Analysis of Silencing Escape of Tomato leaf curl virus: An Evaluation of the Role of DNA Methylation

Abstract

RNA silencing is a sequence-specific mechanism regulating gene expression and has been used successfully for antiviral defense against RNA viruses. Similar strategies to develop resistance against DNA containing Tomato leaf curl virus (TLCV) and some other geminiviruses have been unsuccessful. To analyze this silencing escape, we transformed tomato plants with a hairpin construct from the TLCV C2 open reading frame (ORF). The transgenic plants showed a strong RNA silencing response, and following TLCV inoculation, their infection was delayed. However, the viral infection was not prevented and TLCV DNA accumulated to the levels found in nontransgenic plants. To determine the fate of a transgene carrying homology to the virus, we used transgenic plants carrying the TLCV C4 gene, which induces a distinct phenotype. Upon TLCV infection, the phenotype was abolished and C4 transcript disappeared. Concurrently, TLCV-specific small interfering RNAs were produced. In situ hybridization showed abundant levels of TLCV DNA in phloem cells of TLCV-infected C4 transgenic plants. However, the C4 transcripts were no longer detectable in nonvascular cells. Analysis of the transgene by methylation sequencing revealed a high level of de novo methylation of asymmetric cytosines in both the C4 ORF and its 35S promoter. A high level of methylation also was found at both symmetric and asymmetric cytosines of the complementary-sense strand of TLCV double-stranded DNA. Given the previous finding that methylated geminiviral DNA is not competent for replication, we provide a model whereby TLCV evades host defense through a population of de novo synthesized unmethylated DNA.

BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis

“…The integrated adenovirus type 12 (Ad12) genome was shown to be de novo methylated in hamster cells, and early Ad12 genes were only transcribed to a limited level, while late Ad12 genes were almost completely silenced (Doerfler, 2008). Upon infection by the Tomato leaf curl virus (TLCV), the TLCV C4 transgenic plants lost the phenotype conferred by overexpression of the transgene and the C4 transcript disppeared, and this disappearance was associated with the hypermethylation of both the 35S-C4 and C4 loci in intact TLCV (Bian et al, 2006). Therefore, it is reasonable that BSCTV replication decreased and that pathogenesis was attenuated after C2 was depleted and its ability to counteract the host methylation-related defense was abolished.…”

“…Viral mRNAs can be transcribed bidirectionally from the common region that is conserved in geminivirus genomes, and the resulting two divergent transcripts overlapping in the 39 termini likely form dsRNAs, which can trigger plant host RNA silencing-mediated defense mechanisms (Voinnet, 2005). Therefore, both the dsDNA intermediates and their transcripts can become the targets of plant gene silencing-mediated antiviral defense (Voinnet et al, 1999;Seemanpillai et al, 2003;Bian et al, 2006;Blevins et al, 2006). However, geminiviruses also encode proteins to counteract these defense mechanisms, such as C2/L2/AC2/AL2 proteins, a series of complementary positional homologs that have been reported to function during pathogenesis (Voinnet et al, 1999;van et al, 2002;Trinks et al, 2005;Wang et al, 2005;Kon et al, 2007).…”

Conformation-Selective Methylation of Geminivirus DNA

“…In combination with random hexamer primers, circular DNA can be amplified preferentially from plant samples without any prior knowledge of the sequence. Therefore, it was intriguing to test this technique for the identification of C modifications by restriction analysis and bisulfite sequencing.Geminiviruses induce small interfering RNAs (siRNAs) (2,7,62,73), which may lead to posttranscriptional (PTGS) as well as transcriptional (TGS) gene silencing (4,65). This interplay of PTGS and TGS has been understood as part of the plant defense pathways (reviewed in references 16, 64, 75, and 76).…”

“…Geminiviruses induce small interfering RNAs (siRNAs) (2,7,62,73), which may lead to posttranscriptional (PTGS) as well as transcriptional (TGS) gene silencing (4,65). This interplay of PTGS and TGS has been understood as part of the plant defense pathways (reviewed in references 16, 64, 75, and 76).…”

“…Moreover, both silencing routes may be used to knock down host genes with the help of geminivirus vectors (virus-induced gene silencing [VIGS]) (12,13,20,43,45,51). In this context, the analysis of the methylation status of geminivirus episomal or integrated DNA has revealed important insights (4,11,28,60,62,65).…”