VTechWorks Repository :: Browsing by Author "Lacy, George H."

Browsing by Author "Lacy, George H."

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Characterization and regulation of the speA gene in Escherichia coli
Moore, Robert C. (Virginia Tech, 1990)
In Escherichia coli, the speA gene encodes biosynthetic arginine decarboxylase (ADC), the first enzyme in a putrescine biosynthetic pathway. ADC converts arginine to agmatine, which is hydrolyzed by agmatine ureohydrolase, encoded by the speB gene, to putrescine and urea. ADC is negatively regulated by mechanisms requiring either cAMP and cAMP receptor protein (CRP) or putrescine. A 3,236 base pair (bp) BalI-AccI restriction fragment derived from plasmid pKA5, which contains a 7.5 kilobase (kb) E. coli genomic fragment in pBR322, was subcloned into pGEM-3Z to produce plasmids pRM15 and pRM59. Both pRM15 and pRM59 overexpress ADC and the DNA sequence of the BalI-AccI fragment in each plasmid was determined. A 2,119 bp restriction fragment containing 730 bp 5’ to speA, the speA promoter, and 1,389 bp (463 amino acids) of the 5’-end of speA was used to construct transcriptional (pRM161 and pRM162) and translational (pRM65) speA-lacZ fusion plasmids. The presence of the predicted 160,000 and 157,000 dalton ADC
Characterization of the nifUHD cluster and a new myoglobin-like gene from Nostoc commune UTEX 584
Angeloni, Stephen V. (Virginia Tech, 1992-03-04)
Sequence analysis of the entire 3.5 kb HindIII genomic DNA fragment previously isolated from Nostoc commune UTEX 584 (Defrancesco and Potts 1988), determined the exact locations of the nifU, nifH, and nifD genes and identified two potential stem loop structures, a direct repeat, and an ORF that codes for a protein with a predicted amino acid sequence similar to that of myoglobin from Paramecium caudatum. The N. commune UTEX 584 myoglobin-like protein has a predicted length of 118 amino acids and molecular mass of 12,906 Da. A PCR copy of the gene (glbN) was cloned for overexpression of the protein. The recombinant protein was purified and used for spectral analysis and for the production of polyclonal antisera. Treatment of the recombinant protein with dithionite and CO resulted in spectral shifts characteristic of hemoproteins that bind oxygen. While some of the spectral characteristics are unique to the protein, in general the spectra were more like those of globins than cytochromes. Based on these characteristics and the sequence similarity to the P. caudatum mnyoglobin, we proposed the name cyanoglobin, with the gene designation glbN and the protein designation GlbN. Western analysis of GlbN expression was performed on N. commune UTEX 584 and two species of Anabaena (Anabaena sp. PCC 7120 and Anabaena variabilis). In N. commune UTEX 584 a protein with a molecular mass similar to that predicted for GlbN was detected. This protein was produced in increased amounts under the same growth conditions that resulted in increased production of nitrogenase reductase (the nifH gene product). No proteins of similar size to GlbN were detected in Anabaena sp. PCC 7120 or A. variabilis. A possible function of GlbN may be for oxygen storage, transport, or protection of the nitrogenase system. These functions as well as those of the direct repeat and the potential stem loop structures and their relationship to nitrogen fixation or other physiological processes in N. commune UTEX 584 require further analysis.
Crown Gall of Woody Ornamentals
Lacy, George H.; Hansen, Mary Ann (Virginia Cooperative Extension, 2009-05-01)
This publication covers the symptoms, disease cycle, and control of Botrytis blight of peony.
Determination of the Leak Size Critical to Package Sterility Maintenance
Keller, Scott Wayne (Virginia Tech, 1998-08-18)
This study was divided into four sections: the literature review; the mechanism by which a package defect becomes a leak; and the imposed pressures generated within a package during distribution; comparison of the threshold leak size to the critical leak size and their effect on loss of package sterility; and the relationships between microorganism characterisitics and the threshold leak size, and their effect on the critical leak size. Section II. The mechanism by which a package defect converts to a leaker in an effort to develop a relationship between the threshold leak size and loss of package sterility was studied. The threshold leak size is the hole size at which the onset of leakage occurs. The threshold pressure is that which is required to initiate a leak. Leak initiation was studied in terms of the interaction between three components: liquid attributes of liquid food products, defect size, and pressures required to initiate liquid flow. Liquid surface tension, viscosity, and density were obtained for sixteen liquids. The imposed pressures (Po) required to initiate flow through microtubes of IDs 0, 2, 5, 7, 10, 20 or 50 m, were measured using 63 test cells filled with safranin red dye, tryptic soy broth, and distilled water with surface tensions of 18.69 mN/m, 44.09 mN/m, and 64.67 mN/m, respectively. Significant differences were found between observed threshold pressures for safranin red dye, tryptic soy broth, and distilled water (p < 0.05). Liquids with