2008 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. 3.Progress Report Experiments were designed identify and characterize wheat streak mosaic virus (WSMV) determinant(s) of pathogenicity enhancement and suppression of the host defense RNA silencing pathway; identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite; and develop and evaluate transgenic wheat expressing WSMV non-structural proteins. Twelve deletion mutants of the P1 protein of WSMV were constructed in expression vector plasmids and evaluated in a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. This revealed that the first 17 amino acid residues of P1 are dispensable for suppression of host plant RNA silencing. Several of these deletion mutants have been cloned into a full length infectious clone of WSMV and are being evaluated for effects on virus viability and symptom development. The complete genome sequence was determined for the Nebraska maize chlorotic mottle virus isolate used in disease synergism assays with WSMV. A GUS reporter gene was fused to the amino-terminus of HC-Pro in an infectious clone of WSMV, inoculated to wheat, and exposed to wheat curl mites. Histochemical staining of recovered mites revealed the presence of GUS-HC-Pro fusion protein in mite mouth parts. A study underway will examine whether mites retain the HC-Pro fusion protein following a molt. The P3 and NIa genes of WSMV have been cloned in binary vectors for wheat transformation. Clones of His-tagged HC-Pro and His-tagged P1 have been constructed to aid the study of potential protein-protein interactions and antisera is being produced for nonstructural proteins of WSMV using synthetic peptides. An infectious clone of the type strain of WSMV is being constructed for experiments designed to determine WSMV host range determinants. A new wheat virus, Triticum mosaic virus, was identified in the spring and fall of 2008. A portion of the viral genome has been cloned using PCR and potyvirid-specific primes, and will be sequenced shortly. A span of 4 kbp of the mitochondrial DNA from the wheat curl mite has been sequenced. This segment contains, in order the 12S and 16S ribosomal RNA genes, the gene subunit two of NADH dehydrogenase, the Met tRNA gene, and the genes for cytochrome oxidase subunits 1 and 2. This gene order is unique among mitochondrial DNA of metazoans yet reported. This research is a component of National Program 303 Component 2. Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors. 4.Accomplishments 1. The HC-Pro protein of wheat streak mosaic virus is not required for disease synergism in mixed infection with an unrelated virus. Mixed infections of crop plants with two unrelated viruses may result in disease symptoms far more severe than that produced by the respective viruses in single infections. This phenomenon is referred to as disease synergism. Often, one of the viruses involved is a member of the potyvirus family. Previously, others have shown that the HC-Pro gene of potyviruses is responsible for disease synergism and that pathogenicity enhancement by HC-Pro occurs via suppression of RNA silencing pathways used by the plants as a defense against viral infection. In the experiments reported here, it was demonstrated that a deletion mutant of wheat streak mosaic virus completely lacking HC-Pro was able to produce disease synergism in corn dually infected with maize chlorotic mottle virus. Additional experiments indicated that wheat streak mosaic virus HC-Pro did not function as a suppressor of RNA silencing. Collectively, the experiments indicate that HC-Pro function differs among members of the potyvirus family and that wheat streak mosaic virus likely utilizes a different gene to induce synergistic disease and suppress RNA silencing. This research provides new information on molecular basis of pathogenicity of wheat streak mosaic virus. This accomplishment contributes to ARS Strategic Plan Goal 4 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply), ARS National Program 303 Component 2. Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2B: Plant-Microbe-Vector Interactions. 2. Specific amino acid positions in the HC-Pro protein of wheat streak mosaic virus are critical for mite transmission of the virus. Mutations introduced into the HC-Pro coding region of wheat streak mosaic virus were evaluated for systemic infectivity to wheat and vector transmission by the wheat curl mite. Ten wheat streak mosaic virus mutants bearing point mutations in HC-Pro retained infectivity and wild-type pathogenicity when inoculated to wheat. Cysteine to alanine substitution at three positions within a conserved zinc-finger motif abolished transmission by the wheat curl mite. Mutation at other positions had no effect on vector transmission or resulted in reduced vector transmission efficiency. These results provide evidence that vector transmission of wheat streak mosaic virus requires a functional zinc-finger in the amino-terminal region of HC-Pro. This research provides new information on molecular basis of mite transmission of wheat streak mosaic virus. This accomplishment contributes to ARS Strategic Plan Goal 4 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply), ARS National Program 303 Component 2. Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2B: Plant-Microbe-Vector Interactions. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Review Publications Young, B.A., Hein, G.L., French, R.C., Stenger, D.C. 2007. Substitution of conserved cysteine residues in Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite. Archives of Virology 152:2107-2111. Stenger, D.C., Young, B.A., Qu, F., Morris, T., French, R.C. 2007. Wheat streak mosaic virus lacking HC-Pro is competent to produce disease synergism in double infections with maize chlorotic mottle virus. Phytopathology 97:1213-1221. Stenger, D.C., French, R.C. 2008. Complete nucleotide sequence of a maize chlorotic mottle virus isolate from Nebraska. Archives of Virology 153: 995-997. Folimonova, S.Y., Folimonov, A.S., Tatineni, S., Dawson, W.O. 2008. Citrus tristeza virus: survival at the edge of the movement continuum. Journal of Virology, Volume 82, page 6546-6556.