Publication Date

2021

Document Type

Dissertation/Thesis

First Advisor

Bujarski, Jozef

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Biological Sciences

Abstract

Brome mosaic virus (BMV), Cowpea chlorotic mottle virus (CCMV), and Broad bean mottle virus (BBMV), the members of the genus Bromovirus, are the experimental model viruses in this dissertation research. Packaging of the RNA genome by the viruses is known to be highly selective and specific for the viral RNA but, not 100% accurate. There is evidence that the viruses specifically or accidentally encapsidate viral RNA variants and host (cellular) RNAs. The scope of this dissertation is to identify different types of RNAs that can be packaged by viruses along with the viral RNAs of parental origin.BMV, CCMV, and BBMV were extensively purified by the gradient centrifugation; BMV and BBMV by Cesium chloride gradient ultracentrifugation, whereas CCMV was purified by the sucrose gradient ultracentrifugation. All the virion preparations were treated with nucleases followed by cleaning and concentration by Amicon microfilter tubes. So, virions were free of any occluded or co-precipitated nucleic acids (DNA/RNA). In this study, two types of methods were adopted to infect plants with viruses. First, infection by virus particles, and second by agroinfiltration; BMV R1 and BMV R6. BMV R6 is the BMV mutant with R10P substitution in the N-terminal Arginine Rich Motif (ARM) of the coat protein (CP). Data of next-generation RNA sequencing (NGS RNA-seq) of the RNAs purified from virions were assembled and mapped to the references from the respective viruses and hosts. The second part of the project is the mapping of the recombination events in RNAs purified from BMV and CCMV by using the Virus Recombination Mapper (ViReMA). Finally, BBMV was sequenced and assembled for five strains collected from Libya, Morocco, Syria, Sudan, and Tunisia. BMV particles mechanically inoculated into the barley leaves and Nicotiana benthamiana leaves packaged 0.07 and 0.1% of the host RNAs. Among the host RNAs being encapsidated, mRNAs were the most abundant followed by rRNAs of the nuclear origin. Organellar RNAs like plastid and mitochondrial RNAs were also packaged by the virions. Similarly, wild-type and mutant BMV, BMV R1, and BMV R6, respectively, expressed exogenously through the agroinfiltration systemically infected the Nicotiana plants. RNAs purified from virions were sequenced by the NGS RNA-seq followed by the assembly and mapping of the reads generated to the references. BMV R6 packaged 0.7% and BMV R1 packaged 0.3% of the viral genome with the host RNAs. So, CP mutation affected the number of host RNAs being encapsidated. In the second part, among the RNAs encapsidated by the BMV and CCMV that did not map to the viral RNA, 95.7% and 94.8% of reads were recombinant. Various types of recombinants like segmental cross-over, insertions, deletions, and substitutions were found by the ViReMA. Finally, BBMV genomes from five strains were assembled to the full-length and verified by the Sanger sequencing. The assembled genomes were comparatively analyzed for the nucleotide and translated protein sequence similarities. Despite, the symptomatic phenotypic variation observed among the five strains, they shared an identity of over 95% in the nucleotide sequences of all three RNA segments. The results show that Bromoviruses can encapsidate recombinant viral, as well as the host RNAs along with the parental viral RNAs. The viral CP variant like R10P mutants can contribute to this phenomenon, stressing the role of CP in encapsidation selectivity. Besides, the newly sequenced genome of five BBMV strains can be used for molecular studies of plant RNA viruses.

Extent

169 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

Download

Share

COinS