small surface tensions such as safranin red dye required significantly lower threshold imposed pressures than liquids with large surface tensions such as distilled water (p < 0.05). An equation was developed to quantify the relationship between liquid surface tension, threshold imposed pressure, and defect size. Observed threshold pressures were not significantly different (p > 0.05) than those predicted by the equation. Imposed pressures and vacuums generated within packages during random vibration and sweep resonance tests were measured for brick-style aseptic packages (250 ml), metal cans size 76.2-mm x 114.3-mm (425 ml), quart gable top packages (946 ml), one-half gallon gable top packages (1.89 L) and one-gallon milk jugs (4.25 L). Significant differences were found between packages for observed generated pressures during vibration testing (p < 0.05). An equation to calculate the threshold like size based on liquid surface tension and imposed pressure was established. Section III. The onset of liquid flow through a defect as a result of imposed positive pressures or vacuum were linked to the sterility loss of a package. Five-hundred sixty-three test cells, each with microtubes of 0, 2, 5, 7, 10, 20 or 50 m, manufactured to simulate packages with defects, were biochallenged via an aerosol concentration of 106 cells/cm3 of Pseudomonas fragi Lacy-1052, under conditions of imposed positive pressure or vacuum of 20.7, 13.8, 6.9, 0, -6.9, -13.8, -20.7 kPa, respectively and temperatures of 4 , 25 and 37 C. A statistically significant relationship between loss of sterility due to microbial ingress in test cells and the initiation of liquid flow were found (p < 0.05). Microbial ingress was not found in test cells with microtube IDs of 2 m. Leak sizes critical to the sterility maintenance were found to be different based on the liquid surface tension, and imposed package pressures. The threshold leak size where the onset of liquid flow was initiated, and the critical leak size at which loss of sterility occured were not significantly different (p > 0.05). Section IV. The effects of microorganism size and motility, and the imposed pressure required to initiate liquid flow, on the leak size critical to the sterility of a package were measured. Pseudomonas fragi Lacy-1052, Bacillus atrophaeus ATCC 49337, and Enterobacter aerogenes ATCC 29007 were employed to indicate loss of package sterility. One hundred twenty-six microtubes with interior diameters (I.D.s) of 5, 10, and 20 m and 7 mm in length were used as the manufactured defects. Forty-two solid microtubes were used as a control. An equation was used to calculate imposed pressures sufficient to initiate the flow of tryptic soy broth through all defects. No significant differences were found for loss of sterility as a result of microbial ingress into test cells with microtube ID sizes of 5, 10, and 20 m between the test organisms (p > 0.05). Interactions between the initiation of liquid flow as a result of imposed pressures, and the sterility loss of test cells were significant (p < 0.05).
Development of a Rapid Coliphage Assat
Stanek, James Emmett (Virginia Tech, 1997-01-24)
A rapid coliphage detection assay (RCDA), based on the phage-induced release of b-galactosidase from cells of Escherichia coli (Ijzerman, M., J.O. Falkinham III and C. Hagedorn. (1993) [A liquid, colorimetric presence-absence coliphage detection method. J. Virol. Meth. 45:229-234] was modified to reduce the number of steps required to perform the assay, remove the need for specialized media and buffers, reduce the volumes required, and simplify growth and reaction conditions. Tolerances of the assay were defined at each step of the assay. The number of steps has been reduced from 12 to 7. The b-galactosidase reaction buffer was eliminated. Culture volumes were reduced from 25 ml to 5 ml and reaction volumes were reduced from 10 ml to 0.5 ml. Optimal growth conditions were 37 o C with orbital shaking at 200 rpm, a one hour subculture time and an incubation of subculture with water sample for two hours. Color development occurred at 37 o C in 30 minutes. The changes and modifications of the assay increased the ease of its performance without sacrificing the ability of the assay to detect as few as two phage particles per sample. By understanding the tolerances of the assay, technical support representatives of companies producing kits modeled after the assay will be prepared to answer questions from customers concerning possible kit failures or user error.
Effects of simulated acidic precipitation on the colonization and ice nucleation activity of Pseudomonas syringae pv. syringae and Erwinia herbicola
Murray, Joseph Marshall (Virginia Tech, 1987-03-02)
Precipitation over the eastern United States has been increasing in acidity, particularly within the last three decades. The average annual pH of rain in this area is about 4.2. The foliar surface, or phylloplane, of soybean can be damaged by rain acidified to pH 2.9. Simulated acidic precipitation has an overall inhibitory influence on soil microbial processes. The effect acidic precipitation may have on epiphytic microorganisms has not been examined. Bacteria are among the most numerous residents on the phylloplane.
