The Role Of Small RNA In Interactions Between Whiteflies And Intracellular Symbionts

The whitefly Bemisia tabaci MEAM1,as one member of B.tabaci species complex,and Trialeurodes vaporariorum are globally important pests in the agriculture.Both kinds of the whitefly can bring serious losses to agricultural economy.The whitefly bears Portiera and at least one secondary symbiont in the same bacteriocyte.And they can establish a mutualism relationship with symbiont in bacteriocytes,which is the basis of the infestation of whitefly.However,the genome of intracellular symbionts is reduced and has lost many essential genes.Some whitefly genes related to synthesis of essential amino acids and B vitamins are highly expressed in bacteriocytes,which may compensate for the missing genes in symbionts.Additionally,the genome of intracellular symbionts has lost many transcriptional factors,and lacking of transcriptional regulation.How the symbionts with reduced genome achieve the regulation and expression of genes involved in essential amino acid metabolism largely unknown.The study show that whitefly genes can cooperate with Portiera and secondary symbiont for the synthesis of essential amino acids and B vitamins,respectively.However,the mechanisms underlying how the collaboration between whitefly and symbionts for nutrient synthesis is regulated remain largely unknown.Small non-coding RNAs?sRNAs?impact the transcription,stability or translation of the RNA and function as important post-transcriptional regulatory factors in Bacteria,Archaea,Eukaryotes and eukaryotic organelles.Available studies indicate that host miRNAs or symbiontsRNAs can target its own m RNA.However,the mechanisms underlying the cross-kingdom communications between animal and symbiontsare very limited.This study aimed to explore the role of sRNAs in regulating the expression of genes involved in folate?Vitamin B₉?synthesis in T.vaporariorum and amino acid synthesis,metabolism and transportation in B.tabaci.The main results are as follows.1.Localization and vertical transmission of bacteriocyte symbionts of T.vaporariorium and B.tabaci.The bacterial symbionts Hamiltonella and Arsenophonus have very high prevalence in two globally important pests,the whiteflies B.tabaci and T.vaporariorum,respectively.Both symbionts coexist with the primary symbiont Portiera in the same host cell?bacteriocyte?and are maternally transmitted.Overall,T.vaporariorium was polymorphic for all four microsatellite loci tested.T.vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect.Furthermore,we found that a single mature oocyte of T.vaporariorumwas colonized by multiple bacteriocytes,which mediates the vertical transmission of Portiera and Arsenophonus.2.miR-1 regulates the gene expression of phoAB?Alkaline phosphatase A/B?to affect folic acid synthesis.We had identified 12 conserved miRNAs and 13 precursor miRNAs,and 64 novel miRNAs and 64 precursor miRNAs.We further identified 11 highly expressed sRNAs that target 4066 genes,among which 24 target genes were related to nine KEGG pathways involved infolate biosynthesis,lipoic acid metabolism,nicotinate and nicotinamide metabolism,one carbon pool by folate,pantothenate and Co A biosynthesis,riboflavin metabolism,thiamine metabolism,ubiquinone and other terpenoid-quinone biosynthesis and vitamin B₆ metabolism.Our lab has shown that Arsenophonus could cooperate with T.vaporariorium gene phoAB to synthesize vitamin B₉.Thus,we focused on the role of miR-1in regulating phoAB genes for folate synthesis.Dual-luciferase reporter assay indicated that miR-1 could interact with gene phoAB.After overexpression of miR-1,the expression of phoAB is enhanced,the content of folateis increased.As a result,the titer of Portiera and Arsenophonus and fecundity of the whiteflyis elevated.Therefore,our data revealed that miR-1 can affect folate biosynthesis and the fitness of whitefly and symbiotic bacteriaby regulating phoAB gene expression.3.sRNAs of whitefly and Portiera collaboratively regulates the expression of genes involved in amino acid synthesis,metabolism and transportation.We analyzed the differentially expressed sRNAs with Portiera-cured whiteflies?-PBt?and Portiera-infected whiteflies?+PBt?.We identified 106 whitefly miRNAs were up-regulated and 71 whitefly miRNAs were down-regulated in-PBt whiteflies compared to+PBt whiteflies;14 Portiera sRNAs were up-regulated and 6 Portiera sRNAs were down-regulated in-PBt whiteflies compared to+PBt whiteflies.And we found 220 whitefly m RNAs were up-regulated and 732 whitefly m RNAs were down-regulated in-PBt whiteflies compared to+PBt whiteflies;10 Portiera m RNAs were up-regulated and 23 Portiera m RNAs were down-regulated in-PBt whiteflies compared to+PBt whiteflies.Our analysis showed that sRNAs of whitefly and Portiera can regulate the expression of their own and partner's genes.Portiera can collaborate with the host to synthesize essential amino acids.Therefore,we focused on the mechanism of sRNAs regulating the synthesis,metabolism and transport of essential amino acids.We found that whitefly miRNAs and Portiera sRNAs can jointly and reciprocally regulate the expression of whitefly BCAT?Branched chain amino acid aminotransferase?and Portiera ilv I,his G and car B involved in biosynthesis of Val,Leu,Ile,and His and Arg.Portiera sRNAs can alone regulate the expression of Portiera aro K and whitefly dap B,dap F,lys A,and arg G involved in biosynthesis of Phe,Try,and Lys and Arg.Whitefly miRNAs and Portiera sRNAs can target different location of their shared amino acid synthesis pathways.In general,whitefly miRNAs and Portiera sRNAs can regulate whitefly genes in the final steps and Portiera genes in the early steps of their shared amino acid synthesis.Except that whitefly miRNAs and Portiera sRNAs can regulate the gene expression involved in the metabolism pathways of essential amino acids.Taken together,our findings unravel sRNAs of whitefly and symbionts reciprocally regulate their shared amino acid synthesis in a sophisticated way.This makes the metabolic collaboration between host and symbionts highly coordinated.Meanwhile,Portiera sRNAs can alone regulate large neutral amino acid transporter small subunit 1.And whitefly miRNAs and Portiera sRNAs can negatively regulate the expression of whitefly genes encoding amino acid transporters,including high affinity cationic amino acid transporter 1,excitatory amino acid transporter,proton-coupled amino acid transporter and B?0,+?-type amino acid transporter 1,and Portiera L-lysine exporter protein Lys E.Our study reveals that insect miRNAs and symbiont sRNAs can reciprocally regulate genes encoding amino acid transporters of either host's or symbiont's.4.miR-1791-y affects branched-chain amino acid biosynthesis by regulating the expression of BCAT.qRT-PCR verified the down-regulation of whitefly miR-1791-y and up-regulation of its target gene BCAT in+PBt whiteflies compared to-PBt whiteflies.Dual-luciferase reporter assay demonstrates that BCAT is the putative target gene of miR-1791-y.After overexpression of miR-1791-y,the expression of BCAT is repressed,the content of branched-chain amino acidsis decreased and the titer of Portiera and Hamiltonella is reduced.BCAT silencing led to reduced content of branched-chain amino acids,decreased titer of Portiera and Hamiltonella,and inhibited fecundity of the whitefly.All the data demonstrated that miR-1791-y can affect branched-chain amino acid biosynthesis and the fitness of whitefly and symbiotic bacteriaby regulating BCAT gene expression.Taken together,our findings demonstrated sRNAs play a key role in host-symbiont interactions.This study also provides a new avenue for whitefly control in the future.