Bipartite Geminivirus Movement Protein Function

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The goal of the project is to elucidate how plant virus-encoded movement proteins (MPs) act to coordinate replication of the viral genome with its directional movement through and between plant cells, and to explore the role of MPs in conferring tissue restriction to plant virus infection. Understanding virus-host interactions at this level is important for combating viral diseases, for only from such knowledge can rational anti-viral strategies be developed. Genetic studies show that MPs determine the host range and pathogenic properties of plant viruses. Thus, understanding their functions is essential for developing strategies to engineer plants that will be resistant to a broad range of geminiviruses.The bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) encode two MPs, NSP and MPB. These act cooperatively to move the viral single strand DNA genome within and between cells. NSP is a nuclear shuttle protein that binds viral genomes at their site of replication in the nucleus, and moves these between the nucleus and the cytoplasm. MPB traps NSP-genome complexes in the cytoplasm and moves them along unique endoplasmic reticulum (ER)-derived tubules to and across the walls of developing phloem cells. By site-directed mutagenesis, functional domains have been identified within each MP. The interactions between NSP and MPB are regulated by an activity that is specific to plant cells, and mutational studies suggest that this regulation may involve phosphorylation of the MPs . Use of the green fluorescent protein (GFP) has further shown that SqLCV infection is phloem-limited. To understand the functions of NSP and MPB, the signaling pathways through which they act, and the role of MPB in the phloem restriction of virus infection, now requires biochemical and genetic studies. We also want to understand, and develop new in vivo and in vitro approaches to investigate their functions. The specific goals of the project are to investigate MPB-ER interactions and their roles in the phloem-restriction of virus infection, to investigate the function of AtNSI, an Arabidopsis protein of unknown function that interacts with NSP and to identify host cell proteins that interact with MPB. These approaches will take advantage of different features of cucurbits and Arabidopsis. CLCV expressing free GFP and CLCV-SqLCV recombinants will be studied to determine if CLCV infection is phloem-restricted in Arabidopsis and the role of MPB in the ability to infect Arabidopsis. Fusions of MPB with GFP expressed from tissue-specific promoters in transgenic Arabidopsis wll be used to determine the role of MPB in the phloem limitation to virus infection and the effects of phloem development on virus movement. Determining the subcellular location and tissue-specific and temporal expression patterns of AtNSI will provide information on its function. Direct binding assays with NSP in vitro and a yeast reverse two-hybrid screen will be used to to identify NSP mutants with defects in AtNSI interaction; these will be screened by a PCR-based technique. Identifying host proteins that interact with MPB will define its function and how it targets to ER, and will be accomplished by using the yeast SOS-recruitment system, interaction cloning, and co-purification with MPB.