Postdoctoral Associate (bradelb@ucs.orst.edu)

Current research Minus-strand detection - So far no precise analysis has been done on TYMV minus-strand RNA accumulation in protoplasts. The presence of varying amounts of plus-strand RNA, which is known for several viruses to outnumber the concentration of minus-strand from 10:1 up to 1000:1 at later stages in infection, can give difficulties in the exact determination of minus-strand present. Therefore we apply a modified two-cycle RNase protection method by which in an additional cycle plus- and minus- strands were hybridized and excess amounts of plus-strands were digested by RNase A/T1. Current results - Valylation of the 3’- tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) is known to be important for its efficient replication. Mutations in the anticodon loop of the TLS lead to significant reduction or even loss of replication. These effects correlate with loss of valylation and eEF1A binding to the viral RNA. The stages at which the repression of minus- and/or plus-strand synthesis occur are not yet known. Therefore, Chinese cabbage protoplasts were inoculated with transcripts derived from a wild type (wt) TYMV cDNA clone or from clones containing mutations in their 3’ –TLS and assayed for minus-strand RNA production by two-cycle RNase A/T1 protection assay.  A time course for the infection cycle of wt transcript showed that plus-strand RNA accumulation was characterized by a steady increase beyond 48 h p. i., whereas the accumulation of minus-strand RNA resulted in a plateau reached after about 30 h p. i..  Nucleotide 56 in the anticodon loop of the TLS is the most important nucleotide conferring valine identity. Interestingly, minus-strand RNA of the non-infectious mutant TYMC-G56 (wild type = A56), accumulates in protoplasts to almost wt levels. However, after reaching a peak in minus-strand accumulation 9 h p. i., degradation of minus-strand occurs. Plus-strand RNA accumulation reaches a peak at 18 h p. i. that is 25 times below the wild type accumulation at this time, before being completely degraded. The resulting loss of minus-strand could be attributed to destruction of its plus-strand template. These results support our hypothesis that in the case of insufficient valylation and consequent lack of eEF1A-binding to plus-strand RNA, minus-strand synthesis is allowed to occur unimpeded, interfering with the progression to plus-strand synthesis. Together with the evaluation of other mutants, possible mechanisms of TYMV replication are discussed.

These results support our hypothesis that in the case of insufficient valylation and consequent lack of eEF1A-binding to plus-strand RNA, minus-strand synthesis is allowed to occur unimpeded, interfering with the progression to plus-strand synthesis. Together with the evaluation of other mutants, possible mechanisms of TYMV replication are discussed.

Publications Bradel, B. G., Preil, W. and Jeske, H. 2000.  Sequence analysis and genome organisation of Poinsettia Mosaic Virus (PnMV) reveal closer relationship to marafiviruses than to tymoviruses. Virology 271: 289-297. Bradel, B. G., Preil, W. and Jeske, H. 2000.  Remission of the free-branching pattern of Euphorbia pulcherrima by tetracycline treatment. J. Phytopathology 148: 587-591. Bradel, B. G. 2000. Komplexe Infektionen im Weihnachtsstern (Euphorbia pulcherrima). ISBN 3-86186-303-0. Ph.D. thesis.

Education

	B.S.   1996.   Biological Sciences, Dept. of Molecular Genetics, University of Heidelberg. title of thesis (Diplomarbeit): Charakterisierung des M-Segments von Tahyna-Virus. Characterization of the M-Segment of Tahyna-Virus, a Bunyavirus.
Universität Stuttgart	Ph.D.  2000.  Dept. of Molecular Biology and Virology of the plant, University of Stuttgart (Germany). title of Ph.D. thesis: Complex infections in Poinsettia (Euphorbia pulcherrima) - Komplexe Infektionen im Weihnachtsstern.
	Post-doctoral Associate  5/2000-present. Department of Microbiology at Oregon State University.

 
 
 

updated by B. Bradel (3/6/01)