Dissection of Whitefly-geminivirus Interactions at the Transcriptomic, Proteomic and Cellular Levels

Our project focuses on gene expression and proteomics of the whitefly Bemisia tabaci (Gennadius) species complex in relation to the internal anatomy and localization of expressed genes and virions in the whitefly vector, which poses a major constraint to vegetable and fiber production in Israel and the USA. While many biological parameters are known for begomovirus transmission, nothing is known about vector proteins involved in the specific interactions between begomoviruses and their whitefly vectors. Identifying such proteins is expected to lead to the design of novel control methods that interfere with whitefly-mediated begomovirus transmission. The project objectives were to: 1) Perform gene expression analyses using microarrays to study the response of whiteflies (B, Q and A biotypes) to the acquisition of begomoviruses (Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV). 2) Construct a whitefly proteome from whole whiteflies and dissected organs after begomovirus acquisition. 3) Validate gene expression by q-RTPCR and sub-cellular localization of candidate ESTs identified in microarray and proteomic analyses. 4) Verify functionality of candidate ESTs using an RNAi approach, and to link these datasets to overall functional whitefly anatomical studies. During the first and second years biological experiments with TYLCV and SLCV acquisition and transmission were completed to verify the suitable parameters for sample collection for microarray experiments. The parameters were generally found to be similar to previously published results by our groups and others. Samples from whole whiteflies and midguts of the B, A and Q biotypes that acquired TYLCV and SLCV were collected in both the US and Israel and hybridized to B. tabaci microarray. The data we analyzed, candidate genes that respond to both viruses in the three tested biotypes were identified and their expression that included quantitative real-time PCR and co-localization was verified for HSP70 by the Israeli group. In addition, experiments were undertaken to employ in situ hybridization to localize several candidate genes (in progress) using an oligonucleotide probe to the primary endosymbiont as a positive control. A proteome and corresponding transcriptome to enable more effective protein identification of adult whiteflies was constructed by the US group. Further validation of the transmission route of begomoviruses, mainly SLCV and the involvement of the digestive and salivary systems was investigated (Cicero and Brown). Due to time and budget constraints the RNAi-mediated silencing objective to verify gene function was not accomplished as anticipated. HSP70, a strong candidate protein that showed over-expression after TYLCV and SLCV acquisition and retention by B. tabaci, and co-localization with TYLCV in the midgut, was further studies. Besides this protein, our joint research resulted in the identification of many intriguing candidate genes and proteins that will be followed up by additional experiments during our future research. To identify these proteins it was necessary to increase the number and breadth of whitefly ESTs substantially and so whitefly cDNAs from various libraries made during the project were sequenced (Sanger, 454). As a result, the proteome annotation (ID) was far more successful than in the initial attempt to identify proteins using Uniprot or translated insect ESTs from public databases. The extent of homology shared by insects in different orders was surprisingly low, underscoring the imperative need for genome and transcriptome sequencing of homopteran insects. Having increased the number of EST from the original usable 5500 generated several years ago to >600,000 (this project+NCBI data mining), we have identified about one fifth of the whitefly proteome using these new resources. Also we have created a database that links all identified whitefly proteins to the PAVEdb-ESTs in the database, resulting in a useful dataset to which additional ESTS will be added. We are optimistic about the prospect of linking the proteome ID results to the transcriptome database to enable our own and other labs the opportunity to functionally annotate not only genes and proteins involved in our area of interest (whitefly mediated transmission) but for the plethora of other functionalities that will emerge from mining and functionally annotating other key genes and gene families in whitefly metabolism, development, among others. This joint grant has resulted in the identification of numerous candidate proteins involved in begomovirus transmission by B. tabaci. A next major step will be to capitalize on validated genes/proteins to develop approaches to interfere with the virus transmission.