In vivo analysis of the Tomato Golden Mosaic virus AL 1629 promoter
Tomato Golden Mosaic virus (TGMV) is a member of the Geminiviridae family of single stranded (ss) DNA plant viruses. The focus of my research is on AL2, which activates coat protein expression, and AL3, which enhances replication. Both genes are expressed from a transcript initiating at nucleotide 1629, which is regulated by the AL1629 promoter. Using promoter::beta-D-glucuronidase (GUS) reporter gene fusions in transient leaf assays, specific sequences necessary for promoter activity were identified, and a host factor, Ethylene Response Factor 13 (ERF13), appears to bind the AL1629 promoter. ERFs are transcription factors that regulate gene expression in response to external signals, including viral infection. Further analysis of the AL1629 promoter in transgenic Nicotiana benthamiana plants containing AL1629 promoter::GUS fusions, demonstrated that the AL1629 promoter is inactive in the context of the plant chromosome. This is in contrast to transient leaf infusion assays where the promoter is active. Possible reasons for this difference are: 1) induction of ERF is necessary, as this factor appears to bind the promoter; 2) additional viral sequences are necessary in a chromatin context; 3) the promoter is silenced in a chromatin context; or a combination of these. My analysis has revealed that, when introduced into the chromosome, the promoter is in fact silenced, and that silencing correlates with cytosine methylation of promoter sequences. My analysis also demonstrated that addition of viral sequences upstream of the promoter reverses this effect. The implications for this finding could be that the virus regulates AL1629 promoter activity through methylation.