|Choi, Il-Ryong - FORMER USDA-ARS-5440-20-|
Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 15, 2001
Publication Date: July 21, 2001
Citation: Choi, I., Stenger, D.C., French, R.C. 2001. Wheat streak mosaic virus p3 gene silent mutations affect both replication and movement, revealing an internal rna sequence element. American Society For Virology Meeting. (Not published) Technical Abstract: High sequence conservation in the 3'-proximal half of the P3 gene among divergent wheat streak mosaic virus (WSMV) strains suggested the presence of an internal RNA sequence element. A series of mutants bearing multiple silent mutations in the P3 gene were constructed in an infectious cDNA clone of the WSMV Sidney 81 strain. All P3 silent mutants examined were unable to systemically infect wheat, but did not differ from wild type whe translated in vitro. Multiple silent mutations in the WSMV CI gene did not alter systemic infectivity or in vitro translation properties, whereas a frame shift mutation in the P3 gene abolished infectivity and resulted in altered translation in vitro. To assess replicative and local movement phenotypes, each P3 mutant was placed in a WSMV genome capable of expressing the GUS reporter gene. GUS activity measured in barley protoplasts 36 hours post-transfection indicated that each silent P3 mutant tretained the ability to replicate in plant cells, albeit at 22-80 percent of wild type levels. Minimal GUS activity was detected in protoplasts transfected with the replication-deficient frame shift mutant, even though the GUS reporter gene was inserted upstream of the frame shift mutation. GUS assays conducted 3 days post inoculation (d.p.i.) revealed that each P3 mutant bearing only silent substitutions was able to establish localized infection in inoculated wheat leaves, although infection foci remained limited to small clusters of cells and increased in size only slightly by 5 d.p.i. In contrast, infection foci produced by wild type WSMV expressing GUS were much larger at 3 d.p.i., and by 5 d.p.i. had coalesced to produce continuous zones of GUS expression in inoculated leaves. Collectively, these results confirm the presence of an internal RNA sequence element.