Roles Of Midguts In Whitefly Transmission Of Cotton Leaf Curl Multan Virus And Underlying Mechanisms

Plant viral diseases are major constraints to the world agriculture.In the field,insect vectors such as aphids,planthoppers and whiteflies are responsible for the spread and epidemic of the majority of plant viruses.Among the plant viruses transmitted by insect vectors,begomoviruses(family:Geminiviridae)have emerged as serious constraints to the production of many crops worldwide.In the past decades,cotton leaf curl disease and tomato yellow leaf curl disease caused by begomoviruses have been major constraints to the production of cotton in Pakistan and tomato worldwide.Begomoviruses are transmitted by the whitefly(Bemisia tabaci)species complex.During the last decades,our understanding regarding the whitefly transmission of many begomoviruses has been improved;however,for begomoviruses associated with cotton leaf curl disease,the nature of whitefly transmission of these viruses as well as the influencing factors remained barely explored.In addition,the midgut of whitefly represents one of the major barriers to be crossed by begomoviruses in order to be successfully transmitted,and the mechanisms underlying virus transport across the midgut wall remains unknown.In this study,we investigated the characteristics of whitefly transmission of begomoviruses associated with cotton leaf curl disease and the physiological and molecular mechanisms underlying the transport across the midgut wall of whiteflies by begomoviruses.1)Roles of whitefly midguts in differential transmission of begomoviruses associated with cotton leaf curl disease by different species of whitefliesWe firstly compared the transmission efficiency of Cotton leaf curl Multan viruses(CLCuMV)by four species of whiteflies,including Middle East Asia Minor 1(MEAM1),Mediterranean(MED),Asia ? and Asia? 1,and found that Asia ?1 transmit this virus with highest efficiency,followed by MED,whereas MEAM1 and Asia ? transmit this virus with lowest efficiency.Next,by performing analysis of virus quantity and immune-fluorescence,we found that the differential transmission of CLCuMV was associated with the capacity of this virus to cross the midgut wall of different species of whiteflies.Further,by phylogenetic analysis based on the amino acid sequence of coat proteins of begomoviruses associated with cotton leaf curl disease,we inferred that Asia ? 1 be able to transmit 7 out of 10 known begomoviruses associated with cotton leaf curl disease with high efficiency and MEAM1 and MED whiteflies be able to transmit two of these viruses with high efficiency.2)Screening of proteins in Asia ?1 that interact with the coat protein of CLCuMVWe used the spilt-ubiquitin yeast two hybrid system to screen the cDNA library of Asia ?1 to identify the proteins that interact with the coat protein of CLCuMV.In total,we identified 39 proteins that are involved in various biological processes of the interaction with the coat protein of CLCuMV.Next,we cloned the full length of the genes that encode 8 of these proteins,and verified their interaction with the coat protein of CLCuMV.Finally,we performed GST-pull down assays for three of the proteins to examine the interaction in vitro.3)The role of clathrin mediated endocytosis in Tomato yellow leaf curl virus transport across the midgut wall of whitefliesWe studied the involvement of clathrin mediated endocytosis in begomoviruses transport across the midgut wall of whiteflies by disrupting the clathrin-mediated endocytosis and the endosome network using inhibitor feeding,antibody blocking and dsRNA silencing.We found that following endocytosis and endosome network disruption,the quantity of virus was higher in the midgut relative to that of the whole insect body,and the quantity of virus in the haemolymph was reduced.In addation,the transmission efficiency of TYLCV by the treated insects was reduced.In summary,in this study we compared the transmission efficiency of CLCuMV by four species of whiteflies,and found that the differential transmission of CLCuMV was associated with the differential capacity of CLCuMV to cross the midgut wall of different species of whiteflies.Our phylogenetic analysis of virus coat proteins indicated that most of the begomoviruses associated with cotton leaf curl disease share the same whitefly transmission characteristics to those of CLCuMV.Further,we identified several proteins in Asia ?1 that interact with the coat protein of CLCuMV.In addation,we investigated the role of clathrin mediated endocytosis in the begomovirus transport across the midgut wall of whiteflies.Our findings will not only help to understand and predict the epidemiology of cotton leaf curl disease in the field,but also provide the basis for further unvavelling of the interactions between begomoviruses associated with cotton leaf curl disease and their vector whiteflies.