Characterization of plant-spider mite interactions and establishment of tools for spider mite functional genetic studies

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores feeding on cell contents of over 1100 plant species including more than 150 crops. However, despite its important pest status and a growing understanding of the molecular basis of its interactions with plant hosts, knowledge of the way mites interface with the plant while feeding and the plant damage directly inflicted by mites is lacking. Likewise, while the use of the reverse genetic tools in plants facilitated our understanding of the establishment of defense mechanisms against spider mite herbivory, such tools are lacking for spider mite, preventing the expansion of functional analysis to both sides of the interacting organisms. First, using various microscopy methods, I uncovered several key features of T. urticae feeding. By following the stylet path within the plant tissue, I determined that the stylet penetrates the leaf either in between epidermal pavement cells or through a stomatal opening, without damaging the epidermal cellular layer. Recordings of mite feeding events established that duration of the mite feeding ranges from several minutes to more than half an hour, during which time, mites consume a single mesophyll cell in a pattern that is common to both bean and Arabidopsis plant hosts. In addition, this study determined that leaf chlorotic spots, a common symptom of mite herbivory, do not form as an immediate consequence of mite feeding. Second, using a soaking delivery method of dsRNA, I successfully triggered RNAi response in TuVATPase and TuCOPB2 target genes, resulting in visible phenotypes that correlated with reduced mite fitness and silencing of the VATPase gene. In addition, using RNAi-associated phenotypes, I enhanced RNAi efficiency by mixing dies with dsRNA, to preselect mites that successfully ingested dsRNA, and have established a minimum size of 400 nucleotides of dsRNA to achieve a potent RNAi in spider mite. Overall, my findings established a cellular context of plant-spider mite interactions and contributed to the development of the efficient RNAi protocol, a critical step toward functional characterization in T. urticae