|Redinbaugh, Margaret - Peg|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/13/2001
Publication Date: N/A
Citation: Interpretive Summary: Several years ago, ARS scientists developed a technique, called vascular puncture inoculation (VPI), which allowed us to transmit corn-infecting viruses without using insects. Researchers have benefitted from using the technology for studying virus and virus resistance in corn. It is critical for allowing us to rapidly isolate and characterize new and emerging viruses that infect corn, before the viruses become agronomic problems. VPI involves using a jeweler's engraving tool to push very fine pins through a virus-containing drop into the scutellum of germinating kernels. If the vibrations of the engraving tool make holes in the cell's membranes that allow the virus to enter, then we hypothesized that other molecules, especially RNA and DNA, could enter cells through the same holes. To test the hypothesis, we used VPI to inoculate corn kernels with virus-encoding RNAs and DNAs. We found that both the RNA and DNA were infectious, that is, they produced typical virus symptoms and intact viruses in the inoculated corn seedlings. We also found that the transmission of viral RNA and DNA was not as efficient as the transmission of intact virions, as expected. VIP was more than 100 times more efficient for transmitting viral RNA to corn than leaf rub-inoculation. The development of this efficient technology for transmission of viral nucleic acids will help industry, academic and ARS scientists doing research to identify and characterize virus-resistant maize germplasm, those doing research to investigate molecular aspects of virus biology, as well as those interested in developing virus-based gene expression vectors for corn.
Technical Abstract: Vascular puncture inoculation (VPI) is an effective technique for transmission of maize viruses without using arthropod vectors. The technique involves using a jeweler's engraving tool to push minuten pins through a droplet of virus inoculum and into the scutellum of germinating kernels. Because viruses may enter embryo cells through openings made in cellular membranes by the vibrational wave energy generated from the engraving tool, VPI may be useful for introducing viral RNA and DNA into maize embryos. Maize dwarf mosaic virus (MDMV) virions, MDMV genomic RNA, Foxtail mosaic virus (FoMV) genomic RNA and Maize streak virus (MSV) cDNAs were introduced into kernels by VPI and infection rates determined. Although at high concentrations both produced 100% infection of susceptible maize, the MDMV genomic RNA was transmitted about 100-fold less efficiently than the virion. The FoMV genomic RNA and MSV cDNA were transmitted with less efficiency and the highest transmission rates were about 50%. RNAse (0.1 mg/ml) pretreatment prevented genomic RNA transmission, but not virion transmission. Protease K (2 mg/ml) treatment reduced transmission of both virions and RNA. The data indicated that VPI could be used to transmit viral nucleic acids to germinating maize embryos.