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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION OF HOST-PATHOGEN INTERACTIONS IN BARLEY AND WHEAT Title: Dispensability of the major coat protein of oat blue dwarf virus in genome replication: Substitution of the open reading frame with the enhanced green fluorescent protein gene

Authors
item Edwards, Michael
item Weiland, John

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 3, 2011
Publication Date: N/A

Technical Abstract: Oat blue dwarf virus (OBDV) is a representative marafivirus that infects monocots and a limited number of dicot species and is vectored propagatively by the leafhopper Macrosteles fascifrons.Recently, we reported the generation of clone pOBDV-2r, the first clone of a marafivirus from which infectious RNA can be transcribed. As a preliminary investigation into the production of the two coat proteins thought to be present in OBDV virions, a substitution and several deletion mutations were made in the putative major coat protein open reading frame(ORF)initiating at AUG5712 in the pOBDV-2r clone. Capped, polyadenylated transcripts of pOBDV-2r and these mutants were inoculated to plant protoplasts and assayed for replication at 36 h post-inoculation. OBDV accumulation was readily detected in inoculations with transcripts from pOBDV-2r, demonstrating for the first time the infection of plant protoplasts by a marafivirus. Removal of the major CP ORF (nt 5709 – 6331) resulted in a mutant competent for replication, although accumulating to lower levels relative to the wild type 2r control as detected by Northern blots. In mutant pOBDV-GFP1, the enhanced green fluorescent protein gene replaced the major coat protein gene. Visual inspection by UV-fluorescence microscopy indicated that greater than 50% of the mesophyll protoplasts inoculated with this recombinant were infected. Infection of protoplasts was confirmed by Western blot analysis using anti-GFP polyclonal antiserum and by Northern blot analysis using a probe generated to 500 nt of the 3’-end of the OBDV genome. The combined results indicate that plant protoplasts are suited to the study of marafivirus replication, that the major coat protein is dispensable for OBDV replication, and that the reported phloem-limited nature of OBDV is likely due to a block in virus movement into, rather than replication within, leaf mesophyll cells.

Last Modified: 12/20/2014
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