Inoculation of Maize with Sugarcane Mosaic Virus Constructs and Application for RNA Interference in Fall Armyworms

Abstract

Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (Zea mays), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm (Spodoptera frugiperda) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants. Graphical overview