Stability of AtVSP in the insect digestive canal determines its defensive capability

We have previously demonstrated that Arabidopsis vegetative storage protein (AtVSP) is an acid phosphatase that has anti-insect activity in in vitro feeding assays [Liu et al., 2005. Plant Physiology 139, 1545-1556]. To investigate the functionality of AtVSP in planta as an anti-insect defense protein, we produced AtVSP-overexpressing as well as AtVSP-silenced transgenic Arabidopsis lines, and evaluated impact on the polyphagous American grasshopper Schistocerca americana. Grasshoppers showed no significant difference in weight gain and growth rate when feeding on wild type, overexpressing, or silenced lines, respectively. In addition, AtVSP protein was undetectable in either the midgut or frass of grasshoppers reared on transgenic plants suggesting that AtVSP was unable to withstand proteolytic degradation. To determine the stability of the AtVSP protein in grasshopper digestive canal, midgut extracts from various nymphal stages were incubated with bacterially expressed AtVSP for different periods of time. AtVSP was hydrolyzed rapidly by grasshopper midgut extract, in stark contrast with its fate when incubated with cowpea bruchid midgut extract. Multiple proteases have been detected in the midgut of grasshoppers, which may play important roles in determining the insect response to AtVSP. Results indicate that stability of an anti-insect protein in insect guts is a crucial property integral to the defense protein.