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Journal of Virology, October 2003, p. 11016-11026, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11016-11026.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Tomato Mosaic Virus Replication Protein Suppresses Virus-Targeted Posttranscriptional Gene Silencing

Kenji Kubota,1 Shinya Tsuda,2 Atsushi Tamai,1 and Tetsuo Meshi1,3,4*

Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502,1 Department of Plant Pathology, National Agricultural Research Center, Tsukuba 305-8666,2 Plant Physiology Department, National Institute of Agrobiological Sciences, Tsukuba 305-8602,3 CREST, Japan Science Technologies, Kawaguchi 332-0012, Japan4

Received 3 April 2003/ Accepted 15 July 2003

Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L11 and L11A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L11A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed.

* Corresponding author. Mailing address: Plant Physiology Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Japan. Phone: 81-29-838-8361. Fax: 81-29-838-8347. E-mail: tmeshi{at}nias.affrc.go.jp.

Journal of Virology, October 2003, p. 11016-11026, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11016-11026.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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