Browsing by Author "Grabau, Elizabeth Anne"
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- Functional analyses of tomato 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGR) genes in transgenic plants engineered for altered HMGR expressionYu, Xueshu (Virginia Tech, 1995)3-Hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34) mediates the first regulatory step (HMG-CoA reduction to mevalonate) in isoprenoid biosynthesis. The tomato genome contains at least four differentially regulated hmg isogenes encoding HMGR. Functions of tomato hmg2 in defense responses were studied by promoter analyses of hmg2:GUS gene fusions, overexpression of hmg2 cDNA, and antisense inhibition of hmg1 and hmg2 in transgenic plants. Activity of the hmg2 promoter is developmentally regulated showing expression in seedling cotyledons and hypocotyls, in trichomes, and in reproductive tissues including pollen, stigmas, ovules, petals and mature seeds. hmg2:GUS activity is rapidly induced by wounding or in response to pathogenic viruses or bacteria. hmg2:GUS expression is localized to tissue surrounding lesions generated through interactions with either TMV or the bacterial pathogen, Erwinia carotovora subsp. carotovora (Ecc). Tomato hmg2 cDNA was cloned by PCR, expressed in E. coli to confirm its HMGR activity, inserted behind the double enhanced CaMV 35S promoter, and engineered into tobacco. Southern and northern analyses confirmed transformation and message expression. Enzyme activity was enhanced compared to nontransformed plants. Selected transgenic plants were significantly reduced for Ecc tissue maceration. The size of necrotic lesions induced by TMV was also significantly reduced compared to the nontransformed or vector controls. Thus, genetic manipulation of the rate-limiting step in a major defense pathway provides a novel strategy for enhancing disease resistance. We also generated transgenic tobacco and tomato containing antisense constructs for tomato hmg1 and hmg2 to study their effect on disease resistance. Full-length hmg2 and 5' regions of hmg1 or hmg2 were inserted in the antisense orientation behind a 35S promoter. Tomato expressing the full-length hmg2 antisense showed lower HMGR enzyme activity and were more susceptible to soft rot by Ecc than control plants. In contrast, expression of either antisense hmg/ or antisense hmg2 in the heterologous tobacco system resulted in plants with enhanced resistance to Ecc and reduced TMV lesion sizes. These results may indicate that antisense inhibition is non-specifically exerted on isogenes other than the defense-specific HMGR gene.
- Insertion sequence IS1141: discovery, characterization, and association with Mycobacterium intracellulare colonial variationVia, Laura Ellen Akers (Virginia Tech, 1993)Mycobacterium avium and Mycobacterium intracellulare, (M. avium complex, MAC) are human pathogens causing disease in individuals with acquired immunodeficiency syndrome (AIDS) or with thoracic abnormalities. MAC bacteria are difficult to kill because of the resistance of the pathogens to chemotherapeutic agents. One factor affecting treatment of MAC disease is the presence of interconvertible colonial variants. Transparent (T) variants have greater resistance to antibiotics and higher pathogenicity; opaque (O) variants are more susceptible to antibiotics and less pathogenic. The overall goal of this study was to investigate the mechanism for colonial variation. Based on an observation that T variants of M. intracellulare strain Va14 contained a plasmid which was 6 kb smaller than the 68 kb plasmid in O variants, it had been suggested that a transposable element might be responsible for colonial variation. The first objective was to clone the unique DNA fragment present in the 68 kb plasmid but absent from the 62 kb plasmid. The second and third objectives were to determine if the unique fragment contained a transposable element and to analyze the role of that element in the mechanism of colonial variation in M. intracellulare strain Va14. The fourth objective was to determine the distribution of IS1141 in MAC isolates. Fragments containing copies of the putative element were sequenced and a region 1596 basepairs in length with 23 basepair imperfect inverted repeats was designated as insertion sequence IS1141. IS1141 is the first insertion sequence identified in M. intracellulare. Data base searches using open reading frames (ORF) of IS1141, identified ORFb as significantly similar to the transposases of the IS3 family. The presence or absence of IS1141 in strain Va14 plasmids appeared unrelated to colonial variation, but IS1141 was present in another plasmid and the chromosome of the Va14 variants. Hybridization studies with IS1141 identified three chromosomal copies in O variants and two chromosomal copies in T variants. Va14 T variants each had a common IS1141 restriction fragment length polymorphism (RFLP) pattern which was different than the single RFLP pattern found in opaque variants. Based on these differences, it appears that IS1141 may integrate into the gene(s) responsible for the T phenotype preventing their expression. A survey of 64 James River basin non-AIDS, clinical and James River environmental MAC isolates identified 4 of 24 (17%) M. intracellulare isolates as containing IS1141. IS1141 has not been detected in any clinical or environmental M. avium or Mycobacterium species X isolates and may be limited to M. intracellulare.
