Mechanism Of Begomoviruses Infection In Primary Salivary Gland And Ovary Of Whitefly

In the past decades,begomoviruses have emerged as important insect-borne plant viruses of agricultural significance.For now.multiple begomoviruses,such as tomato yellow leaf curl virus(TYLCV),papaya leaf curl China virus(PaLCuCNV)and tomato yelloxw leaf curl Clhina virus(TYLCCNV)are responsible for the substantial losses in the production of many crops in China.In the field,begomoviruses are transmitted by whitefly Benisia tabaci(Gennadius)(Hemiptera:Aleyirodidae),which is a species complex containing over 36 putative cryptic species,and the epidemics of begomoviruses in the field is closely associated with the prevalence of xwhiteflies.Begomoviruses are transmitted in a circulative persistent manner by whiteflies.The route of begomovirus movement in whitefly body is as follows:begomoviruses are firstly ingested,and then the viruses pass along the food canal and reach the midgut;next,they cross the midgut and enter the haemolymph,from where they translocate into the primary salivary glands;finally begomoviruses are secreted along with the saliva into plants.Further,begomoviruses may also invade other organs,e.g.,ovary,to achieve transovarial transmission.During these processes,begomoviruses need to cross epithelial cells of the midgut and primary salivary gland,or follicular cells of the ovary to reach the developing eggs.Therefore,a detailed understanding of the process whereby viruses achieve successful transport within whitefly will surely help to decipher the nature of whitefly transmission of begomoviruses.However,so far researches regarding begomovirmses transcytosis mainly focused on the whitefly midgut,the trafficking route of begomoviruses in the whitefly primary salhvary gland and ovary are hitherto largely unexplored.In addition,transovarial transmission of begomoviruses by whitefly was mainly investigated in invasive whiteflies,and little is known about the vertical transmission of begomoviruses by indigenous whiteflies.Here,we investigated the molecular mechanisms whereby begomoviruses infect whitefly primary salivary glands or ovarnes.We also compared the transovarial transmission efficiency of a begomoviruses,namely TYLCV,by whiteflies of seven indigenous cryptic species,and investigated the transovarial transmission of PaLCuCNV by MEAM1 whiteflies.The results are summarized as follows:1)Clathrin-mediated endocytosis is involved in the entry of begomoviruses into whitefly primary salivary glands(PSGs)The majority of viruses are internalized into the host or vector cell by clathrin-mediated endocytosis(CME).By disrupting endocytosis using RNA silencing,and harnessing a novel research system which entails the injection of purified virions directly into the haemolymph thus bypassing the midguts,we found CME was involved in virus entry into PSGs as evidenced by the decreased virus quantity in whitefly PSGs.The results of immunofluorescence snd transmission assays further validated the role of CME in virus entry’into the PSGs.2)Early endosome plays an important role in begomoviruses transport in whitefly PSGBy disrupting the vesicle trafficking in PSGs using inhibitor injection,virus transmission efficiency by whitefly was decreased,indicating intracellular vesicle trafficking is involved in virus passage through PSGs.Through knock down of the genes related to endosome network by dsRNA injection,we found that only early endosome is involved in virus transport in PSGs.Silencing of early endosome compartments significantly reduced the virus quantity in whitefly PSGs,and virus transmission efficiency by whiteflies as well.However,RNA silencing of genes belonging to the other whitefly components that have been shown to be involved in the virus transport in midgut cells,resulted in no significant difference of the virus quantity in the whitefly PSGs when compared to the control,suggesting that some whitefly components involved in virus transport within cells may differ in midgut and PSGs.3)Early endosomes are involved in TYLCV entry into whitefly ovaries While knock down of early endosome related genes significantly decreased the virus quantity in whitefly ovaries,silencing of genes related to late endosome and recycling endosome did not affected the virus quantity in the ovaries,suggesting the entry of TYLCV into whitefly ovaries entails early endosome trafficking4)TYLCV transovarial transmission varies among different whitefly species Transovarial transmission of TYLCV may be partly responsible for its widespread in China,and so far it is explored only in two invasive species of the B.tabaci complex Here,transovarial transmission of TYLCV by seven whitefly species indigenous to China,including Asia 1,Asia Ⅱ 1,Asia Ⅱ 3,Asia Ⅱ 6,Asia Ⅱ 7,China 1 and China 2.were analyzed.TYLCV DNA was detected in eggs of all species except Asia Ⅱ 7,and in nymphs of Asia 1.Asia Ⅱ 3,China 1,but in none of the adult offsprings of all seven species.Our results suggest that these whiteflies indigenous to China are unable to transmit TYLCV,a begomovirus alien to China,via ova to produce future generations of viruliferous adults,although most of the species exhibit varying ability to carry over the virus to the eggs/nymphs of their offspring via transovarial transmission5)PaLCuCNV is not able to be transovarially transmitted by MEAM1 whitefliesTransovarial transmission of PaLCuCNV by MEAM1 whiteflies was examined.The results showed that PaLCuCNV was not transmitted vertically to the offspring eggs.nymphs or adults of whitefly via ova.Then we constructed mutant viruses using PaLCuCNV and TYLCV,the latter of which is known to be transovarially transmitted readily by MEAM1 whiteflies,by partially exchanging the coat protein gene,and subjected them to transovarial transmission assays.Our results showed that mPaLCuCNV,in which coat protein gene of PaLCuCNV was partially replaced by that of TYLCV,could be transovarially transmitted to the whitefly offspring eggs,nymphs and adults,and correspondingly mTYLCV could not be transovarially transmitted.Taken togenther,this thesis studied the endocytosis and intracellular trafficking pathways of begomoviruses exploited to infect ovary and PSGs of whiteflies.Our findings suggest that CME and early endosome network play important and conserved roles in virus entry into multiple organs of whitefly.Further,we found that the efficiencies of transovarial transmission of TYLCV may vary for different whitefly species.and coat protein is the sole determinant of the transovarial transmission of begomoviruses.Our findings provide new insights into the nature of begomoviruses transmission by whitefly,and will also serve as theoretical basis for the development of novel tactics targeting those whitefly-borne plant viruses.