Functional analysis of the virion-sense encoded proteins of Beet curly top virus and studies of the virus-beet leafhopper vector (Circulifer tenellus) interaction

Beet curly top virus (BCTV; Genus Curtovirus , Family Geminiviridae) causes curly top disease in a wide host range of dicotyledonous plants and is transmitted by the beet leafhopper (Circulifer tenellus). A full-length infectious clone (BCTV-W4), associated with a recent curly top disease outbreak in peppers and tomatoes in New Mexico, U.S.A., was generated and shown to be an isolate of the Worland strain of BCTV. To investigate the role of BCTV-W4 capsid protein (CP) in viral pathogenesis and biology, a frameshift mutant and a series of alanine scanning mutants were generated. Most N-terminal CP mutants were infectious in Nicotiana benthamiana, formed virions and were leafhopper-transmissible. In contrast, the frameshift mutant and most C-terminal mutants were not infectious in N. benthamiana and did not form virions. Virion formation was positively correlated with infectivity; however, one N-terminal mutant was infectious and did not form virions, suggesting that BCTV can move long distance in plants in a non-virion form. Another N-terminal mutant was infectious and formed virions, but was not insect-transmissible, revealing a domain involved in virus movement through the insect.; The BCTV genome encodes three proteins on the virion-sense DNA: V1 (CP), V2 (ss-ds DNA regulator) and V3 (a putative movement protein). A functional analysis of these proteins was conducted by determining the subcellular localization and cell-to-cell movement properties of each protein fused to the green fluorescent protein (GFP). Transient expression experiments performed in tobacco protoplasts and epidermal cells of N. benthamiana leaves and bean hypocotyls revealed that CP-GFP localized to the nucleus and formed ring-like structures, whereas V2- and V3-GFP localized to the endoplasmic reticulum (ER). In many cells expressing V3-GFP vesicle-like structures were observed around the nucleus, in the cytoplasm and at the cell periphery. V2- and V3-GFP, but not CP-GFP, showed a limited capacity for cell-to-cell movement in epidermal cells.; Finally, to further investigate the BCTV-vector interaction, a PCR-based method for detection of BCTV in viruliferous leafhoppers was developed. This method was used to determine the spatial and temporal distribution of BCTV in leafhoppers. Results obtained from these studies were consistent with a persistent but non-propagative mode of circulative transmission.