The major aim is to investigate the key mechanisms for the systemic action of dsRNA and the success of RNAi in vivo in pea aphids. The specific objectives are:
- to monitor RNAi in vivo for a number of well selected RNAs that target specifically key genes for normal insect growth, development and homeostasis. To investigate the systemic distribution, we selected targets that are present at different locations in the insect body (gut, nervous system, endocrine system, epidermis, fat body, gonads), and also in different developmental stages (larva, adult). Here the focus is on species- and tissue-specific aspects of systemic RNAi, and the results can then help to evaluate RNAi as a novel strategy in the control of pest insects, specifically important aphids like A. pisum.
- to investigate the presence and impact of physical barriers in the insect body and the kinetics of cellular uptake in different tissues for successful RNAi in vivo. For this purpose, live cell imaging microscopy with labeled dsRNAs will be employed.
- to determine the orthologs of transport proteins (e.g., scavenger, SID, pattern recognition receptors) involved in the uptake process for RNAi (cf. data from 1 & 2) by cloning, sequencing and expression analysis.
to investigate the function of the transport proteins (from part 3) in insect cells:
- to screen a library of cell lines (i.e. primary and continuous cells from different species and tissues, including the insect midgut) for competence/refractiveness in uptake of dsRNA;
to engineer refractory cell lines to over-expression of the identified transport proteins and evaluation of systemic spread of RNAi;
- in the cell lines competent for dsRNA uptake, knockouts will be made of the transport proteins that are responsible for the uptake process.