Study On The Role Of Peptidoglycan Recognition Protein In The Interaction Between Bemisia Tabaci And Symbiotic Bacteria

Symbiotic bacteria in insects can be divided into primary symbiont and secondary symbiont.Insect-borne symbionts affect insect biology and ecology.Innate immune response is the first line of defense against pathogens,where peptidoglycan recognition protein(PGRP)plays a very important role.Insects recognize the peptidoglycan in the cell wall of the pathogen by PGRP,initiate the IMD or Toll pathways to synthesize antimicrobial peptides,and kill the bacteria.Many insects encode multiple PGRP proteins;different insect species encode PGRPs varying in the types,number,and function.Previous studies showed that PGRPs contribute to the interacions between insects and their symbionts.Two invasive pests whitefly Bemisia tabaci species MEAM1 and MED house the primary symbiont Candidatus P or tier a aleyrodidarum(P or tiera)and the secondary symbiotic symbiont Candidatus Hamiltonella defensa(Hamiltonella)in the specialized cells,called bacteriocytes.Most individuals of MEAM1 and some individuals of MED contain secondary symbiosis Rickettsia sp(Rickettsia).These two B.tabaci cryptic species encode only one PGRP protein.The role of this pgrp gene in the interaction between B.tabaci and Portiera and Hamiltonella(localized in bacteriocytes)and Rickettsia(localized in the whole body cavity except bacteriocytes)is unknown.In this study,the pgrp gene was silenced by dsRNA microinjection to detect the symbiont titer and whitefly fitness.Furthermore,Rickettsia was transfected into whiteflies to examine pgrp expression and whitefly fitness.All these are aimed to to reveal the role of pgrp gene in the interaction between whiteflies and three species of symbiosis.The results are as follows:1.The evolutionary analysis of B.tabaci PGRPThe genomes of seven B.tabaci species(MEAM1,MED,SSA1,SSA2,SSA3,Uganda and ASIAII5)encode only one peptidoglycan recognition protein.The amino acid sequence similarity of the seven peptidoglycan recognition proteins is as high as 94%.The similarity of amino acid sequences among B.tabaci PGRP,G.morsitans PGRP-LB,D.melanogaster PGRB-LB-C,D.melanogaster PGRB-LB-A,D.melanogaster PGRB-LB-D,D.melanogaster PGRB-LC,D.simulans PGRP-SA,D.simulans PGRP-SB1,D.melanogaster PGRP-SC2,and D.melanogaster PGRP-SB2 is 23.51%.PGRP of all seven B.tabaci species clustered within the same clade.Whitefly PGRP clustered with PGRP-LB,PGRP-SB,and PGRP-LF in the same clade.It indicates that whitefly PGRP may have the similar function of PGRP-LB,PGRP-SB and PGRP-LF.2.Silencing pgrp gene in B.tabaci ME AM1 reduces the titers of the symbiont Hamiltonella and RickettsiaHamiltonella and PGRP protein was found in bacteriocytes of B.tabaci MEAM1.and Rickettsia and PGRP protein was found in midguts of B.tabaci MEAM1,and PGRP was mostly close to Rickettsia in midguts.After pgrp gene of B.tabaci MEAM1 was silenced by dsRNA microinjection,the titers of both Hamiltonella and Rickettsia were significantly reduced.3.Silencing pgrp gene in B.tabaci MED reduced the titers of the symbiont Hamiltonella and Rickettsia and whitefly fecundityBy dsRNA microinjection,the pgrp gene was silenced in the MED species carrying Portiera,Hamiltonella and Rickettsia,and the MED species carrying only Portiera and Hamiltonella.When the pgrp of these two species are silenced,the titers of Hamiltonella and Rickettsia were decreased significantly,and the degradation of cell walls for these two symbionts were observed.These evidences suggest that pgrp acts to protect the secondary symbionts Hamiltonella and Rickettsia in B.tabaci MEAM1 and MED.LysA expression and content of lysine was significantly reduced after pgrp silencing.Additionally,silencing pgrp gene reduces whitefly fitness.4.Transfection with Rickettsia incresed pgrp gene expression and whitefly fecundityB.tabaci MED population lacking Rickettsia has lower pgrp gene expression than B.tabaci MED harboring Rickettsia.Rickettsia was isolated and transfected from B.tabaci MED harboring Rickettsia into MED species carrying only Portiera and Hamiltonella.Rickettsia was detected at the 14th d after transfection.Moreover,Rickettsia can be continuously transmitted to the next generation and colonized.The titer of Rickettsia was only decreased in F1 generation and F2 generation,but Rickettsia bacteria began to spread stably in F3 generation and later.The above results demonstrate that Rickettsia can be successfully transfected between different strains of B.tabaci.As the Rickettsia titers increased,the expression of whitefly pgrp also began to increase.Also,Rickettsia transfection didn’t affecctthe survival rate of MED species but increase whiefly fecundity.In conclusion,this study demonstrated the role of pgrp in the interactions between whiteflies and secondary symbionts.