|Molecular and functional characterization of the movement protein of Ourmia melon virus|
P. MARGARIA (1), C. T. Anderson (1), M. Turina (2), C. Rosa (1). (1) The Pennsylvania State University, University Park, PA, U.S.A.; (2) CNR, Torino, Italy
<i>Ourmia melon virus </i>(OuMV) has a genome composed of three +ssRNA molecules, each coding for a single protein. Our work focuses on the RNA2-encoded movement protein (MP). We selected 11 amino acids for alanine scanning mutagenesis, either conserved in the MP of ourmia-, tombus- and aureoviruses (all phylogenetically distantly related), or charged and solvent-exposed. We infected <i>Nicotiana benthamiana</i> via agroinfiltration with OuMV infectious clones carrying the desired mutations. Viral movement was impaired for five of the mutants: the MP (and CP) were detected by Western blot analysis at the site of agroinfiltration for all the mutants, whereas no MP (or CP) was present in systemic tissues of the same plants or in new plants inoculated with tissues from the agroinfiltated area, suggesting that these substitutions impaired both systemic and local movement. EGFP-wtMP fusion was expressed through agroinfiltration in puntate spots at the cell periphery of infected <i>N. benthamiana</i> and <i>Arabidopsis thaliana</i> Col 0 and leaf staining with propidium iodide and aniline blue showed that the wt MP co-localizes at the cell wall, with plasmodesmata. So far, three eGFP-mutant MPs did not localize in foci. Isolated protoplasts infected with eGFP-mutant MPs showed absence of the tubule protrusions from the plasma membrane, typical of cells infected with eGFP-wtMP. We are currently using Arabidopsis mutants to test the requirements of specific cellular components for viral movement.