|Miller, W. Allen|
Submitted to: Entomological Society of America Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 10/19/2012
Publication Date: N/A
Citation: N/A Interpretive Summary:
Technical Abstract: Pierce’s disease is a common and devastating disease of grapevines caused by the bacterium Xylella fastidiosa. The bacterium is often transmitted to plants by the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Hemiptera: Cicadellidae) which has become a high profile pest insect targeted for control efforts. Novel strategies are under investigation for modifying or enhancing the insecticidal activity of a naturally occurring dicistrovirus (Homalodisca coagulata virus-1 or HoCV-1) infective to GWSS. In vitro transcribed RNA from three plasmid clones carrying wild-type (pT7-HoCV1-3’Rz, pT7-Rz-HoCV1-3’Rz) or mutated (pT7-Rz-mutHoCV1-3’Rz) HoCV-1 viral sequences was transfected into GWSS-Z15 cells derived from H. vitripennis. Active viral RNA replication was not apparent for Rz-HoCV1-3’Rz or Rz-mutHoCV1-3’Rz RNA transfections. RNA generated from the pT7-HoCV1-3’Rz construct caused cytopathic effects (CPE) and was fully and persistently infective in GWSS-Z15 cells. Transcripts generated with or without a 5’ CAP on the sequence were fully infectious for the pT7-HoCV1-3’Rz construct with an up to 200 fold increase in positive and negative sense HoCV-1 viral RNA transcript quantities detected by day 2 post-transfection. Relative levels of negative sense RNA were approximately 10 fold lower than positive sense RNA at their peak on day 2 post-transfection. Supernatant from infected cells (day 2 post-transfection) was overlayed onto new GWSS-Z15 cells and both positive and negative sense viral RNA strands were detected suggesting that naturally infective and stable particles were generated following transfection of the HoCV1-3’Rz RNA transcript. We are currently examining HoCV1-3’Rz infected GWSS cells by electron microscopy and investigating the infectivity of clone-generated HoCV-1 particles in H. vitripennis insects. Future experiments will target recombinant HoCV-1 particles for enhanced infectivity and lethality in the leafhopper host.