Environment risk assessment for toxic chemicals and genetically-engineered microorganisms: a microcosm approach
Scanferlato, Vjera Sostarec (Virginia Tech, 1990)
Microcosms were used in this research to assess the environmental risk associated with two types of stressors: genetically-engineered microorganisms (GEMs) and toxic chemicals. Approaches used to evaluate the potential environmental impact of these two stressors are fundamentally different, and arise mainly from the fact that the GEMs are living and capable of replication. Risk assessment for genetically-engineered Erwinia carotovora strain L-864 included investigations of its persistence and effect on structure and function of aquatic microcosms. Densities of genetically-engineered and wild type E. carotovora declined at the same rate in water or in sediment, falling in 32 days below the level of detection by viable counts. Selective media, antibiotic resistance, and most probable number analysis were used to enumerate genetically-engineered E. carotovora in environmental samples. This technique was able to detect as few as 1 to 10 target cells/10 g soil. In thermally-perturbed aquatic microcosms, genetically-engineered E. carotovora persisted significantly longer than in unperturbed microcosms, suggesting the vulnerability of stressed ecosystems to colonization by GEMs. Competition study showed that the genetically-engineered E. carotovora did not displace the wild type strain. Effects of genetically-engineered and wild type strain on indigenous bacteria belonging to specific functional groups important in nutrient cycling were similar: inoculation of either strain caused a temporary increase in densities of total and proteolytic bacteria, while it did not affect amylolytic and pectolytic bacteria. Treatment with engineered bacteria did not change biomass values of the receiving community, but caused a transitory increase in its metabolic activity. The inability of genetically-engineered E. carotovora to persist, displace resident species, and affect metabolic activity of a community indicates a low risk of adverse ecological effects in aquatic systems. Microcosms were also used to assess environmental risk for toxic chemicals. A study was conducted to assess the fate of sediment-associated copper and to investigate its effects on the structure and function of the aquatic community. Most of the added copper was bound to sediment particles. In microcosms containing 100 µg Cu/kg sediment, chlorophyll a content and respiration significantly decreased compared to the control. Addition of 1000 µg Cu/kg sediment caused a decrease in production, respiration, respiration/biomass ratio, ATP, and chlorophyll a. The last study compared responses of Simocephalus exspinosus (Daphniidae) to copper during a single-species test to responses of S. exspinosus populations in a microcosm test. Responses of S. exspinosus were similar in both test systems: there was an increased production of young at 30-46 µg Cu/l, while the organisms did not survive exposure to concentrations > 100 µg Cu/l. In these studies, microcosms showed a potential to predict fate and effects of chemical and biological contaminants released into the biosphere.
Environmental risk assessment of a genetically-engineered microorganism, Erwinia carotovora
Orvos, David R. (Virginia Polytechnic Institute and State University, 1989)
Environmental use of genetically-engineered microorganisms (GEMs) has raised concerns over potential ecological impact. Development of microcosm systems useful in preliminary testing for risk assessment will provide useful information for predicting potential structural, functional, and genetic effects of GEM release. This study was executed to develop techniques that may be useful in risk assessment and microbial ecology, to ascertain which parameters are useful in determining risk and to predict risk from releasing an engineered strain of Erwinia carotovora. A terrestrial microcosm system for use in GEM risk assessment studies was developed for use in assessing alterations of microbial structure and function that may be caused by introducing the engineered strain of E. carotovora. This strain is being developed for use as a biological control agent for plant soft rot. Parameters that were monitored included survival and intraspecific competition of E. carotovora, structural effects upon both total bacterial populations and numbers of selected bacterial genera, effects upon activities of dehydrogenase and alkaline phosphatase, effects upon soil nutrients, and potential for gene transfer into or out of the engineered strain. No significant difference was found in survival of the engineered strain as compared to its wildtype parent. Both strains survived for over two months in microcosms. The effects of both strains upon populations of total bacteria and selected bacterial genera were determined; while some effects upon community structure were observed, they were not significant. The engineered strain was not found to be a superior competitor compared to its parent; three different doses of engineered and wildtype strains were used. ln addition, neither strain affected activities of dehydrogenase or alkaline phosphatase in soil. Likewise, no effects were observed upon the nutrients monitored. However, transfer of the kanamycin resistance gene that had been inserted into the engineered E. carotovora strain may have occurred. Five species of indigenous bacteria displayed kanamycin resistance 15 days after being exposed to the engineered Erwinia. DNA from these strains was isolated, purified, and hybridization experiments executed to determine if any homology existed between these DNAs and the kanamycin resistance gene that had been inserted into E. carotovora. Using biotin-Iabeled probes and Iow-stringency washing conditions, homology was observed. However, before gene transfer can be proven, additional studies, including amplification and sequencing, may be required. Although a simple microcosm design was employed, it yielded sufficient information to conclude that release of the engineered Erwinia carotovora will not affect any of the microbial measures of integrity that were studied in a manner different from that of the wildtype. Effects upon plant material and other higher taxa will be the focus of future studies.
