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Subject: Plant Pathology × Author: Borah, Munmi × End date: 2018 ×
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    ThesisItemOpen Access
    Development of RNA based vaccine against Cucumber mosaic virus infecting Bhut Jolokia (Capsicum chinense Jacq.) crop and Citrus tristeza virus infecting citrus plantations of Assam
    (AAU, 2016) Borah, Munmi; Nath, P. D.
    “Development of RNA based vaccine against Cucumber mosaic virus infecting Bhut Jolokia (Capsicum chinense Jacq.) crop and Citrus tristeza virus infecting citrus plantations of Assam” were carried out at and Department of Plant Pathology, Assam Agricultural University, Assam, India and Laboratory of Plant Breeding and Biometry, Department of Crop Science, AUA, Athens, Greece. Utilizing virus genome properties enabled the design of novel, safe, and efficacious vaccines against different viral diseases infecting plants. In this study, it was shown that, dsRNA derived from viral sequences could interfere with cognate virus infection in a sequence-specific manner by delivering dsRNA to plant cells. In dsRNA-mediated protection, a dsRNA homolog of a viral silencing suppressor gene expressed in plants, which interferes with or prevents various stages of the viral life cycle, resulting in attenuated disease symptoms or resistance. It was aimed to produce CMV specific RNA vaccine to manage CMV infecting Bhut Jolokia crop of Assam. Application of these RNA based vaccines at the seedling stage could effectively reduce CMV infection at the later stage of the crop. These virus-free seedlings of Bhut Jolokia crop could give rise to a healthy crop growth. Taking it as a model system, it was further aimed to produce CTV specific RNA vaccine and to carry out a proof-of-concept to substantiate the same concept further in management of CTV infecting citrus plantations of North East India. A protocol for the synthesis of dsRNA using T7 RNA polymerase was utilized to produce RNA based vaccine against Cucumber mosaic virus infecting Bhut Jolokia (Capsicum chinense Jacq.) crop and Citrus tristeza virus infecting citrus plantations of Assam. CMV-encoded 2b gene based dsRNA was produced and tested against CMV infecting Bhut Jolokia (Capsicum chinense Jacq.) plants of Assam. The infection of CMV in Bhut Jolokia pepper plants was successfully interfered, demonstrating the applicability of RNA-based vaccination. In this study, double-stranded RNA derived from CMV-2b silencing suppressor gene sequence in Escherichia coli, could interfere with cognate virus infection. When dsRNA CMV-2b exogenously applied, along with CMV strain, onto Bhut Jolokia plants resulted in suppressing CMV infection. DAS-ELISA was used to identify the presence of CMV in the inoculated plants. Bhut Jolokia Pepper plants infected with CMV became severely stunted, nonproductive with dull light green foliage having a leathery appearance. In contrast, plants that received dsRNA of CMV-2b were less symptomatic and remained healthy as compared to those infected by CMV. Four experiments were conducted where; disease incidence was 15%, 5%, 29.5% and 0% when dsRNA of CMV-2b molecules were co-applied with CMV, as compared to 55%, 55%, 92% and 70% when only CMV was infected. As a result of dsRNA mediated resistance crop canopy increased, which is necessary for improved yields of the crop. This study constitutes a non transgenic approach of protection of Bhut Jolokia pepper plants against CMV. With the success of CMV specific RNA vaccine, the investigation further aimed towards production of a dsRNA construct coding for the three silencing suppressors of CTV to generate RNA-based resistance and to conduct a proof-of-concept of specific protection against viral infection. It was aimed to get more insight on the role of the CP, p20 and p23 genes as silencing suppressors of CTV in pathogenesis through topical application of these dsRNA molecules. The CP, p20 and p23 gene sequences of the North East India strain of Citrus tristeza virus was folded into a double-stranded (ds) RNA structure. dsRNA of sufficient quantities (several micrograms) obtained using in vitro transcription protocols for CP, p20 and p23 genes of the virus. The proof-concept experiment on application of these dsRNA against CTV infected citrus plants revealed that, while applied topically over leaf surface against the cognate virus, all three dsRNA constructs (CTV-CP, CTV-p23 and CTV-p20), could suppress the virus replication. This results successfully interpreted the proof -of -concept about the suppression of viral titre locally up to 10 days of topical application, through RNAi based method in citrus crop infected with CTV-North East India strain. The results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of post transcriptional gene silencing in natural virus infections, triggering the viral RNA for degradation. A dsRNA construct encoding silencing suppressors could be significantly suppressed the replication of viruses and confer potential resistance against the virus.