Start Date: Dec 01, 2007
End Date: Dec 01, 2012
To better understand the linkages between aphid genotype, the cellular mechanisms for virus recognition and transport, and vector efficiency, microscopy and a biological assay for virus infectivity will be used to identify the transmission barrier locations and their modes of action in nonvector genotypes. Immunolabeling studies will identify the tissues (gut or salivary) associated with virus accumulation, while ultrastructural TEM studies will determine the cellular mechanisms operating to prevent virus movement, e.g. virus recognition or transport in the cytoplasm. An injection recovery bioassay will determine if non-vector aphids that successfully acquire virus into the hemolymph have an immunological response that degrades virions in the hemolymph. Proteomic and genomic studies will be used to identify aphid proteins and genes that facilitate virus transmission pathway in vectors or prevent virus movement in nonvectors. Protein targets would include virus receptors that facilitate virus binding and uptake or proteins that facilitate virus transport through gut and salivary tissues. We will use three complementary approaches in parallel: (a) Differential Gel Electrophoresis (DIGE) allows a direct comparison of vector and non-vector proteomes using differential protein extraction, (b) DIGE comparison of vector and nonvector aphid proteins that bind specifically to virus particles and (c) a quantitative shotgun approach employing isotope coding (iTRAQ) to compare tissue-specific proteomes from aphids that have tissue-specific barriers for viral transmission to those tissues in aphids that are efficient vectors with no identified barriers. To directly identify aphid genes involved in virus transmission we will expand our preliminary studies that showed a yeast two-hybrid system transformed with aphid cDNA libraries and screened for components that bind to the RPV-CP or RPV-RTD can identify aphid proteins that interact with luteovirid structural proteins. We plan to continue screening the libraries to identify any additional candidate genes beyond the 32 currently in hand, and then begin a more in-depth characterization of each candidate. In situ labeling of mRNAs will be used to identify differential tissue expression of genes coding for virus-binding proteins between vector and nonvector genotypes and determine if they are localized only to tissues associated with virus transmission or if they are expressed in a variety of tissues. All of the experiments outlined above will identify a set of aphid genes and proteins that are correlated with the ability of aphid genotypes to transmit circulative viruses. Our collection of F2 aphid genotypes and knowledge of the location and mechanism of virus transmission barriers within each genotype provides a unique resource to directly correlate (in multiple genotypes) the presence or absence of an aphid gene or protein with a transmission phenotype localized to a specific tissue or mechanism. We acknowledge that this is still only correlative data and we will not have proof of functionality. This will come, but is not within the scope or timeframe of this grant.