Erwinia carotovora extracellular proteases: characterization and role in soft rot
Kyöstiö, Sirkka R. M. (Virginia Tech, 1990-12-03)
Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene encoding a Prt, prt1, was cloned from cosmid pCA7 containing Ecc genomic DNA into plasmid pSK1. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produced intracellularly a 38 kDa Prt with the same pI (4.8) as the secreted Ecc Prt. Prt1 activity produced by E.coli/pSK23 was inhibited by phenanthroline, which inhibits Zn-metalloproteases, but not by Ecc intracellular proteins. Analysis of deletion mutants indicated a 1.2 kb region necessary for Prt1 production. Sequencing of the pSK1 insert revealed a 1,041 bp open reading frame (ORF1) corresponding to the prtl region. ORF1 encodes a putative polypeptide of 347 amino acids with a total molecular mass of 38.8 kDa. The location of the prt1 promoter was determined to be 173 to 1,173 bp upstream from ORF1 by constructing transcriptional fusions to lacZ in plasmid pCD267. Primer extension revealed the start of prt1 mRNA 205 bp upstream of ORF1. The deduced amino acid sequence of the prt1 was compared to other proteases; it is similar to several bacterial Zn-metalloproteases. Prt1 production by Ecc was not observed during growth in rich broth; however, Northern analysis showed prt] mRNA accumulation in Ecc grown in planta. The role of prt1 in soft rot was determined by constructing a Prt1-deficient Ecc; prt1 insertionally inactivated by a kanamycin resistance gene was used to replace wildtype prt11 in the Ecc genome by homologous recombination. This mutant (L-957) had approximately 60 to 80% reduced Prt activity suggesting the presence of a second Prt (Prt2). Prt2 was purified from Ecc culture supernatant. This protease, also a metalloprotease, has a molecular mass of 45 kDa and pI of 4.8. Its amino terminal sequence had Significant sequence identity to metalloproteases from Erwinia chrysanthemi and Serratia marcescens, but not to Prt1. Further, unlike Prt1i, Prt2 was inhibited by Ecc intracellular proteins. The effect of proteases in potato tuber maceration was measured using L-957 and L-763, a Tn5 transposon mutant constructed previously. L-763 had no extracellular protease activity and may have been mutated in a regulatory region. Both mutants macerated significantly less tuber tissue than the wildtype Ecc. Reduced maceration of L-957 and L-763 was correlated with slower in planta growth. This suggests Prt1 production provides a nutritional advantage for Ecc growth on potato.
Evolution of a gene for pathogenicity: endo-pectate lyase
Allen, Caitilyn (Virginia Polytechnic Institute and State University, 1987)
Erwinia carotovora subsp. Carotovora (Ecc) and Erwinia carotovora subsp. Atroseptica (Eca) are plant pathogenic bacteria that cause soft rot disease of many plant species and blackleg disease of potatoes, respectively. Ecc and Eca attack plants by means of a group of extracellular plant tissue-degrading enzymes. which rapidly breaks down the pectic polymers that form a structurally important part of the plant cell wall, is considered central to soft rot pathogenesis. In this work, I isolated and studied the genes encoding this enzyme from Ecc and Eca. A clone library of Ecc strain EC14 was constructed using cosmid PLAFR3. This library contains 2,200 clones with an average insert size of 27 kilobases of DNA and included a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. The proteolytic clone was used to complement a Tn5-induced protease mutant of Ecc; the complemented mutant was restored to near-wild type phenotype. Six of the pectolytic clones hybridized to a probe from a. previously isolated extracellular endo-pectate-pectate lyase gene from Ecc; one pectolytic clone had homology to a previously isolated clone encoding endo-polygalacturonase: three clones showed no relationship to either of the previously characterized Ecc pectolytic enzyme genes. A clone encoding the major endo-pectate lyase gene from Ecc was chosen for subcloning and further study. I used the plasmid vector pBR322 to construct a clone bank of Eca strain SRB; of the 1700 clones screened, five were pectolytic. Two of the Eca pectolytic. clones had homology to the Ecc endo-pectate lyase gene; upon examination, they proved to contain the same insert in opposite orientations. The Ecc endo-pectate lyase had a pI of 9. 5 and a molecular weight of 33,000; the analogous Eca endo-pectate lyase had a pI of 9.2 and a molecular weight of 31,000. Both enzymes required a divalent cation for activity (preferring Ca2+ over Mg2+ over Mn²⁺. The restriction endonuclease maps of the two clones did not have any tested sites in common. These differences suggest that although these two genes may have originated from a common ancestral gene, considerable divergence has taken place. I analyzed the fine structure of the Ecc endo-pectate lyase gene by DNA sequencing. The coding region of the gene is preceded by E. coli-type -10 and -35 sequences and encodes an unmodified protein of 281 amine acids. A typical secretion signal peptide is not present.
Farnesyltransferase: Gene Expression in Plants and Role in Plant Development
Zhou, Dafeng (Virginia Tech, 1997-03-14)
Protein farnesyltransferase (FTase, E. C. 2.5.1.21) post-translationally modifies regulatory proteins involved in controlling cell growth, division, and differentiation. Recently, a cDNA clone (PsFTb) encoding a pea (Pisum sativum) FTase b subunit was isolated. Initial studies led to the hypothesis that FTase plays a role in the regulation of plant cell division. To gain insight into FTase function in plants, a detailed study of the expression pattern of FTase genes was carried out. A cDNA (NgFTb) encoding the b subunit of tobacco FTase was cloned from a Nicotiana glutinosa cDNA library to initiate studies in tobacco. In tobacco BY-2 suspension culture, levels of NgFTb mRNA and FTase activity transiently increased at the early log phase of cell growth and rapidly declined before cells entered stationary phase. These data, along with inhibitor studies in the BY-2 system, support our hypothesis. To understand the expression and regulation of pea FTase subunit genes, 5'-upstream sequences of both pea FTase subunit genes (PsFTb and PsFTa) were cloned from a pea genomic library. The 5'-upstream sequence (~2 kb) of PsFTa was fused to GUS (b-glucuronidase) and GFP (green fluorescent protein) reporter genes and introduced into tobacco plants. This 2 kb upstream region appears insufficient to provide PsFTa promoter function. On the other hand, 3.2 kb of PsFTb 5'-upstream sequence expressed as a PsFTb:GUS construct is fully functional in transgenic tobacco plants. GUS expression was most prominent in actively growing cells supporting FTase involvement in plant cell cycle control. GUS activity was also found in mature and imbibed embryos but not premature embryos, consistent with the role of FTase in abscisic acid (ABA) signaling. An unexpected pattern of GUS activity, not correlated with dividing cells or ABA signaling, was also observed in the transgenic plants. GUS activity was detected in vascular bundles adjacent to actively-growing tissues and in regions that connect two organs, e.g., junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls. Auxin promotes PsFTb expression while light and sucrose inhibit expression. These spatial and temporal expression patterns strongly suggest that FTase has a broader role associated with regulation of nutrient transportation or allocation in plants.
