Detecting, Cloning, and Screening for Suppressors of RNA Silencing in \u3ci\u3eMaize Chlorotic Mottle Virus\u3c/i\u3e and \u3ci\u3eSugarcane Mosaic Virus\u3c/i\u3e

Maize lethal necrosis disease (MLND) is one of the most important viral diseases of maize. MLND occurs when Maize chlorotic mottle virus (MCMV) co-infects the same plant with one of several potyviruses, including Sugarcane mosaic virus, Wheat streak mosaic virus or Maize dwarf mosaic virus. Originally prevalent in the Midwest and Peru in the 1970s, the disease was called corn lethal necrosis (CLN) and was controlled through breeding and sanitation. Recently, the disease has re-emerged in East Africa and is rapidly spreading and threatening the food sources of subsistence-farming populations. This re-emergence has raised several questions about the unknown molecular mechanisms of MLND. RNA silencing is a prominent antiviral defense system in plants that may be involved in viral synergism. In single and double infections, MCMV and SCMV activate maize antiviral RNA silencing machinery, resulting in the accumulation of virus-derived small RNAs. Most plant viruses encode proteins called viral suppressors of RNA silencing (VSRs) to inactivate RNA silencing and overcome the host defense system. VSRs have been identified in several potyviruses but no silencing suppressor has been identified in MCMV. In this project, protocols to detect both SCMV and MCMV in plant tissue were established and optimized. A clone of the MCMV Nebraska isolate (MCMV-NE) and each open reading frame (ORF) of MCMV and SCMV were constructed for Agorbacterium infiltration. To identify silencing suppressors in MCMV and SCMV, individual proteins were cloned into binary vectors for transient expression in Nicotiana benthamiana and candidate proteins with silencing suppression activity have been identified. Identification and characterization of VSRs in MCMV and SCMV establishes the foundation to further study the molecular mechanisms involved in MLND. Advisor: Hernan Garcia-Rui