Graduation date: 1993

Some positive-strand RNA plant viruses possess a transfer RNA-like structure (TLS) at the 3'-terminus of their genomic RNAs. The closest mimicry to tRNA is exhibited by the valylatable TLSs from tymoviruses and furo-like viruses, which are able to interact with key cellular tRNA enzymes: [CTP, ATP]:tRNA nucleotidyltransferase (CCA NTase), valyl-tRNA synthetase (ValRS), and translation elongation factor 1A (eEF1A). In this thesis, I report the discovery of two new roles of the Turnip yellow mosaic tymovirus TLS, in translation enhancement (Chapter 2) and repression of minus strand initiation (Chapter 4). Placement of the 3'-terminal 109 nts of TYMV RNA in a luciferase reporter RNA with a generic 5'-UTR enhanced translation by about 20-fold in cowpea protoplasts. Exhibiting a synergistic relationship with the 5'-cap, the 3'-translation enhancement was largely dependent on the aminoacylatability of the TLS and apparently on eEF1A interaction. In the presence of the 5'-UTR from genomic TYMV RNA, translation of both the overlapping proteins p69 and p206 was strongly dependent on a 5'-cap structure, and was enhanced by the 3'-enhancer. These in vivo results contradict the proposed model in which translation initiation of p206, but not p69, is cap-independent and TLS-dependent (Barends et al. Cell 112(2003):123-9). In vitro experiments with a partially purified preparation of TYMV replicase have investigated the phenomenon of minus strand repression. Interaction of purified eEF1A���GTP specifically with the valylated TLS decreased the template activity for minus strand to near-background levels. eEF1A���GTP acts by making the 3'-CCA minus strand initiation site unavailable to the replicase. The influence of eEF1A in simultaneously enhancing translation and repressing minus strand synthesis can be considered a regulation that ensures robust translation early in the infection and that offers a coordinated transition from translation to replication. Previously shown to be critical for TYMV infectivity, a valylatable TLS was investigated for its role in the replication and infectivity of the bipartite Peanut clump pecluvirus. A valylatable TLS provided a small competitive advantage in protoplasts and whole plants. The advantage was more apparent in protoplasts than in whole plants, and more so in the replication protein-encoding RNA1 than in the trans-replicating RNA2. / Graduation date: 2004

Coreplication experiments have revealed that replication of turnip yellow mosaic virus (TYMV) RNA in turnip protoplasts is cis-preferential. Genomes encoding mutant p141 or p66, proteins essential for virus replication, were inefficiently rescued by a helper genome. One model for the cis-preferential replication of TYMV is that p66 and p141 form a complex that associates with the RNA from which they are translated, limiting their availability in trans. Three types of experiments were used in this study in an attempt to obtain physical evidence for the hypothetical interaction between p66 and p141. Immunoprecipitations from in vitro translation reactions using antiserum that recognizes p66 (and its progenitor, p206) coprecipitate p141, indicating that the proteins form a complex in vitro. The results of coimmunoprecipitations of translation products with in-frame deletions did not lead to definitive information about interaction domains. p66 and the helicase domain of p141 do not detectably interact in the yeast two-hybrid system or in GST fusion interaction assays. Problems with the expression of full length p141 fusions make conclusions about the interaction of other p141 domains with p66 not possible at this time. Since the helicase domain of p141 does not appear to interact with p66, future experiments will focus on obtaining expression of smaller domains of p141, outside the helicase domain, and determining if they interact with p66. Variations to the model that do not necessitate the direct interaction between p66 and p141 are also considered. / Graduation date: 1997

Turnip yellow mosaic virus is a monopartite, plus sense RNA virus infecting the Cruciferae, and is a model system for the study of RNA virus replication. A cDNA clone (pTYMC) representing an infectious RNA genome of the European isolate of TYMV was constructed and used to assess the importance of virus genes in virus infectivity. Derivatives of pTYMC with alterations in open reading frame 69 (ORF- 69) were made. The mutations disrupted the expression of ORF-69 in vitro as predicted. Although the ORF-69 mutants were competent for replication in protoplasts, none of the mutants detectably infected turnip or Chinese cabbage plants, except where reversion mutations led to the restoration of an uninterrupted ORF-69. The data suggest a role for ORF-69 expression in the cell-to-cell movement of the virus. Mutant RNAs with a deletion or frameshift in the coat protein ORF infected protoplasts and plant leaves. No systemic infection symptoms were generated by these mutants, and no viral products were detected in young, expanding tissue of infected plants. When the coat protein deletion mutant and an ORF-69 mutant were co-inoculated onto plants, only a virus producing a coat protein of wild type size was detected in symptomatic, systemic tissue in these inoculations, emphasizing a requirement for the expression of native size coat protein for the systemic translocation of TYMV infection. The role of ORF-206 expression in TYMV replication was examined. Three classes of mutants were made in ORF-206: those affecting the synthesis of the 150 kDa protein, those affecting the synthesis of the 70 kDa protein, and those affecting the synthesis of both the 150 and the 70 kDa proteins. All ORF- 206 mutations eliminated RNA infectivity. Protoplast inoculations using mixtures of individual ORF-206 mutant RNAs and a helper genome demonstrated that co-replication of defective genomes could occur. Moreover, inoculations in which individual 150 kDa and 70 kDa protein mutant RNAs were combined showed that complementation between these two classes of mutants was possible. The data indicate that RNAs expressing wild type 150 kDa protein are favored replication substrates in mixed infections, and suggest that the 150 kDa protein functions preferentially in cis. / Graduation date: 1993

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues(R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA was used for relative quantification of viral protein in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity.