Gene regulation in a pathogen-plant interaction: soft rot erwinias versus potato tubers
Yang, Zhenbiao (Virginia Tech, 1990)
Erwinia soft rot is a widespread disease destructive to numerous important crop plants. Damage to plants is primarily due to celldegrading enzymes (CDEs) secreted by the bacteria. I am interested in potato (Solanum tuberosum) soft rot because it is of agricultural importance and it represents an ideal model system for understanding molecular events in plant-pathogen interactions. Much has been learned in vitro about the molecular genetics of CDEs in the past decade; however, little is known about their expression in plantae To study expression of genes for these enzymes during pathogenesis and plant responses to erwinias or their enzymes, I developed a membrane-separated system for simultaneous studies of potato and bacterial gene expression. This system facilitates the isolation of plant tissue-free bacterial cells and bacteria-free plant tissue for subsequent analysis of gene expression by RNA blot hybridization. Using this system, I demonstrated that in compatible interactions, rnRNAs for three Erwinia carotovora subsp. carotovora (Ecc) CDE genes were induced to high levels and were induced sequentially: exo-pectate lyase (PL), endo-PL, and then endopolygalacturonase (PG) with maximal mRNA accumulations at 6, 9, and 12 hr, respectively. Induction of these mRNAs was well correlated with tissue maceration. In the incompatible interaction, however, induction of all three Ecc genes was reduced several-fold compared to the compatible interaction. The kinetics of mRNA accumulation during pathogenesis were distinct from those of in vitro accumulation induced by polygalacturonic acid. My results confirm that in planta expression of these genes was induced by exo-PL reaction products as suggested by other researchers. In studies of plant genes correlated with plant responses to pathogens and environmental stresses [plant defenseresponse (PDR) genes], I also showed Ecc triggered active responses distinct from wound responses. I used gene probes for phenylalanine ammonia- lyase (PAL) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), key genes in the biosynthesis of phenylpropanoid- and terpenoidderived compounds believed to be important in plant defenses. Ecc inoculation caused much more rapid and greater increases in PAL mRNA and enzyme activity levels in potato tuber than wounding alone. Escherichia coli, a non-plant pathogen, carrying a plasmid which encodes Ecc endo-PL, also induced PAL mRNA accumulation. Ecc induced a specific HMGR isogene (HMGR1) not activated by wounding. My results support the existence of an HMGR mul-ci-gene family. Wounding resulted in a rapid and transient accumulation of HMGR2 mRNA followed by a slower accumulation of HMGR3 mRNA. These isogenes are distinct from the Ecc-induced HMGRI gene.
Inheritance of reactions to gray leaf spot and maize dwarf mosaic virus in maize and their associations with physiological traits
Donahue, Patrick J. (Virginia Polytechnic Institute and State University, 1989)
Gray leaf spot, caused by Cercospora zeae-maydis, can be a yield-limiting factor in maize where continuous minimum tillage practices are followed. Commercial corn hybrids were evaluated for response to gray leaf spot for seven years at two Virginia locations (Shenandoah and Wythe Counties) and one year at a third location in Virginia (Montgomery County). Yield losses, when comparing resistant to susceptible classes, were approximately 2,000 kg ha⁻¹ at Wythe County in 1982, 750 kg ha⁻¹ at Shenandoah County in 1984, and 2,150 kg ha⁻¹ at Montgomery County in 1988. The inheritance of reaction to gray leaf spot was studied using a 14 inbred diallel in Montgomery and Wythe Counties, Virginia in 1987 and 1988 planted in randomized complete block designs. Resistance was found to be highly heritable and controlled by additive gene action. Inbreds producing high yielding, resistant, and agronomically superior hybrids were identified (B68, NC250, Pa875, Va14, Va17, and Va85); and several hybrids between these lines had high levels of resistance, high yield, and good general agronomic characters (B68 x KB1250, KB1250 x Pa875, and NC250 x Pa875). Currently available inbreds could be used to produce hybrids with higher levels of resistance than hybrids currently available to growers, and these could serve as a basis for gray leaf spot breeding programs. Lesion size measurements were not correlated with disease scores. Late-season photosynthesis rates were associated positively with resistance. The hybrids of some inbreds were found to produce high levels of pigment (believed to be anthocyanins) around the gray leaf spot lesions. These did not limit the size of the individual lesion later in the season. Some pigment(s)-producing genotypes were found to be resistant when the pigment character was expressed. This type of resistance must prevent or inhibit infection of the leaf but not later colonization, once established. Maize dwarf mosaic virus (MDMV) also limits maize production in some areas where johnsongrass (Sorghum halepense L.) is a problem. Resistance to MDMV was found to be mainly additive and highly heritable. However, a strong specific combining ability component was found, indicating that the background of the material receiving resistance genes may have a strong effect on the expression of resistance. Inbreds capable of producing high-yielding, resistant, and agronomically acceptable hybrids are available (B68, NC250, A632, Pa875, Va17, and Va85); and several hybrids between these lines have high levels of resistance, high yield, and good general agronomic characters (B68 x KB1250, KB1250 x Pa875, and NC250 x Pa875).
