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Journal of Virology, October 2009, p. 10472-10479, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.00665-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences{triangledown} ,{dagger}

Pavan Kumar,1,{ddagger} Sandra Uratsu,2 Abhaya Dandekar,2 and Bryce W. Falk3*

Plant Biology Graduate Group, University of California, Davis, California,1 Department of Plant Sciences, University of California, Davis, California,2 Department of Plant Pathology, University of California, Davis, California3

Received 31 March 2009/ Accepted 24 July 2009

Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3' of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3' 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3' portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.

* Corresponding author. Mailing address: 212 Hutchison Hall, Department of Plant Pathology, University of California, Davis, Davis, CA 95616. Phone and fax: (530) 752-0302. E-mail: bwfalk{at}ucdavis.edu

{triangledown} Published ahead of print on 29 July 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{ddagger} Present address: Somagenics Inc., 2161 Delaware Avenue, Santa Cruz, CA.

Journal of Virology, October 2009, p. 10472-10479, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.00665-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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