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

• Published in: Molecular plant-microbe interactions Vol. 19; no. 6; pp. 614 - 624
• Main Authors: Bian, X.Y, Rasheed, M.S, Seemanpillai, M.J, Rezaian, M.A
• Format: Journal Article
• Language: English
• Published: St Paul, MN APS Press 01.06.2006; The American Phytopathological Society
• Subjects: Base Sequence; begomoviruses; Biological and medical sciences; disease course; disease resistance; disease severity; DNA Methylation; Fundamental and applied biological sciences. Psychology; gene expression; gene expression regulation; gene silencing; Genes, Viral; Immunity, Innate - genetics; Lycopersicon esculentum; messenger RNA; Molecular Sequence Data; Nicotiana - anatomy & histology; Nicotiana - genetics; Nicotiana - microbiology; Nicotiana tabacum; nucleic acid hybridization; nucleotide sequences; phenotype; phloem; Phytopathology. Animal pests. Plant and forest protection; plant viruses; Plant Viruses - genetics; Plant Viruses - pathogenicity; Plant viruses and viroids; Plants, Genetically Modified - cytology; Plants, Genetically Modified - microbiology; RNA; RNA Interference; RNA silencing; sequence alignment; sequence analysis; Solanum lycopersicum - genetics; Solanum lycopersicum - microbiology; tobacco; Tomato leaf curl virus; Transgenes - physiology; transgenic plants; transgenic resistance; Virus Replication; Plant pathogen; Tomato leaf curl virus; Homology; Begomovirus; Mechanism; transgenic resistance; Messenger RNA; Gene; Dicotyledones; Angiospermae; Lycopersicon esculentum; begomoviruses; Antiviral; Geminiviridae; Transgenic plant; Solanaceae; Nucleotide sequence; gene silencing; Transposable element; Gene expression; Open reading frame; IS element; Virus; Resistance; Vegetable crop; Spermatophyta; Methylation
• ISSN: 0894-0282; 1943-7706
• DOI: 10.1094/MPMI-19-0614

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.