Insertion sequence IS1141: discovery, characterization, and association with Mycobacterium intracellulare colonial variation
Via, 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.
Isolation and characterization of rice (Oryza sativa L.) β-Glucosidases
Muslim, Choirul (Virginia Tech, 1995-04-14)
The objectives of this study) are: (1) partial purification and characterization of rice β-glucosidase, (2) determination of the physiological role of the enzyme during rice germination, and (3) histochemical localization of the enzyme. The method for partial purification of the enzyme was based on that of Schliemann (1984), which included differential solubility, cryoprecipitation, and cation exchange chromatography. The enzymes were characterized with respect to their molecular weights, pI value, pli and temperature profile of activity and stability, activity in the presence of selected denaturants and organic’ solvents, substrate specificity, and inhibition by several known β-glucosidase inhibitors. To examine the physiological role of rice β-glucosidase, histochemical localization of the enzyme in dry seeds and application of inhibitors of the enzyme to the germinating seeds were carried out. The seeds were soaked in the presence or absence of β-glucosidase inhibitors, and the number of germinating seeds, growth and development of coleoptile and roots, and enzymatic activity of β-glucosidase and α-amylase were studied. To study histochemical localization of rice β-glucosidase, the chromogenic substrates were used. The substrates were incubated with cross and longitudinal sections of whole seeds and seedlings, tissue sections, protoplast and plastid preparations from 5-6-day-old coleoptiles. The development of the colors were observed under the light microscopes. Among the cation exchange chromatography fractions, two distinct peaks of oNPGase and pNPgase activity were found: fraction-1 (Fr-1) and fraction-2 (Fr-2) forms. It was found that the two forms of rice β-glucosidase are different with respect to susceptibility to denaturation by SDS, substrate specificity and some physico-chemical properties. Fr-1 is susceptible to denaturation by SDS, and catalyzes specifically the hydrolysis of several β-galactosides (pNPGal, X-gal, and 6-BNGal) but not gentiobiose and cellobiose, and is stable over pH range (4 to 10). Fr-2, on the other hand, is more resistant to denaturing agents, catalyzes the hydrolysis of gentiobiose and cellobiose, but not any of the β-galactosides mentioned above; it is relatively stable at pH 9, and less stable at high temperatures than Fr-1. Both Fr-1 and Fr-2 are 120 kD native dimers, made up of 60 kD monomers. In rice dry seeds, β-glucosidases were distributed in the aleurone layers and embryo parts. β-glucosidase inhibitors suppressed germination at the activation stage. The inhibitors Suppressed the expression of α-amylase and β-glucosidase during germination detected at the activity level. It is proposed, therefore, that the pre-existing f-glucosidase is involved in the regulation of availability and activity of a hormone (gibberellin) at the early step of germination that controls expression of hydrolytic enzymes such as α-amylase. In mature seeds, the Fr-1 is found mainly in the scutellum region and aleurone layers, while the Fr-2 form is in the axis of the embryo. In the seedling, the Fr-1 form is found in the scutellum, shoot and coleoptile, while the Fr-2 form is in the root. In young tissue of shoot and coleoptile, the enzyme is localized in the epidermis and vascular bundles. At the subcellular level it is localized to the plastids.
Molecular cloning, characterization, and expression of 3-hydroxy-3-methylglutaryl coenzyme a reductase gene from tomato
Park, Hee-Sung (Virginia Tech, 1990)
In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme regulating biosynthesis of phytosterols, plant growth regulators, carotenoids, antimicrobial defense compounds, and numerous other isoprenoids. To initiate molecular studies of HMGR in relation to defense responses in plants, we utilized yeast HMGR cDNA sequences to isolate tomato genomic sequences encoding HMGR. The nucleic acid sequence and gene structure was determined. The tomato HMGR gene (HMG2) contains four exons separated by three introns and encodes a polypeptide of 602 amino acid residues (about 64,714 Da). Two membrane-spanning regions are contained in the NH₂-terminus of the HMGR polypeptide. The COOH-terminus shares significant homology with HMGR sequences from different species. Genomic Southern hybridization analyses reveals that tomato contains 3 to 4 HMGR genes. The HMGz2 gene cross-hybridizes to mRNA of about 2.7 kb which is highly induced in tomato cells treated with fungal elicitors and in stems, leaves, or roots stressed by wounding suggesting that the HMGz2 is a defense-related gene in tomato. Hybridization with a gene specific probe indicates that the HMG2 gene is induced specifically during defense responses and is distinct from the gene(s) expressed during fruit development and ripening.
