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Investigation of Histone Methylation of Tomato Curly Stunt Virus (TOCSV) in Resistant and Susceptible Tomato Lines
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Investigation of Histone Methylation of Tomato Curly Stunt Virus (TOCSV) in Resistant and Susceptible Tomato Lines
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Investigation of Histone Methylation of Tomato Curly Stunt Virus (TOCSV) in Resistant and Susceptible Tomato Lines
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Dissertation
Investigation of Histone Methylation of Tomato Curly Stunt Virus (TOCSV) in Resistant and Susceptible Tomato Lines
2022
Overview
Tomato Curly Stunt Virus (ToCSV) is one of the most catastrophic tomato diseases witnessed in South African fields since 1997. It is one of the utmost economically important begomoviruses reported in South Africa with infected crops portraying symptoms such as yellowing, leaf curling, stunting and fruit set reduction. The molecular mechanisms employed by the ToCSV-tomato pathosystem has not been fully elucidated yet, thus this study aims to contribute to the current body of knowledge using this pathosystem. In this study, an infectivity study of mock and ToCSV-inoculated resistant (R, NIL 396) and susceptible (S, NIL395) near isogenic tomato lines was investigated at 8-, 15-, and 35-dpi. Thereafter, to investigate if epigenetic defence takes place, Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (qPCR) was used to identify presence of activation mark H3K9Ac and H3K9me2 at 15- and 35-dpi. This was conducted as a pilot study since this is the first report of using ChIP to identify host induced histone modification as a TGS defence mechanism in ToCSV. The infectivity study portrayed results revealing that the S line accumulates larger viral load compared to the R line at all time points throughout viral progression with a fold change of 1.06 between 8 and 15 dpi, and a fold change of 2.43 between 15 and 35 dpi. In addition, mild symptoms were observed for ToCSV-inoculated R plants along with the viral load, tolerant like behaviour is observed in R plants rather than that of a resistant phenotype, thus the R line will be referred to as the tolerant (T) line from hereon. Bokhale et al., (2020) reported elevated levels of RNA dependent RNA polymerase 1 (RDR1) was observed in R plants as compared to S plants in addition to lower viral load when compared to the S line. Therefore, NIL396 line may be linked to tolerance, however, this hypothesis requires further characterization and investigation.ChIP is one of the most commonly used methods with high sensitivity that is used when detecting the presence of epigenetic modifications, especially histone post translational modifictions such as acetylation and methylation. Histone 3 lysine 9 modifications are common in geminivirus infected crops with H3K9Ac and H3K9me2 one of the most common marks identified during epigenetic defence using transcriptional gene silencing (TGS). Unlike TYLCV that has been extensively studied globally characterizing and understanding the various molecular processes governing epidemiology, ToCSV is a relatively new virus identified in South Africa with little research been completed using this pathosystem, therefore, this study was conducted as a pilot study aiming to improve research quality on the overall study. Hence, this study is aimed to identify the presence of epigenetic activation and repressive marks H3K9Ac and H3k9me2, respectively. This is the first time ChIP has been used in the ToCSV-tomato pathosystem to detect the presence of epigenetic defence ii at the TGS level. Post crosslinking, sonication, antibody assay and DNA purification, immunoprecipitated DNA was used to detect presence of ToCSV using conventional PCR. Gene expression of DNA and histone methyltransferases CMT2, MET1, KYP/SUVH4 and AGO4 were investigated using qPCR. It was observed that ToCSV-inoculated immunoprecipitated DNA associated with marks of activation and repression was present in both T and S lines at 15 and 35 dpi; however, the ChIP negative control, Normal Mouse IgG, produced a viral amplicon when compared to the mock ChIP negative IgG control. To determine if this observation was due to plant DNA, 18S rRNA was amplified in mock and ToCSV-inoculated T and S plants suggesting plant DNA co-precipitates along with viral DNA. This data highlights the presence of activation and repressive epigenetic marks of acetylation and methylation in the ToCSV-tomato pathosystem suggesting the role TGS has as a defence mechanism against ToCSV; however, the data remains inconclusive to report if epigenetic defence does occur during infection. In addition, it was observed that T plants displayed upregulation of all DNA and histone methyltransferase genes whereas S plants show downregulation except for AGO4. Hence, the gene expression data portrays methylation occurring in both T and S plants between 15 and 35 dpi, thereby substantiating the H3K9me2 repressive mark observed in ChIP. Thus, several aspects relating to the experiment should be considered such as the downstream application, choice of IgG used, and method of viral DNA isolation, as well as additional epigenetic methods such as DNA methylation used in conjunction with ChIP allowing further accurate results of ChIP related experiments.
