Mechanisms Underlying The Movement And Replication Of Begomoviruses In Whitefly Bemisia Tabaci

Geminiviruses are a large family of DNA viruses that cause serious crop losses worldwide.The whitefly Bemisia tabaci is a species complex that contains more than 36 cryptic species.Begomoviruses,which constitute the largest geminivirus genus,are exclusively transmitted by the whitefly B.tabaci in a persistent-circulative manner There are multiple barriers during the circulative transmission of viruses,including midgut infection and dissemination barriers,salivary gland infection and escape barriers,and transovarial transmission barrier.Passage of viruses through these barriers requires specific interactions between virus and vector components.However,the whitefly ligands that participate in begomovirus transport across these barriers are largely unknown.In addition,whether begomoviruses replicate within whiteflies remains unclear.In this study,we focused on these important questions,and the results are summarized as follows:1)Transovarial transmission mechanism of begomovirusesOur previous study found that Tomato yellow leaf curl virus(TYLCV)entry of the reproductive organ of its vector mainly depended on the developmental stage of the whitefly ovary,and the transovarial transmission of TYLCV to offspring increased with whitefly adult age.In contrast,another begomovirus,Papaya leaf curl China virus(PaLCuCNV)was never detected in the ovary of viruliferous whiteflies,and could not be transovarially transmitted by whiteflies.As vitellogenin(Vg)plays a critical role in insect yolk formation and oogenesis,we assumed that Vg is involved in the entry of TYLCV into ovary.Here,we showed that the interaction between TYLCV coat protein(CP)and whitefly Vg was vital for virus entry into whitefly ovary.The entry of TYLCV virions into the oocyte shares the same route of Vg transport into the oocyte.When knocking down the expression of Vg,the entry of TYLCV into ovary was inhibited and the transovarial transmission efficiency decreased.In contrast,PaLCuCNV CP did not interact with whitefly Vg.We further showed that TYLCV could be maintained for at least two generations in the absence of virus-infected plants,and the adult progenies were able to infect healthy plants.2)The replication site of TYLCV in whitefliesThe above results that TYLCV could be vertically transmitted in whiteflies for at least two generations in the absence of virus host plants,strongly implying replication of TYLCV in progeny of viruliferous whiteflies.Quantification of virus abundance in the adult offspring,which developed from eggs deposited on cotton by viruliferous whiteflies,found that the amount of TYCV increased during the first 11 days after eclosion.which adequately proved replication of TYLCV in the progeny population.Fluorescence in situ hybridization(FISH)and immunofluorescence assays showed that TYLCV virions were only detected in cells in the central region of primary salivary glands(PSG)of adult offspring derived from viruliferous whiteflies.Moreover,TYLCV specifically accumulated in cells in the central region of PSG of whiteflies during long-term retention following a 48-h acquisition access period(AAP),while no such accumulation was found for PaLCuCNV,Examination of virus complementary-sense DNA strand and transcripts also suggested that TYLCV mainly replicate in the central region of PSG.In contrast,PaLCuCNV could not replicate in whiteflies.These results demonstrated that TYLCV is able to replicate in whiteflies and that specific cells in the central region of whitefly PSG are the major place of replication.3)The mechanism of TYLCV replication in whitefliesTo gain insight into the molecular mechanisms underlying the replication of TYLCV in whitefly PSG,we used RNA-Seq to investigate the transcriptional response of MEAM 1 whitefly PSG to TYLCV infection.We found that TYLCV induces DNA synthesis machinery,proliferating cell nuclear antigen(PCNA)and DNA polymerase δ(Po1δ),to establish a replication-competent environment in whiteflis.TYLCV replication-associated protein(Rep)interacts with whitefly PCNA,which recruits DNA Polδ for replication.In contrast,PaLCuCNV did not change DNA synthesis machinery and PaLCuCNV Rep could not interact with whitefly PCNA.4)The mechanism of TYLCV across the midgut wallA total of 76 proteins(including Vg)were identified as putative TYLCV interacting proteins in whitefly midgut using a midgut-specific immunoprecipitation followed by high-throughput mass spectrometry proteomic analysis method.Among these candidates,we chose Vg for further investigation,because of our previous findings that TYLCV interacts with whitefly Vg and this interaction mediates virus entry into whitefly ovary.The expression of Vg in whitefly midgut was confirmed by several evidences.Localization of TYLCV and Vg in the midgut of viruliferous whiteflies showed that TYLCV colocalized with Vg in the midgut epithelial cells.Moreover,Vg accompanied TYLCV across the midgut epithelial cells,starting at the lumen side of microvillar membrane,passing through the cytoplasm and ending in place near to the basal membrane,suggesting that Vg is involved in the entry and intracellular transport of TYLCV in the midgut epithelial cells.When knocking down the expression of Vg or disrupting the interacting between TYLCV CP and Vg in the midgut using anti-Vg antibody,the transport of virus across the midgut epithelial cells was inhibited.These results suggest that Vg is a mediator of TYLCV across the midgut wall of whiteflies.Taken together,we found that the whitefly Vg plays a critical role in facilitating the transport of TYLCV across the midgut and transovarial transmission barriers.Moreover,we proved that TYLCV is able to replicate within whiteflies,and that specific cells in the central region of whitefly PSG are the major place of replication.We also revealed mechanisms that TYLCV have evolved to replicate in whiteflies.These novel findings give better understanding of the complex interactions between viruses and their insect vectors and provide valuable clues for designing strategies to block virus transmission.In addition,our study demonstrated that insect Vg is also synthesized in the midgut,a tissue previously known as non-Vg producing.