Poster abstracts

Poster number 94 submitted by Cody Buchmann

Suppression of transcriptional gene silencing by geminivirus AL2 and L2 proteins

R. Cody Buchmann (Dept. of Molecular Genetics, Ohio State Univ.), Shaheen Asad (National Inst. for Biotechnology and Genetic Engineering, Faisalabad, Pakistan), Gireesha Mohannath (Dept. of Molecular Genetics, Ohio State Univ.)

Abstract:
Geminiviruses replicate ssDNA genomes through dsDNA intermediates that associate with histone proteins to form minichromosomes. Like most viruses, geminiviruses are targets of RNA silencing (post-transcriptional gene silencing; PTGS). In addition, we have learned that siRNA-directed methylation of geminivirus chromatin, including inhibitory events such as cytosine methylation and histone methylation (e.g. H3K9), also acts as key host defense against geminiviruses. Such chromatin methylation typically leads to transcriptional gene silencing (TGS). In earlier work, we found that AL2 from Tomato golden mosaic virus (TGMV), and the related L2 protein of Beet curly top virus (BCTV), can inactivate host adenosine kinase (ADK). ADK activity is needed to sustain the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus we hypothesized that AL2 and L2 can suppress TGS by inhibiting methylation. To test this, N. benthamiana plants containing a transcriptionally-silenced GFP transgene were inoculated with PVX vectors expressing AL2 or L2. We found that TGS was reversed by both proteins. TGS could also be reversed by VIGS-induced knock-down of endogenous ADK expression. In another approach, transgenic Arabidopsis lines containing AL2 or L2 driven by a dexamethasone (dex)-inducible promoter were constructed. Using RT-PCR, we observed that dex-induced AL2 or L2 expression leads to the expression of endogenous transposons that are transcriptionally silenced by methylation. Bisulfite sequencing confirmed that AL2-mediated TGS reversal at an AtSN1 locus (SINE type retrotransposon) correlates with a significant reduction in cytosine methylation. In addition, we measured incorporation of labeled dNTP by Taq polymerase following digestion of genomic DNA with MspI (C'CGG; sensitive to CNG methylation). We observed that dex-induced AL2 expression results in increased (1.5 to 2-fold) incorporation, and thus concluded that AL2 causes a genome-wide decrease in methylation. These data provide strong support for the idea that methylation (and TGS) acts as a defense against geminiviruses, which is countered by AL2 and L2.

Keywords: Transcriptional gene silencing, geminivirus, dna methylation