Intracellular Trafficking Route Of Begomoviruses In Whitefly

The whitefly Bemisia tabaci(Hemiptera:Aleyrodidae)is a specie complex consisting of more than 36 cryptic species.Whitefly cause serious damage by transmitting begomoviruses including Tomato yellow leaf curl virus(TYLCV)and Cotton leaf curl Multan virus(CLCuMV).The emergence of begomoviruses as important pathogens is closely related to the prevalence of whiteflies worldwide.As begomoviruses are transmitted in circulative manner,viruses have developed complex interactions with their insect vectors,in which viruses need to be transported across the epithelial cells in gut and salivary gland.The extensive use of transmission electron microscopy observations have shown that the transcytosis process is responsible for the transport of luteovirids across midgut and salivary gland.However,there are still several questions remain to be answered.(1)What are the genes and vesicles that are needed during the transport from the apical to the basal of epithelial cells and which membranous compartment is responsible for secreting virus into hemolymph?(2)Does transcytosis process help the transport of other circulative transmitted virus?To address these questions,we used specific inhibitors,dsRNA-mediated gene silencing,and immunofluorescence to study the importance of intracellular vesicle trafficking in the transmission of begomoviruses by whitefly.The results are summarized as follows:(1)The intracellular transport of begomovirus depend on vesicles.In this study,we showed that most of viral particles of begomoviruses accumulate in a lectin HPA-labeled vesicles,which were later demonstrated to be endosomal system.Virus acquisition can be inhibited by Brefeldin A,which can disrupt intracellular vesicle transport by pleiotropic effect on the entire endosome system and Golgi apparatus.Our data suggested that vesicle trafficking was critical for the transport of TYLCV into vector hemolymph.(2)Early endosome plays important role in virus transport.By disrupting different kinds of vesicles,we further showed that the early steps of endosomal trafficking is important for intracellular transport of begomoviruses.Whereas,Golgi apparatus,late endosome,lysosome,and recycling endosome were shown unrelated to the transmission.(3)Begomoviruses may be transported to hemolymph by endosomal tubular network.Viral particles were also found in tubular-shape vesicles,which may be part of endosomal tubular network.And silencing sorting nexins that are responsible for membrane tubulation resulted in fewer viruses in hemolymph suggesting the involvement of endosomal tubular network.(4)Ubiquitin-proteasome may suppress viral titer in whitefly.Disrupting ubiquitin-proteasome system lead to a significant increase of begomoviruses in whitefly.However,it is showed that the structural protein of begomoviruses will not be uniquitinated,indicating that ubiquitin-proteasome system may influence begomoviruses by activating immunal response,disabling receptor-mediated endocytosis or ubiquitinating non-structural protein of begomoviruses.Taken together,our data suggest that early endosome play an important role in virus transport.Ubiquitin-proteasome system can help whitefly by reducing viral titer in whitefly.Understanding the intracellular transport of viruses in their insect vectors can help us to develop better strategies for virus control.On the other hand,the transcytosis of viral particles can also be exploited for delivering toxins,such as neurotoxins from spiders,into the insect hemocoel and insect control.