Identification And Preliminary Functional Characterization Of Whitefly Proteins That Interact With The Coat Protein Of Geminivirus

Geminiviruses are a group of single-stranded circular DNA plant viruses.Plant diseases caused by geminiviruses are now major constrants to the crop production in many region of the world.The genus Begomovirus contains the majority of described species within the family Geminiviridae and members of this genus are exclusively transmitted by the whitefly in a persistent-circulative manner.The worldwide spread of Middle East Asia Minor 1(MEAM1)and Mediterranean(MED)whiteflies has been accompanied by the epidemics of geminiviruses during the last 20 years.There are multiple barriers in whitefly for viruses before they can be efficiently transmitted,including midgut infection barrier,midgut escape barrier,salivary gland infection barrier and salivary gland escape barrier.Passage of viruses through these barriers requires specific and complex interactions between virus coat protein and vector components.The whitefly receptor proteins that participate in the begomovirus transport across cells are largely unknown.In this study,we focus on the molecular mechanism by which geminiviruses cross the midgut and salivary gland barrier of whitefly.1)Preliminary study on function of the selected proteins during the geminivirus acquisition by MEAM1 whiteflyIn previous study from our laboratory,a total of 76 proteins were identified as putative tomato yellow leaf curl virus(TYLCV)interacting proteins in whitefly midgut using a midgut-specific immunoprecipitation followed by high-throughput mass spectrometry proteomic analysis.Among these candidates,we chose an Unknown protein(Unpro)and a Solute carrier family 2,facilitated glucose transporter member 8(Sl2ca8)for further functional investigation.Thses two ptroteins both have transmembrane domains as determined by in silco prediction.Silencing of Unpro and Slc2a8 genes significantly increased the amount of virus acquired by MEAM1 whiteflies.The gene transcript level in TYLCV-infected whiteflies was significantly lower than that in uninfected whiteflies.Considering their previously reported function,we propose that Unpro and Slc2a8 may be involved in vesicular transport of TYLCV and more specifically virus degradation,which warrants further investigation.According to the quantitative proteomic data,the expression of integrin alpha-PS2(Integrin)and laminin subunit alpha(Laminin)were significantly up-regulated in the TYLCV-and PaLCuCNV-infected whiteflies compared to un-infected whiteflies.Therefore,we chose Integrin and Laminin for further functional investigation.Thses two ptroteins both have transmembrane domains.Silencing of Integrin significantly decreased the amount of virus acquired by MEAM1 whiteflies.Laminin gene transcript level in TYLCV-and PaLCuCNV-infected whiteflies were lower than that in un-infected whiteflies,but no significant changes of Integrin.Considering their previously reported function,we propose that Integrin and Laninin may synergistically regulate the endocytosis of begomovirus in whitefly,but the detailed moleculer mechanism warrants further investigation.2)Screening of proteins in the midgut and salivary gland of MEAM1 that interact with the coat protein of PaLCuCNV by yeast two hybridWe use the yeast two hybrid system to screen the cDNA library of MEAM1 midgut and salivary gland to identify the proteins that interact with the coat protein(CP)of PaLCuCNV.In total,we identified 10 proteins that interacted with PaLCuCNV CP.Next,we cloned the full length of the genes that encode these proteins,and found that protein transport protein Sec61 subunit beta(SEC61?)and eukaryotic translation initiation factor 5A(eIF5A)can interact with PaLCuCNV CP.Next,for SEC61?,we performed GST-pull down assay to verify its interaction with PaLCuCNV CP in vitro.In addition,the amount of PaLCuCNV acquired by whiteflies was significantly reduced by silencing of SEC61? gene.SEC61? gene transcript level in PaLCuCNV-infected whiteflies were lower than that in un-infected whiteflies.Considering its previously reported function,we propose that SEC61? may be involved in transport of PaLCuCNV or the replication of PaLCuCNV in whiteflies,but the detailed moleculer mechanism warrants further investigation.To sum up,the transport pathways of geminivirus in whiteflies is complex.The results suggest that Intergin and SEC61? promote begomoviruses acquisition by whiteflies,and Unpro and Slc2a8 inhibite begomoviruses acquisition.And the role of Laminin in begomoviruses transmission is unknown.These novel findings contribute to a better understanding of the complex interactions between geminiviruses and whitefly,and provide valuable basis for further study of the mechanism of the transmission of geminiviruses by whitefly.