Delivery of Maize mosaic rhabdovirus into Peregrinus maidis by microinjection for functional analysis of genes involved in response to virus infection

The corn planthopper, Peregrinus maidis, is a major pest of agronomically-important crops (corn and sorghum), has a large geographical distribution, and transmits Maize mosaic rhabdovirus (MMV). Acquisition of MMV by feeding on infected corn plants often results in low infection and transmission rates in P. maidis and the midgut has been proposed as a significant barrier to virus infection. To increase infection and transmission efficiency for vector functional genomics experiments, we have by-passed the midgut and directly injected purified MMV into the vector. We found that injection of high doses of MMV (10ng-12.5ng) resulted in 100 % infection and 93 % transmission efficiency. In dose-response experiments, MMV titer and transmission efficiency decreased as the amount of virus inoculum injected was reduced. The utility of virus inoculation by microinjection for studying virus-vector interactions was explored by examining the impact of gene knockdown via RNA interference (RNAi) on virus titer in insects inoculated by microinjection. Previously, we found that phosphoinositol-4, 5-bisphosphate-3-kinase (PI3K) δ transcript was less abundant in virus-infected P. maidis. Co-injection experiments with MMV and dsRNA of PI3K δ were conducted and we found that virus titers were higher in the insects in which PI3K δ transcript abundance was reduced by RNAi. These findings demonstrate that injection of virus is a robust method for obtaining high MMV infection efficiency of the planthopper vector, and in combination with RNAi, will enable functional analysis of insect genes that may recognize or respond to MMV.