Analysis of silencing escape of tomato leaf curl virus: an evaluation of the role of DNA methylation.

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.

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs.

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA beta satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.