Molecular mechanisms of pathogenesis incited by Erwinia carotovora subsp. carotovora
Roberts, Daniel Paul (Virginia Polytechnic Institute and State University, 1985)
Erwinia carotovora subsp. Carotovora (Ecc) incites soft-rot on many plants. It is believed that soft-rot is due to the concerted activity of extracellular enzymes. Recombinant DNA techniques were used to study the molecular basis of pathogenesis incited by Ecc. Specifically, a clone library of Ecc strain EC14 DNA in plasmid pBR322 was constructed and transformed into Escherichia coli strain HB101. Some of the E. coli strains that contain these hybrid plasmids produce pectinases or cellulase(s). Plasmid pDR1 contains a 3.4 kilobase (kb) EC14 DNA fragment and mediates the production of endo-pectate lyases with isoelectric points (pI) of 9.5 and 7.5 in strain HB101. The pI 9.5 enzyme is believed to be the major extracellular pectolytic enzyme in soft-rot while the pI 7.5 enzyme has no documented counterpart in EC14. Subclone and transposon tn5 analyses of pDR1 indicate that 1.5 kb is necessary for the production of the pI 9.5 and pI 7.5 enzymes and that these enzymes are produced independently of other EC14 pectate lyase enzymes. Plasmid pDR30 contains a 2.1 kb EC14 DNA insert that mediates the production of an endo-polygalacturonase and an exo-pectate lyase in HB101. The exo-pectate lyase encoded by pDR30 produces an inducer of endo-pectate lyase synthesis as a reaction product. The endo-polygalacturonase encoded by pDR30 is thought to play a role in plant cell wall pectic polymer degradation. Restriction endonuclease and Southern hybrididizatian analyses indicate that the EC14 genes on plasmids pDR1 and pDR30 are not part of the same operon. Escherichia coli strain HB101 containing plasmid pDR1 or plasmid pDR30 is unable to macerate potato tuber slices. However, HB101 containing plasmids pDR1 and pDR30 can cause limited maceration of potato tuber slices. There appears to be a genetic interaction between plasmids pDR1 and pDR30 in maceration of potato tuber tissue. However, the EC14 gene(s) contained on plasmid pDR1 are transcribed independently of the EC14 genes contained on plasmid pDR30. It is possible that transcription of certain pectolytic enzymes independent of other pectolytic enzymes provides a flexible system for plant cell wall pectic polymer degradation.
Molecular Studies on Soybean Mosaic Virus-Soybean Interations
Qusus, Saba J. (Virginia Tech, 1997-04-18)
In the U.S., soybean mosaic virus (SMV) is classified into seven strain groups, designated G1 to G7, based on their different responses on resistant soybean [Glycine max (L.) Merr.] cultivars. These responses are: symptomless or resistant (R), necrotic (N), and mosaic or susceptible (S). The gene-for-gene model has been proposed for SMV-soybean interactions. In the majority of cultivars, a single dominant gene, Rsv1, confers both the R and N responses. In the first part of this study, the coat protein (CP) genes of two SMV strains, G1 and G6 were isolated, cloned, and sequenced. Gene isolation was done by reverse transcription-polymerase chain reaction (RT-PCR) on partially purified virus preparation without prior RNA extraction. Amplified products were blunt-end ligated into pNoTA/T7 vector and transformed into competent cells. Sequencing was performed in both directions on heat-denatured double-stranded plasmids. The predicted 265 amino acid sequence of the CP of G1 and G6 strains were 98.9% identical, with only two amino acid differences. Correlating the CP sequences of G1, G2, G6, and G7, with their virulence on resistant soybean cultivars indicated that the CP is not likely to be the R- and/or N-determinant in the SMV-soybean system. The second part of the study involved studying the pathogenesis of G1, G6, and G7 strains on inoculated leaves of R, N, and S soybean cultivars by leaf imprint immunoassay. Results indicated four types of reactions: i) susceptible, showing unrestricted replication and spread; ii) immune, where no virus was detected; iii) systemic spread, showing unrestricted replication but limited spread along the veins; and iv) restricted replication and spread, where infection was restricted to few foci along the veins. Results of this study indicated that Rsv1-mediated resistance is a multicomponent type of resistance that involves both inhibition of virus replication as well as cell-to-cell movement. The third part of the study aimed at investigating Rsv1-mediated resistance at the cellular level. For this purpose, an SMV-soybean protoplast system was developed. Protoplast isolation was based on a combined cellulase-pectolyase Y-23 digestion and metrizamide-sorbitol gradient purification protocol. Virus inoculation of protoplasts was facilitated by either polyethelene glycol (PEG) or poly-L-ornithine (PLO), and method of detection was by Western blotting using antiserum to whole virus. Inoculation by PEG was successful, but results were irreproducible because of the adverse effect of PEG on protoplast viability. Inoculation by PLO was inconclusive because of the high background from residual inoculum. Additional research is needed before a protoplast system can be used to study the mechanism of Rsv1 resistance to SMV at the cellular level.
