Molecular Mechanisms Underlying The Infection Of Whitefly Midgut Cells By Tomato Yellow Leaf Curl Virus

Tomato yellow leaf curl virus(TYLCV,genus Begomovirus,family Geminiviridae)is a single-stranded circular DNA virus.TYLCV is transmitted by whiteflies of the Bemisia tabaci cryptic species complex in a persistent circulative manner.During the last decades,TYLCV has caused extensive losses to the production of many crops such as tomato and tobacco worldwide.B.tabaci ingests virions when sucking phloem sap of TYLCV-infected plants.Thereafter viruses go through a sequential path of stylet-midgut-haemolymph-salivary gland in B.tabaci,and finally are transmitted into new host plant with vector saliva secretion.In this process,B.tabaci midgut is the first barrier viruses encounter,thus the capacity of viruses to move across vector midgut wall is closely related to virus transmission efficiency.Previous research shows that TYLCV hijacks clathrindependent endocytosis to pass through the vector midgut apical membrane(midgut invasion barrier),after which the intracelluar trafficking of TYLCV from the midgut apical surface to basal surface relies on vesicles via Rab5-labeled early endosome.Although the role of clathrin-dependent endocytosis and vesicle trafficking systems of B.tabaci in virus movement have been recognized,the molecular mechanisms underlying endocytosis and vesicle trafficking,such as the endocytic receptor(s)and factors related to vesicle trafficking,remain unknown.In this study,we used the coat protein(CP)of TYLCV as bait protein,and GST pulldown coupled with LC-MS/MS or split-ubiquitin yeast two hybrid system,to screen proteins of the whitefly Middle East-Asia Minor 1(MEAM1),a species of the B.tabaci cryptic species complex,that may interact with TYLCV CP.(1)B.tabaci proteins BtCUBN and BtAMN compose a receptor complex(BtCubam)to aid TYLCV in overcoming vector midgut invasion barrier.We used TYLCV CP as bait protein,and native protein extracts of MEAM1 as prey proteins for GST pull-down and LC-MS/MS analysis.We captured one receptor protein of MEAM1 named Cubilin(BtCUBN).Due to the lack of transmembrane region in BtCUBN,we performed further analysis and found that BtCUBN formed an endocytic receptor complex with one transmembrane protein of B.tabaci named amnionless(BtAMN).qPCR analysis showed that the transcription level of BtCUBN or BtAMN in MEAM1 midgut was significantly higher than that in the carcass.Immunogold or immunofluorescence revealed the presence of BtCUBN and BtAMN in the microvilli and cytoplasm of MEAM1 midgut.GST pull-down assay indicated that TYLCV CP bound to BtCubam receptor complex by interacting with the 12-19 CUB domains of BtCUBN.These results,together with our findings obtained from co-localization analysis via immunofluorescence,suggest that TYLCV is internalized together with BtCubam via clathrin-dependent endocytosis.Next,BtCubam may dissociate from TYLCV in early endosome,wherein BtCubam moves back to the membrane through recycling endosome.In addition,both in vitro and in vivo experiments indicated that TYLCV CP exhibited no affinity with CUBN of a non-vector insect Trialeurodes vaporariorum,and this lack of afffinity may be an important reason why TYLCV is unable to overcome the midgut invasion barrier of T.vaporariorum.(2)Interruption of BtCubam leads to significant reductions of TYLCV acquisition and transmission by B.tabaci.qPCR,western blot and immunofluorescence analysis demonstrated that TYLCV infection up-regulated the transcription and translation of BtCUBN and BtAMN.Adminstration of anti-CUBN rabbit mAb to interfere with the interaction between BtCUBN and TYLCV CP or dsRNA micro-injection to suppress the expression of BtAMN reduced the capacity of viral acquisition and transmission by MEAM1.(3)Vesicle-associated membrane protein-associated protein B-like was identified as a TYLCV CP-binding protein.We used TYLCV CP as bait and MEAM1 cDNA library with three open read frames as prey.Vesicle-associated membrane protein-associated protein B-like(VAPB),an abundantly expressed protein in MEAM1 midgut,was identified as putative TYLCV CP-binding protein by split-ubiquitin yeast two-hybrid system.The interaction between VAPB full length and TYLCV CP was confirmed by split-ubiquitin yeast two-hybrid system.GST pull-down analysis showed that their interaction depended on the SCS2 domain of VAPB but was independent of VAPB transmembrane region.Immunofluorescence showed a clear co-localization of VAPB and TYLCV CP in vivo,but the phase when the interaction of VAPB with TYLCV CP occurs in the process of viral trafficking in MEAM1 midgut is yet to be defined.(4)VAPB negatively regulates the movement of TYLCV.TYLCV infection up-regulated the transcription and translation of VAPB in B.tabaci whole body and midgut.And ds VAPB adminstration led to higher viral amount in MEAM1 haemolymph and salivary gland,as well as higher viral transmission efficiency by MEAM1.However,the mechanisms underlying this negative regulation warrant further investigations.In conclusion,our study indicates that,B.tabaci proteins BtCUBN and BtAMN on the surface of cell membrane combine to form a receptor complex BtCubam.When TYLCV has reached the midgut lumen following ingestion by B.tabaci,TYLCV binds to BtCubam via interacting with specific domains of BtCUBN.The virus is then internalized together with BtCubam via clathrin-dependent endocytosis.These observations preliminarily reveals the molecular mechanisms underlying the movement of TYLCV from the intestinal tract to inside the cells of vector midgut.Based on these results,we proposed a model illustrating the process by which TYLCV moves across the infection barrier of B.tabaci midgut.In addition,we found that VAPB plays an inhibitory role in TYLCV release from B.tabaci midgut cells to haemolymph.These novel findings not only provide further insight into the path of TYLCV movement across the vector midgut wall and the underlying molecular mechanisms,but also offer important information for the research and development of methods of blocking TYLCV transmission via interference with the virus movement.In view of the common path of begomovirus movement in the body of B.tabaci,our results also provide valuable reference for the research on the interactions between all viruses of the genus Begomovirus and their insect vectors.