The topic of this thesis is focused on the testing of resistance of selected Brassica species to the black rot infection and viral mosaics caused by economically important pathogens of Brassicaceae family. The theoretical part describes characteristics of causal pathogens - Xanthomonas campestris pv. campestris (Xcc), Turnip mosaic virus (TuMV) and Turnip yellow mosaic virus (TYMV), and summarize the current state of a resistance study of these pathogens in the Brassicaceae family. The thesis also describes modern molecular methods used for the detection of bacterial and viral pathogens. In the experimental part, the detections of Xcc, TuMV and TYMV pathogens were optimized by PCR and RT-PCR. For bacterium Xcc, the Real-time PCR targeting a part of the zur gene sequence was designed using a TaqMan® probe. This detection system was subsequently processed in the form of a certified methodology for use in diagnostics. To increase the specificity, Real-time PCR targeting zur gene was involved in the Multiplex Real time PCR reaction. Then the dynamics of the Xcc infection was monitored in 6 hybrid cabbage cultivars. The testing of resistance to the black rot disease was optimized by the procedure including artificial inoculations using the suspension of the Xcc isolates HRIW 3811, 3971A and 1279A and the SU1 isolate originated from the Czech Republic. In a four-year experiment, the total of 42 homozygous breeding lines and 4 hybrid cultivars were tested, where 5 lines were recommended for breeding for resistance to the black rot disease. For the detection of TuMV and TYMV viruses, Real-time RT-PCR approaches based on the TaqMan® probe and SYBR Green dye were tested. The target region of both detections was the coat protein. The TuMV detection has been optimized for SYBR Green approach; for the TYMV detection, the use of the TaqMan® probe has been recommended. Detection systems were used to evaluate artificial inoculations of 6 cabbage cultivars by individual viruses. The tested plants did not show visual symptoms of infection therefore the presence of viruses was evaluated by Real-time RT PCR. The system designed for TYMV detected the presence of virus in all tested samples, TuMV was detected only in two samples. Negative detection results are probably in connection with the absence of TuMV symptoms which indicates unsuccesful plant inoculation. For both detection systems, it was recommended the verification on a wider range of viral isolates prior to standard use in diagnostics

Plant viruses have been instrumental in our understanding of the biophysical properties pertaining to non-enveloped icosahedral virus particles. A substantial amount of research has been performed over five decades on Turnip yellow mosaic virus (TYMV), arguably one of the most extensively studied icosahedral plant viruses and the type-member of the Tymovirus plant virus genus. Even with a substantial body of published scientific literature, little is known about the role of specific coat protein (CP) residues in TYMV assembly, disassembly and disencapsidation. We have shown through our mutagenesis studies that the N-terminal region of the CP that is involved in the formation of an annulus structure and is disordered in A-subunit pentamers is not essential in vivo, but annulus-forming residues are critical in ensuring virion stability and low accessibility after virus is purified (Chapter 2). We have shown that a range of amino acid residue types is tolerated within the CP N-terminus in vivo, although they can greatly affect the stability of virions and empty particles, most notably at low pH (Chapter 3). Unlike full-length CP, N-terminal deletion and substitution mutants fail to reassemble into particles in vitro (Chapter 2, 3) suggesting a critical determinant for the N-terminus in reassembly (discussed Chapter 7). This is the first documented in vitro reassembly reported for a member of the Tymoviridae family and should provide a framework for further studies. We have identified a new way to create empty artificial top component (ATC)-particles through treatment with EDTA (Chapter 6) and we also show that tymoviruses can be engineered with altered pH-dependent enhanced stability (Chapter 4). In collaboration with the Qian Wang laboratory from the University of South Carolina we have shown that an RGD (Arg-Gly-Asp) motif can be genetically engineered within the CP of TYMV, resulting in infectious particles with attractive stem-cell adhesion properties (Chapter 5). With focus on basic viral mechanisms, we have crystallized the TYMV virion and ATC particle at pH 7.7 and collected data to less than 5 Å resolution (Chapter 4, supplementary). These structures represent the first tymovirus-based structures solved above pH 5.5 and will provide insight into the N-terminal conformations within the TYMV particle. Finally, we have characterized an N-terminal CP cleavage seen after ATC formation (Chapter 4) suggesting an additional and yet uncharacterized feature associated with decapsidation. / Graduation date: 2012