In Lactuca sativa, the dominant gene Tu confers resistance to infection by turnip mosaic virus (TuMV). Tu and Dm5/8, a gene for resistance to Bremia lactucae, are linked in L. sativa. The area surrounding Dm5/8 on the genetic map of L. sativa contains restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. The orientation of Tu relative to Dm5/8 was not known. Locating Tu would indicate which markers are on the map of lettuce close to Tu. To locate Tu on the L. sativa genetic map, F$ sb3$ families from recombinant F$ sb2$ in the Dm5/8 area of a cross between TuMV-resistant (Cobbham Green) and susceptible (Calmar) cultivars were inoculated with TuMV and phenotyped for Tu by indirect enzyme-linked immunosorbent assay. Polyclonal antibodies for immunodetection were produced using turnip mosaic virus coat protein expressed in E. coli. Phenotypic ratios within F$ sb3$ families were used to determine individual F$ sb2$ genotypes for Tu. With these genotypes, Tu was located on the genetic map of L. sativa relative to data present for Dm5/8 and surrounding markers, between OPM18 and OPY13. Using bulked segregant analysis, bulks created for the Dm5/8 locus were screened for genetic polymorphisms by the RAPD technique. Five new RAPD markers, UBC346, UBC517, UBC563, UBC599, and UBC675 were found linked to Tu after mapping relative to F$ sb2$ genotypes for Tu and other RAPD markers. The resulting three-point mapping information indicates that Tu is flanked by two markers, OPM18/OPL08 and UBC346, at respective genetic distances of 0.4 and 0.7 cM.

The response of Arabidopsis thaliana (Columbia) to infection with turnip mosaic virus (TuMV) was characterized at the level of: disease symptom expression, cell content and protein composition. Visual symptoms observed were chlorotic and mottled leaf colouring, severely stunted growth, distortion of leaf blades and delayed bolting. All plants died before seed cases dehisced. Electron microscopy revealed three types of cylindrical inclusion bodies: pinwheels, scrolls and laminated aggregates, in the cytoplasm of infected plants similar to those observed in other plants infected with TuMV. Inoculation of Arabidopsis with TuMV resulted in quantitative changes in several proteins in both soluble and membrane proteins, as revealed by electrophoresis on 12% polyacrylamide gels. Antibodies were made to both infected membrane and soluble proteins. Western blots of infected and uninfected, soluble and membrane proteins probed with antibodies revealed quantitative changes in the same proteins identified by polyacrylamide gels. A CNBr 4B activated sepharose column was used to make infection-specific antibodies to infected soluble proteins. No infection-specific host proteins were detected in Arabidopsis infected with TuMV.

In lettuce (Lactuca sativa), the dominant gene Tu confers resistance to turnip mosaic virus (TuMV) infection. In order to eventually clone and characterize the Tu gene using a map-based cloning strategy, the chromosome region in which Tu is located needs to be saturated with molecular markers. Random polymorphic DNA (RAPD) markers were screened using bulked segregant analysis. Nine new RAPD markers, UBC431$ rm sb{420}, UBC431 sb{940}, UBC434 sb{360}, UBC434 sb{1000}, UBC439 sb{520}, UBC448 sb{685:750}, UBC135 sb{240}, OP108 sb{410} and OP108 sb{1305},$ were identified as linked to Tu. Each marker was mapped relative to Tu using F$ sb2$ individuals previously known to be recombinant in the area surrounding the Tu locus. Three new markers, UBC431$ rm sb{420}, UBC439 sb{520} and UBC135 sb{240}$ are within a 5 cM area of Tu. As the number of DNA markers on the map increased map expansion and difficulties in determining a unique order were encountered. To increase the confidence in the estimate of genetic distances, a population of 500 F$ sb2$ plants was screened in order to identify more recombinant individuals around the Tu locus. The population was screened using markers UBC431$ sb{420}$ and UBC135$ sb{240}.$ Thirty-three recombinants were identified in an interval of 6.6 cM. Two markers, UBC346$ sb{1067}$ and OP108$ sb{634},$ tightly flanking Tu were converted to sequence characterized amplified regions (SCAR 346 and SCAR L08). No polymorphism was detected among the SCARs generated. The area surrounding Tu now includes 24 RAPD markers in an interval of 44 cM.

Interactions between plant and virus proteins play pivotal roles in many processes during the viral infection cycle. Analysis of protein-protein interactions is crucial for understanding virus and host protein functions and the molecular mechanisms underlying viral infection. Several interactions between virus-encoded proteins have been reported. However, few interactions between viral and plant proteins have been identified so far. To examine interactions between Turnip mosaic potyvirus (TuMV) proteins and plant proteins, recombinant proteins were produced and used in ELISA-type assays and in in vitro co-immunoprecipitation experiments. An interaction between TuMV P1 proteinase and wheat poly(A)-binding protein (PABP) was identified. An interaction between P1 protein and the plant Arabidopsis thaliana eukaryotic initiation factor (iso)4E [eIF(iso)4E] was also found. Finally, potential interactions between both TuMV CI and P1 proteins and between TuMV CI protein and eIF(iso)4E were identified.

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

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

Magister Scientiae (Biodiversity and Conservation Biology) / Heavy metal contamination tends to be a problem in inner city agricultural areas and gardens. High levels of certain heavy metals have been found in the soil and vegetables in the Cape Town Metropolitan area. The aim of this project was twofold. Firstly to ascertain whether water (ground or surface) was responsible for the heavy metal problem found in vegetables in the Philippi and Kraaifontein-Joostenbergvlakte farming areas in Cape Town; and secondly to evaluate the efficacy of two possible remediation methods, namely chelation (with EDTA) and precipitation (using phosphate), aimed at tackling the problem. In order to achieve this a water survey and greenhouse experiment were conducted. The water survey involved collecting a number of samples; both from surface dams and boreholes, from the two farming areas. Results showed minimal heavy metals in both, and therefore ruled this out as the source of the heavy metal problem. The greenhouse sand culture experiment tested the effects of the two remediation methods on the growth, development and elemental content of turnip and spinach plants treated with two cadmium and lead concentrations. In summary, Cd reduced growth more than Pb; Cd accumulated in roots and leaves, and Pb in roots. Several treatments, both metal and mitigation enhanced the chlorophyll content. The difference between the EDTA and phosphate mitigation treatments were not significant in the case of cadmium but in the case of lead, high phosphate resulted in increased growth. The large variation of results in this study, and indeed those of the available literature, indicate that the remedial treatments investigated here are not necessarily the most effective and that other treatments should be investigated to control the uptake of either cadmium or lead, as agricultural soils in future become more contaminated with either or both of these heavy metals.

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