DEFINING THE MOLECULAR DETERMINANTS OF THRIPS VECTOR COMPETENCE TO TRANSMIT ORTHOTOSPOVIRUSES

<b>Project Methods</b><br> Objective 1. Characterize the fine-scale, co-localization patterns of TSWV-interacting proteins (TIPs) with viral structural proteins in thrips.Our aim for Obj.1 is to characterize F. occidentalis proteins that interact directly with GN and other TSWV structural proteins (GC and N) during the virus infection cycle (i.e., binding, internalization, replication, virion morphogenesis and exocytosis).Our published and preliminary supporting data focus our efforts on four TIPs for further validation and localization in insect cells,and in thrips tissues. With the exception of one novel thrips protein, these proteins have been shown to be associated with various virus processes in other arthropod-virus systems. Two of these interacting proteins also exhibited differential expression at the transcript-level in first instar larval guts of F. occidentalis after exposure to TSWV. Through complementary, state-of-the-artprotein-protein interaction approaches [colocalization, BiFC, and co-IP experiments] performed in vivoand in situ,we plan to generate a comprehensive physical interaction map that defines the F. occidentalis TIPs – virus structural protein interactions in the context of their cellular co-occupancy at the confocal and TEM levels as a means to define the biological roles of the four TIPs. The outcomes of Obj. 1 will provide a contextual framework for identifying cellular components associated with the novel, non-annotated TIP and other TSWV-responsive genes examined for Obj. 2, and will generate hypotheses to be addressed by Obj. 3 regarding thrips gene function in relation to virus transmission. Experiments will be robustly repeated (biological, experimental treatment and technical replication),and data analyzed using appropriate parametric and nonparametric statistical analysis methods and models depending on data types and distributions.Objective 2. Functionally annotate TSWV-responsive TIPs with regards to cell biology and larval growth and development.Preliminary data sheds light on two F. occidentalis proteins that may be the gateway to unraveling the molecular basis of thrips vector competence. One appears to be a novel F. occidentalis protein that has no known orthology to publicly-available proteins, and the other is a CPR-RR1 type cuticle protein, however its functional role has yet to be determined. Network analysis (WGCNA and membership analyses) of larval gut transcripts perturbed by TSWV exposure and infection (differentially-expressed transcripts, DET) placed these two proteins as the most tightly connected members within separate modular networks of co-expressed transcripts, many of which have known functional roles in other metazoan systems. Given the novelty of these proteins, and their possibly large influence on other gut-responsive genes, for Obj. 2 we plan to annotate these two TIPs by interrogating the validity of the predicted DET co-expression network connections Network analysis using WGCNA provides a pathway towards identification of candidate biomarkers of tissue system function and response to stress. Working on the premise that highly connected DETs (hubs) summarize and/or represent the gut response to virus by the collection of co-expressed connecting genes, and that interconnectivity between module members may indicate similar roles or cross-talk between pathways, we plan to perform reciprical, gene silencing experiments to monitor larval gut expression patterns of other genes in their network, and examine associated phenotypes (larval survival, body size, development (molting) and nutrient reserves (bio-energy: protein, lipid and glycogen content). As a supplemental approach, we will use our newly-developed, proof-of-conceptthrip gene-editing protocol (CRISPR/Cas9) to produce knock-out mutants for further examination of phenotypes and experimentation. Based on a priori annotations we have for the connecting genes, and the phenotypic response of guts to silencing,we hope to reveal the unique and/or collaborative roles of the DETs with regards to larval thrips biology. As with Obj. 1, all experiments will be robustly repeated and analyzed using the most suitable statistical methods.Objective 3. Functionally characterize the biological role(s) of TIPs during TSWV infection of the thrips vector.The combined knowledge of the TIP/viral protein physical interaction maps and TIP cellular co-localization with viral structural proteins (Obj. 1), and functional annotations of the TSWV-responsive (gut DETs) TIPs in the context of other responsive gut genes and thrips health (Obj. 2) will be used to design experiments aimed to determine the respective roles of these proteins in virus acquisition and/or replication in thrips. To fully interrogate protein function, we will use an array of experimental methods that are customized for the protein of interest. We will usean array of functional annotation tools [gene knockdown/knockout, pharmacological inhibition, and competitive binding assays] to thoroughly characterize the functional role of TIPs in the virus infection cycle in thrips. Defining protein function in F. occidentalis will be a springboard for future experiments with other vectors (thrips isolates and species) to explore their utility as biomarkers of vector competence and targets for disrupting transmission.