Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:gradschool_diss-1440 |
| Date | 01 January 2005 |
| Creators | Zhang, Chunquan |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of Kentucky Doctoral Dissertations |
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