Identification of a transcriptionally-responsive, TSWV-interacting protein in Frankliniella occidentalis first instar guts

Tomato spotted wilt virus (TSWV) and other thrips-borne orthotospoviruses are some of the biggest threats to production of food and ornamental crops. Frankliniella occidentalis, the principal thrips vector, transmits TSWV in a circulative-propagative manner, i.e., the virus enters the body (gut-entry), replicates and disseminates to insect tissue systems, and persists over the lifespan of the vector. To capture a wide array of F. occidentalis proteins that interact directly with the TSWV attachment protein, G_N, during the virus infection cycle over development, a membrane-based yeast two-hybrid assay (MbY2H) was performed with TSWV G_N cDNA cloned into a bait vector and one amplified, non-normalized F. occidentalis cDNA library representing all post-embryonic stages into the prey vector. The MbY2H screen resulted in 229 non-redundant sequences with moderate to very high confidence scores in their interactions with G_N. Blastn analysis of these putative interactors against an assembled first instar (L1) gut transcriptome of F. occidentalis significantly identified 116 transcripts. One novel transcript sequence, A220, was also identified to be an up-regulated, highly-connected hub gene (network analysis) in L1 guts in response to TSWV infection. A220 was amplified and subjected to 5’ and 3’ RACE to generate a full-length clone. The sequence was confirmed, encoding a hypothetical protein that contains several predicted eukaryotic linear motifs indicative of putative functional sites in the peptide. Given the dual implication of A220 as a putative G_N-interactor and transcriptionally responsive to TSWV, this sequence is a top candidate to move forward for functional analysis.