Roles Of Whitefly Salivary Glands And Ovary In Begomovirus Transmission

Geminiviruses have unique twin particle morphology and either monopartite or bipartite single-stranded DNA genomes. Geminiviruses form the second largest plant virus family, Geminiviridae and cause devastating diseases to a number of economically important crops such as tomato, cotton and cassava worldwide. Geminiviruses are mainly transmitted by the whiteflies in the Bemisia tabaci species complex, which colonizes many plant species and vectors over 200 different plant viruses. The increase in vector populations has often resulted in the prevalence of geminiviral diseases. In this study, we investigated the transmission mechanisms of two begomoviruses Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl China virus (TYLCCNV) by the invasive species in the B. tabaci complex, i.e. Middle East Asia Minor 1 (MEAM1) and Mediteranean (MED) whiteflies. The results are summarized as follows:(1) Localization of TYLCV in MEAM1 whiteflyImmunofluorescent localization of TYLCV in midgut showed that viral particles were enriched in the filter chamber. In the ovary, viral particles were only traced in mature eggs, not in immature ovarioles. In salivary glands, viral particles were only traced in several cells in the central region of salivary gland and only very few viral signals were detected in other regions of the gland.(2) The specificity of TYLCV and TYLCCNV transmission by MEAM1 and MED whitefliesWe examined the ability of MEAM1 and MED whiteflies to transmit TYLCCNV and TYLCV. MEAM1 whiteflies transmitted both viruses, while MED whiteflies only transmitted TYLCV but not TYLCCNV. In addition, MED whiteflies accumulated and retained more viruses than did MEAM1 whiteflies, which suggested that the failure of MED whiteflies to transmit TYLCCNV was not due to its inability to ingest and retain the virus. We next examined whether potential barriers inside the whitefly body blocked TYLCCNV passage by tracing viral DNA in different whitefly tissues along the virus transmission pathway. The results demonstrated that (i) the efficiency of TYLCCNV penetration into primary salivary glands (PSGs) of MED whiteflies was low; and (ii) even if TYLCCNV virions could penetrate the PSGs of MED whiteflies, they failed to move out of the salivary duct and thus could not be secreted to saliva. To investigate whether viral CP is implicated in the specificity of virus transmission, we exchanged a partial CP sequence of TYLCV which has lower similarity with that of TYLCCNV and examined the transmissibility of the two recombinant viruses by MEAM1 and MED whiteflies. This exchange resulted in successful transmission of recombinant TYLCCNV by MED whiteflies while almost no transmission of recombinant TYLCV. Salivary glands were also involved in the transmission specificity of the two recombinant viruses. Immunofluorescence staining of TYLCV and TYLCCNV in the salivary glands of MED whiteflies indicated that TYLCV virions occupied the entire salivary glands during 3-h to 12-h virus access acquisition period (AAP). Interestingly, during 12-h to 24-h AAPs, virions gradually accumulated to the central region of PSGs. From 36-h to 72-h AAPs, the viral CP was observed only in a few specific cells along the salivary duct, and little signal was traced in other regions of PSGs. Strikingly, during 4-h to 12-h AAPs, TYLCCNV virions were always distributed in circumference cells of MED PSGs. From 24-h to 72-h AAPs, virions were scarcely localized in PSGs. The accumulation dynamics of TYLCV and TYLCCNV in the salivary glands of MEAM1 were similar with that of MED whiteflies. These results suggest that begomovirus transmission specificity is determined jointly by the whitefly salivary glands and viral CP.(3) Transovarial transmission of TYLCV by MEAM1 and MED whitefliesImmunofluorescence showed that TYLCV virions were located only in some mature ovariles, and never in immature ovariles. Furthermore e, the efficiency of transovarial transmission of TYLCV increased with the whitefly age. Those observations indicate that the capacity of vertical transmission of TYLCV by whitefly is determined by the developmental stage of the ovary. The synthesis of Vg in ME AMI whitefly was low within 5 d after eclosion, and then increased rapidly until 11d after eclosion. Immunofluorescence showed that TYLCV CP and Vg colocalized with each other in viruliferous MEAM1 and MED whiteflies. Next, we tested whether TYLCV CP interacts with Vg by co-immunoprecipitation (CO-IP) of TYLCV CP with anti-Vg specific antibody and vice versa. Our results showed that both antibodies co-immunoprecipitated the other interacting protein as detected by Western blotting, suggesting a specific interaction between CP and Vg. Feeding whiteflies with anti-Vg antibody decreases the vertical transmission rates of TYLCV. These data demonstrated that the vertical transmission efficiency of TYLCV was associated with the developmental stage of whitefly ovary, and Vg is involved in this process.In summary, our study showed the localization of two begomoviruses in the body of two invasive species of the whitefly B. Tabaci and revealed important cellular and molecular mechanisms of transmission specificity as well as the vertical transmission of two viruses by the two species of whitefly B. Tabaci. The study has advanced our understanding of the interactions between whiteflies and begomoviruses, and provided new knowledge for further divulging the molecular mechanisms of geminivirus transmission by whiteflies and developing novel methodology aimed at blocking virus transmission by whiteflies.