- Rhizosphere competence, antibiotic and siderophore biosynthesis in Pseudomonas chlororaphis: implications for the biological control of cotton seedling disease pathogensMulesky, Melinda Anne (Virginia Tech, 1995)Cotton seedling disease caused by Pythium ultimum Trow and Rhizoctonia solani Kühn occurs worldwide in soils ranging from pH 4.5 to 8.5. Studies with cotton have not yet established the relative importance of two classes of secondary metabolites synthesized by soilborne pseudomonads, siderophores (sid) (low molecular weight Fe⁺³ chelators) and antibiotics (ant), in the suppression of these pathogens. Greenhouse bioassays to screen for rhizosphere competent strains identified a single strain of Pseudomonas chlororaphis (L-850), that produced siderophores and multiple antifungal antibiotics, including one or more phenazines. A Tiff Image Analyzer (TIA) software program was developed that allowed assessment of wild-type (wt) L-850, and (ant) and (sid) mutant populations as a function of cotton root surface area (cm²) in the absence of soil irrigation. Bacterial density and distribution patterns on roots evaluated 22, 36, and 50 DAP, in two pathogen-free soils (pH 5.7, high Fe⁺³, high phosphorus (P); pH 8.0, low Fe⁺³, low P) indicated that populations of both wt and mutants persisted after day 22 at levels between log 4.6 (lower laterals) to log 6 cfu/cm² (upper tap) even as total root area increased 122% from day 22 to 50. Population densities of all strains were consistently 1/8 to 3/4 log unit lower in the pH 5.7 soil on the lower tap and upper lateral roots, respectively. The loss of siderophore production appeared to enhance the rhizosphere competence of strain L-850. For greenhouse trials with three pathogen inoculum densities (low, intermediate, high) protection against preand postemergence damping-off (phase 1) and hypocotyl/root rot of young plants (phase 2) by the (sid) and wt strains was similar (P = 0.05) whereas, protection by seed treatment with the (ant) mutant was reduced. The level of suppression provided by L-850 was equivalent (P = 0.05) to the standard fungicide at low and intermediate pathogen pressure. These studies demonstrated a minimum contribution of siderophores in the biological control of cotton seedling disease and established a significant role for antibiotic biosynthesis over a range of soil physical and chemical characteristics.
- Secretion of active recombinant phytase from stably transformed soybean cellsLi, Jia (Virginia Tech, 1995-12-15)The objective of this research was to express a fungal phytase gene in transgenic soybean cells to to study the potential for improving phosphorus utilization in soybean meaL A simple and inexpensive particle inflow gene gun was constructed and bombardment was optimized as assayed by β-glucuronidase reporter gene expression. A somatic embryogenesis approach was used for soybean regeneration from culture. The efficiencies of embryo induction and embryo conversion to form roots and shoots were compared in commercial soybean cultivars to identify optimal cultivars for recovery of transgenic plants. To study the expression of a recombinant fungal phytase gene (PhyA from Aspergillus niger), four expression vectors were constructed in soybean transformation vectors. PhyA was placed under the control of either a constitutive cauliflower mosaic virus 35S promoter or a soybean seed specific β-conglycinin promoter, each with or without a patatin endoplasmic reticulum (ER) signal sequence. All four vectors were sequenced and introduced into 'Williams 82' suspension culture cells by particle bombardment. Stably transformed cell lines were selected and tested for stable integration by Southern analysis. The presence of the phytase protein product was detected by immunoblotting. Activity of recombinant phytase was characterized by enzyme assay. Cell lines containing the phyA gene under control of the CaMV 35S promoter and ER signal sequence secreted active phytase into the culture medium. The pH and temperature optima were determined for recombinant phytase.