Transcriptional Silencing of Geminiviral Promoter-Driven Transgenes Following Homologous Virus Infection

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Abstract
Promoters isolated from the Tomato leaf curl virus (TLCV) drive both constitutive and tissue-specific expression in transgenic tobacco. Following systemic TLCV infection of plants stably expressing TLCV promoter:GUS transgenes, transgene expression driven by all six TLCV promoters was silenced. Silencing in the TLCV coat protein promoter:GUS plants (V2:GUSdeltaC) was characterized in more detail. Transgene silencing observed in leaf, stem, and pre-anthesis floral tissue occurred with the continued replication of TLCV in host tissues. Infection of the V2:GUSdeltaC plants with heterologous geminiviruses did not result in transgene silencing, indicating that silencing was specifically associated with TLCV infection. Nuclear run-on assays indicated that silencing was due to the abolition of transcription from the V2:GUSdeltaC transgene. Bisulfite sequencing showed that silencing was associated with cytosine hypermethylation of the TLCV-derived promoter sequences of the V2:GUSdeltaC transgene. Progeny derived from V2:GUSdeltaC plants silenced by TLCV infection were analyzed. Transgene expression was silenced in progeny seedlings but was partially reactivated in the majority of plants by 75 days postgermination. Progeny seedlings treated with the nonmethylatable cytosine analog 5-azacytidine or the histone deacetylase inhibitor sodium butyrate exhibited partial reactivation of expression. This is the first report of the hypermethylation of a virus-derived transgene associated with a DNA virus infection.