Investigation of aspects of Impatiens necrotic spot virus associated with a new disease of lettuce in California and factors involved in long-distance movement of Cucurbit leaf crumple virus in pumpkin plants

In the 2006 and 2007, outbreaks of a viral disease of lettuce ( Lactuca sativa) in Monterey County California characterized by stunted and distorted plant growth and necrotic spots and yellowing of leaves was suspected to be caused by Tomato spotted wilt virus (TSWV). However, tests performed on representative symptomatic leaves indicated that the disease was caused by the related tospovirus Impatiens necrotic spot virus (INSV). Surveys conducted in 2008 and 2009 revealed that this new disease was widespread and reached incidences as high as 27%. The disease affected all types of lettuce and the highest incidences were later in the growing season. Molecular characterization of the INSV isolates from lettuce revealed that these were nearly identical to isolates from other hosts in the Salinas Valley and other geographic locations. Disease outbreaks were associated with moderate to high thrips populations and the predominant species was the western flower thrips (Frankliniella occidentalis). An RT-PCR assay was developed that allowed for detection of INSV in thrips. Surveys for reservoir hosts revealed crop plants (e.g., radicchio and faba beans) as well as certain weeds, especially cheeseweed and shepherd's purse, are potential reservoirs of INSV. A functional analysis of the INSV NSs and NSm genes, was performed using Agrobacterium-mediated transient expression. The NSs was localized to the cytoplasm and nuclear and cell peripheries, and this protein was an effective suppressor of gene silencing. The NSm protein localized to the cell periphery and more specifically to punctate bodies in the cell wall, which were identified as plasmodesmata based on co-localization with the Tobacco mosaic virus movement protein (MP). Thus, the INSV NSs and NSm proteins function as a silencing suppressor and movement protein of the virus, respectively. The long-distance movement of the bipartite begomovirus Cucurbit leaf crumple virus (CuLCrV) was then investigated. Mutational analysis of the CuLCrV capsid protein (CP) revealed that the CP was not required for viral replication, but that the intact CP was required for long-distance movement in pumpkin plants. These results indicated that the CP is required for systemic infection of CuLCrV, perhaps indicating that virions are the form in which the virus moves long distance. To identify host factors involved in CuLCrV long-distance movement, we expressed and purified a GST-CP fusion protein and used it as a probe in protein overlays with proteins from pumpkin phloem sap. Six candidate proteins were identified that interacted with the CuLCrV CP.