2007 Annual Report 1a.Objectives (from AD-416) Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non-structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 1b.Approach (from AD-416) Experiments will be conducted using a cloned cDNA copy of the wheat streak mosaic virus (WSMV) genome from which infectious viral RNA is generated in vitro and tested for biological activity in wheat and other cereal species. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the abilty to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done Yeast two hybrid methodology will be used to determine if Potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC-Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant-negative interference effects on WSMV infection. 4.Accomplishments Wheat streak mosaic virus P1 protein is a suppressor of plant RNA gene silencing. Plants have developed basal defenses against viral attack by degrading viral RNA, termed RNA silencing. Aphid transmitted relatives of wheat streak mosaic virus use their HC-Pro protein to inhibit host RNA silencing. However, the HC-Pro gene of wheat streak mosaic virus can be entirely deleted with little effect on virus multiplication. Each of the wheat streak mosaic virus encoded proteins was tested for suppression of RNA silencing revealing that the P1 protein of wheat streak mosaic virus fulfills this role. This research provides new information on molecular basis of pathogenicity of wheat streak mosaic virus. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors). 5.Significant Activities that Support Special Target Populations None.