Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Plant resistance; Arabidopsis thaliana; turnip crinkle virus. Includes bibliographical references (p. 69-72).

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Turnip crinkle virus; arabidopsis; thaliana; TCV; avrRpt2; avrRpm1; avrRps4; systemic acquired resistance; virulence; Avr gene; R gene; pseudomonas syringae. Includes bibliographical references (p. 60-66).

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

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.

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

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Turnip crinkle virus; Hypersensitive response. Includes bibliographical references (leaves 52-61).

La socavación es una de las operaciones críticas dentro del proceso productivo en una mina explotada por hundimiento de bloques o paneles. El conocimiento del proceso de socavación proviene en gran medida de la experiencia operativa, sin un mayor conocimiento teórico. Por lo tanto, resulta fundamental mejorar el entendimiento de este proceso, y apuntar hacia la confección de guías de diseño ingenieril. La revisión del estado del arte a la fecha mostró que la tendencia actual en el diseño se orienta a la selección del hundimiento avanzado como estrategia de socavación, en general optando por geometrías de corte basal alto, en particular corte inclinado o hundimiento frontal. En el presente trabajo se muestra una comparación geotécnica relativa entre las técnicas de socavación que fueron consideradas con mayor potencial de aplicación dentro de las identificadas en la revisión de diseños: por un lado, hundimiento convencional, y por otro, las variantes de hundimiento avanzado, corte plano e inclinado. Modelación numérica elástica mediante el software Map3d fue realizada para entender el impacto de las distintas geometrías de corte basal, sobre dos aspectos críticos: los esfuerzos inducidos en la frente de socavación y la condición de los pilares de los niveles de producción y hundimiento. La estrategia de modelación desarrollada fue del tipo paramétrica, modificando una variable de interés a la vez: la secuencia de socavación, la geometría del corte; la razón entre los esfuerzos horizontal y vertical in situ y la profundidad. Para comparar entre las variantes, se utilizaron dos indicadores: la razón entre el esfuerzo vertical inducido, medido en los pilares del nivel de hundimiento posterior a la socavación, y el esfuerzo vertical in situ; y un factor de seguridad, asumiendo el criterio de falla de Mohr-Coulomb, aplicado sobre los pilares de los niveles de producción y hundimiento. Los resultados obtenidos muestran que la estrategia que parece ser más robusta corresponde a la de corte inclinado avanzado, debido a que en la mayoría de los escenarios analizados, ofrece las condiciones de diseño más benignas: mayores factores de seguridad sobre los pilares y bajos esfuerzos inducidos en el nivel de hundimiento, en particular con respecto al caso de corte plano.

TCV (Turnip crinkle virus) coat protein is required for the resistance response in Arabidopsis thaliana Di-17 plants. An aspartate to asparagine mutation at amino acid four of the coat protein is sufficient to result in resistance-breaking. To determine the essential chemical properties responsible for the induction of resistance, a series of site-directed mutants were produced. Serine as well as asparagine at amino acid four induces systemic disease on both Di-3 and Di-17 plants; however, replacement of aspartate with glutamate retains the ability to induce the HR (hypersensitive response) and resist TCV infection with rapid and strong induction of PR-1 gene. These data suggest that the negative charge at the fourth amino acid of the coat protein is critical for the induction of resistance. Taken together with other mutagenesis research, the N-terminus of the coat protein appears to be the sole viral recognition element. The A. thaliana TIP protein is suggested to be involved in resistance, mainly through its C-terminus. Interestingly, one of the resistance-breaking mutants (D4N) produces a HR on Di-3 plants that are normally susceptible. The Di-3 TIP protein has several differences from the Di-17 TIP. To detect whether the delayed HR is related to interaction between Di-3 TIP and D4N mutation, a yeast two-hybrid assay was attempted. Interactions have not yet been detected. There are a number of possible explanations.