of B vitamins in whitefliesHamiltonella in B.tabaci and Arsenophonus in T.vaporariorum were specifically eliminated in whiteflies by antibiotic treatment.All four B vitamins(Riboflavin,Nicotinic acid,Pyridoxine and Biotin)had significantly lower levels in symbiont-cured whiteflies compared to symbiont-infected whiteflies.It appears that Hamiltonella and Arsenophonus may provide these B vitamins for the host.Additionally,the level of pantothenate in B.tabaci was significantly reduced after Portiera were eliminated by Rifampicin treatment.It suggests that Portiera is involved in pantothenate synthesis.3.Evolutionary origin of horizontally transferred biotin and pantothenate genes in whitefliesHorizontally transferred bioA and bioD were duplicated,of which,one of these duplicated genes was pseudogenized,and bioB and panBC have acquired intron.We found that all the whitefly B.tabaci species were investigated possess these horizontally transferred biotin and pantothenate genes.The amino acid sequence identity was high among all B.tabaci species but low among whiteflies,Hamiltonella,Buchnera,and E.coli.Furthermore,whitefly BioA and BioD clustered with Wolbachia strains of the bedbug and planthoppers and Cardinium associated with the whitefly parasitoid and planthopper.BioB fell within the clade of Rickettsia,and clustered with Wolbachia strains of the bedbug and planthoppers and Cardinium associated with the whitefly parasitoid and planthopper.Collectively,these data suggest that horizontally transferred BioA,BioD and BioB share a common evolutionary origin in whiteflies.Additionally,Pan BC fell within the clade of Pseudomonas.4.Functional complementation of E.coli biotin and pantothenate auxotrophs with whitefly bioA,bioD,bioB and panBCTo test the hypothesis that bioA,bioD,bioB and panBC of the whitefly function in biotin synthesis,the E.coli K-12 bioA,bioD,bioB and panBC knockout mutant were generated using the Lambda Red protocol and functionally complemented E.coli K-12 mutant with whitefly bioA,bioD,bioB and panBC,respectively.Compared to wild type E.coli,E.coli K-12 knockout mutants did not grow on vitamin B7-deficient Difco biotin assay media(B7DB)or vitamin B5-deficient Difco pantothenate assay media(B5DP).Although whitefly bioA,bioD,bioB and panBC shared low amino acid sequence similarities with E.coli homolog genes,complementation with whitefly bioA,bioD,bioB and panBC rescued E.coli K-12 knockout mutants on B7 DB or B5 DP media.In contrast,cells transformed with the p MD19-T empty vector grew poorly on B7 DB or B5 DP media without biotin or pantothenate supplementation.The results show that bioA,bioD,bioB and panBC have the functions for biotin and pantothenate synthesis.5.Horizontally transferred bioA,bioD and bioB are involved in biotin synthesis and confer whitefly fitness benefitsAfter Hamiltonella was eliminated,expression of horizontally transferred bioA,bioD or bioB was significantly elevated,indicating that horizontally transferred biotin genes are functional in the biotin synthesis of whiteflies.The recombinant proteins of BioA,BioD and BioB were successfully generated.Immunofluorescence microscopy showed that BioA and BioD were mainly located in the peripheral regions of bacteriocytes in contact with external medium,while BioB was distributed both in the peripheral regions and around bacteriocyte nuclei in whiteflies infected with Hamiltonella,which is different from the distribution of Hamiltonella.After Hamiltonella was cured,the protein expression levels and patterns were maintained in whiteflies,confirming that BioA,BioD and BioB were not encoded by Hamiltonella.These data further suggest that BioA,BioD and BioB likely perform some functions in bacteriocytes.Expression of whitefly bioA,bioD and bioB were reduced after feeding on ds RNAs for 3 days.In addition,the protein expression levels of BioA,BioD and BioB were significantly reduced in bacteriocytes after the RNAi treatment of these whiteflies.Silencing whitefly biotin genes significantly reduced biotin levels.As a result,gene silencing significantly increased the mortality of female adult whiteflies.After biotin supplementation in the artificial diet,the mortality of dsbioA-fed whiteflies was decreased over three days,which is close to that of ds GFP-fed whiteflies.Additionally,gene silencing significantly repressed the fecundity of female adult whiteflies.Meanwhile,after biotin supplementation in the artificial diet,the fecundity of dsbioA-fed whiteflies was restored.Furthermore,the abundance of Hamiltonella was not changed in RNAi treated whiteflies,suggesting that Hamiltonella couldn't complement the depleted roles of horizontally transferred biotin genes in whiteflies.These findings show that horizontally transferred bioA,bioD and bioB are involved in biotin synthesis and confer whitefly fitness benefits.6.Horizontally transferred panBC cooperates with Portiera for pantothenate synthesis,thereby facilitating the coordination of whitefly and symbiont fitnessImmunofluorescence microscopy showed that PanBC was located in the cytoplasmic region of bacteriocytes and had some clusters.Pan BC and Portiera were localized in bacteriocytes,indicating that Pan BC could be transported into Portiera cells.After Portiera was cured,the Pan BC protein level decreased significantly.To study the metabolic function of horizontally transferred panBC,whitefly panBC was silenced by microinjection with dspanBC.The level of Pan BC and the titer of Portiera were significantly reduced in bacteriocytes after microinjection with dspanBC.As a result of gene silencing,the pantothenate level was decreased significantly in dspanBC-injected compared to ds GFP-injected whiteflies.In addition,silencing the horizontally transferred panBC significantly increased the mortality of female adult whiteflies and reduced female fecundity after microinjection with ds RNAs.Supplementation with pantothenate restores symbiont titer,Pan BC level,and fitness of RNAi whiteflies.Therefore,horizontally transferred panBC cooperates with Portiera for pantothenate synthesis,thereby facilitating the coordination of whitefly and symbiont fitness.The symbionts in some insects,mainly blood-feeding insects,are able to provision B vitamins for insects.This study demonstrated that Hamiltonella and Arsenophonus in whiteflies feeding on plant phloem can provide some B vitamins.Furthermore,we revealed that horizontally transferred bioA,bioD and bioB are involved in biotin synthesis and confer whitefly fitness benefits.Horizontally transferred panBC cooperates with Portiera for pantothenate synthesis,thereby facilitating the coordination of whitefly and symbiont fitness.The results of this study uncovered a third mechanism in that horizontally transferred genes in whiteflies can synthesize B vitamins alone or by cooperation with Portiera.Our results support the hypothesis that intron gain and duplication of HTGs may help them become functional.The ubiquity of horizontally transferred biotin or pantothenate genes in various whitefly populations indicates that they are essential for whitefly evolution,which may lead to the loss of biotin and pantothenate synthesis genes in whitefly symbionts,including Hamiltonella.Thus,this study extends our understanding on the basis of complex host-symbiont interactions.These findings also provide excellent genetic targets for insect control by selective interference.