Graduation date: 1993

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: plant pathogen interactions; turnip crinkle virus. Includes bibliographical references (p. 59-61).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 50-52).

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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

Turnip crinkle virus (TCV) has been implicated in the suppression of the hypersensitive response (HR), a type of programmed cell death induced during active resistance in Arabidopsis thaliana. In order to investigate the involvement of individual viral components in mediating suppression, TCV genes were cloned for use in an Agrobacterium tumefaciens mediated transient expression in Nicotiana benthamiana. Agroinfiltration of the HR-inducing avrPto/Pto system in conjunction with individual TCV genes has identified the p38 gene, which encodes the viral coat protein, as the gene responsible for the cell death suppression phenotype. The extent of cell death suppression by coat protein was quantified and found to be equal to the level of suppression by the whole virus and AvrPtoB, another cell death inhibitor from bacteria. Thus, the coat protein alone is sufficient to inhibit the HR in plants. Further, the effect of TCV on HR initiation by an avirulence factor from an unrelated bacterial pathogen was investigated. The presence of TCV does not affect the production, secretion or cellular processing of the bacterial avirulence factor.

Most plants are capable of mounting resistance responses to various pathogen attacks. For a hypersensitive response (HR) to occur, a dominant or semi-dominant resistance (R) plant gene is required to recognize a dominant avirulence (Avr) factor of the pathogen. Three types of Arabidopsis thaliana, Dijon-17 (Di-17), Dijon-3 (Di-3), and Columbia-0 (Col-0), are significant in understanding the genetics of Turnip crinkle virus (TCV) resistance. It has been shown that three genes are needed for successful resistance to TCV in A. thaliana: the dominant R gene HRT, the recessive gene rrt, and a third gene, TIP. Crosses of Di-17 and Di-3 plants, and crosses of Di-3 and Col-0 plants are being analyzed to determine the genotype of the F1 progeny. Using cleaved amplified polymorphic sequence (CAPS) markers, it is possible to determine the genotype of the progeny compared to the wild-type parents at the HRT and TIP loci. Additionally, protein analysis tools will be employed to compare the Di-3 and Di-17 TIP alleles to determine if there are any significant differences in the protein.

Upon detection of a pathogen, plants initiate specific signaling events designed to prevent host colonization and pathogen proliferation. Appearance of the hypersensitive response (HR), a type of programmed cell death signifies activation of active defenses in response to a one-to-one recognition of host, Resistance or R gene, and pathogen, avirulence or avr gene, encoded products. Turnip crinkle virus (TCV), however, has been shown to suppress the ability of Col-0 Arabidopsis thaliana plants to produce the HR in response to an avirulence factor. The extent of suppression was quantified by measuring cellular electrolyte leakage resulting from programmed cell death. Interestingly, cellular ion leakage levels were significantly lower in TCV-infected plants when challenged with bacteria expressing either of two bacterial effectors avrRpt2 or avrRpm1, suggesting that TCV can suppress the HR to a range of HR-inducing avirulence factors. In order to determine the viral component(s) responsible for mediating this suppression, each of the five TCV open reading frames (ORFs) was tested using an Agrobacterium tumefaciens-mediated transient expression assay in Nicotiana benthamiana. Though sequencing of the five TCV clones revealed mutations in the p28, p88, and p9 clones, Agro infiltration of an HR-inducing system in conjunction with individual TCV ORFs, or combinations of, was used to gather data to determine the role each may possess in the suppression phenotype. Full-length TCV was also expressed in the presence of AvrPto/Pto to establish suppression phenotype in Nicotiana. To assay for suppression of cell death in a heterologous system, both the mutant and wild-type clones were also tested in yeast for cell-death suppression induced by hydrogen peroxide exposure.

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

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: protein interaction; coat protein; resistance; arabidopsis; turnip crinkle virus. Includes bibliographical references (p. 58-62).