PCR-Based Test for Differentiating Varieties of Gaeumannomyces graminis, The Take-All Pathogens
Rachdawong, Sansanalak (Virginia Tech, 1999-11-19)
Take-all is the most devastating root disease of wheat worldwide. The causal agent is Gaeumannomyces graminis (Sacc.) Arx & Olivier. Based on morphological characteristics and host ranges, three varieties of G. graminis have been recognized. G. graminis var. tritici Walker (Ggt) is the major causal agent of take-all of wheat and barley and the most economically important take-all pathogen. G. graminis var. avenae (Turner) Dennis (Gga) attack oats and causes take-all patch of turf grasses while G. graminis var. graminis (Ggg) is pathogenic on turf grasses but is non-pathogenic on wheat. Conventional diagnosis of take-all pathogens is based on field symptoms such as blackened roots, stunted growth, and white-heads and morphological characteristics such as hyphopodia type, size of perithecia, asci, and ascospores. These procedures are time-consuming, laborious, and often inconclusive. The objective of this study was to develop a rapid, simple, and specific method for differentiation of G. graminis varieties using PCR and molecular-based technology. Exploitation of genes associated with pathogenicity of G. graminis as markers for the test was proposed. Metabolic activities of G. graminis associated with pathogenesis were investigated, namely, the abilities to produce avenacinase and to oxidize manganese. Avenacinase, an avenacin detoxifying enzyme, was associated with Gga pathogenicity for oats but this enzyme is not important in Ggt pathogenicity for wheat. Manganese oxidation was also correlated with Ggt virulence. In this study, avenacinase-like genes were discovered in Ggt and Ggg and manganese oxidation was confirmed for Ggt, Gga, and Ggg. All isolates of Ggt except isolate ATCC 28230 were manganese oxidizers. Ggg and Gga isolates could oxidize manganese but their precipitation patterns were not as intense or closely correlated with mycelial growth as for Ggt. Pathogenicity assays on oats for Ggt, Gga, and Ggg isolates confirmed that Ggt isolates could not cause disease on oats aside from occasional slight root damage. Root weight was reduced for oat seedlings inoculated with Gga isolates. Comparison of partial sequences of avenacinase-like genes from Ggt and Ggg showed strong homology to that of Gga (94.8% identity to Ggt and 94.6% identity to Ggg). However, the Ggt gene was more closely related to that of Ggg (99.2% identity) than to Gga. DNA restriction endonuclease polymorphisms of the genes supported DNA sequencing information and revealed that there were variations within the genes among Ggt, Gga, and Ggg. Variety-specific electrophoretic patterns were obtained when the genes were digested with HaeIII. Ggt, Gga, and Ggg upstream (5') variety-specific primers and a downstream (3') universal primer were designed from the avenacinase and avenacinase-like DNA sequences. PCR amplification with Ggt-, Gga-, and Ggg-specific primers generated fragments of 870, 617, and 1,086 bp, respectively. Each 5'-specific primer showed high specificity for its own DNA template in mixed populations of DNA templates. The optimized PCR procedure was sensitive to DNA template concentration as low as 100 pg. Genomic DNA of sixteen Ggt isolates, seven Gga isolates, and five Ggg isolates were tested. Although all Ggt isolates were originally isolated from wheat, seven isolates produced Ggg-specific fragments. This result corresponded well with HaeIII DNA polymorphisms, pathogenicity assay, and manganese oxidizing ability. All but one Gga isolates produced the variety-specific fragment. Ggt- and Gga- specific products were generated from Gga isolate RB-W. Although Ggg-specific fragments were produced from all Ggg isolates, non-specific products were also observed from isolates that were not from wheat origin suggesting some genetic variations due to host ranges. Additionally, no non-specific amplification was obtained from any closely related fungi such as Gaeumannomyces cylindrosporus or Phialophora spp. The test developed in this study is the first test capable of identification of Ggt, Gga, and Ggg in a single PCR tube with a basic PCR protocol. The test is rapid and specific. Interpretation of results is simple and conclusive based on differences in size of each variety-specific fragment.
Relationship of plasmids in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum
Jucker, Markus Thomas (Virginia Tech, 1991-12-05)
Bacteria of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum group (MAIS) are opportunistic human pathogens and widespread in the environment. The first objective of this study was to demonstrate that plasmids from clinical isolates are closely related to plasmids from environmental MAIS isolates. A 12.9 kb plasmid, pVT2, from a clinical M. avium isolate, MDl, was cloned and used a a DNA probe to examine the relationship of MAIS plasmids. The pVT2 probe hybridized with plasmids isolated from MAIS strains from the environment, from patients without AIDS with pulmonary infections, and from AIDS patients with disseminated MAIS infections. Similar results were seen with a second probe derived from pLR 7, a 15.3 kb plasmid from clinical M. intracellulare strain LR 113. The similarity of plasmids from environmental and clinical isolates supports the hypothesis that the environment is a source of MAIS infection in humans.

Browsing by Author "Lacy, George H."

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