Browsing by Author "Gary Payne, Committee Member"

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The Development of Analytical Strategies for the Quantitative Proteomic Analysis of Biological Systems Impacting Agriculture and Human Health.
(2010-10-22) Collier, Timothy; David Muddiman, Committee Chair; Gary Payne, Committee Member; Edmond Bowden, Committee Member; Morteza Khaledi, Committee Member
Evolutionary History and Population Dynamics of the M2 Double-stranded RNA of Rhizoctonia solani
(2008-04-26) Charlton, Nikki De-Roi; Marc Cubeta, Committee Chair; David Shew, Committee Co-Chair; Mike Benson, Committee Member; Gary Payne, Committee Member
M2 is a 3.57 kb double-stranded RNA (dsRNA) mycovirus first identified and sequenced from the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3). This dsRNA has been hypothesized to regulate metabolic pathways associated with the parasitic and saprobic activity of the fungus. Isolates from a well-characterized field population of R. solani AG- 3 obtained from potato were examined for the M2 dsRNA. The detection frequency was 51.3%. The M2 dsRNA was isolated from 59 isolates and reverse-transcription PCR products were obtained for three genomic regions of the M2 dsRNA to examine genetic diversity, reconstruct their evolutionary history, and identify distinct evolutionary lineages. Site compatibility analysis performed using SNAP Workbench for clades in the M2 dsRNA strict consensus tree identified recombination events among clades. Recombination limited the genealogical analyses to specific haplotypes with non-recombining partitions in each data set. The reconstructed genealogies indicated differential selection pressures among the genome. Analysis of the ratio of nonsynonymous substitutions to synonymous substitutions concordantly revealed that regions within the genome are undergoing differential selection. Isolates from closely related fungi in the Rhizoctonia species complex were examined for the M2 dsRNA. M2 was detected in representative isolates belonging to three AG of R. solani (AG-1-IA, AG-4, and AG-6; teleomorph=Thanatephorus) and four AG of binucleate Rhizoctonia (AG-A, AG-F, AG-R, and AG-U; teleomorph=Ceratobasidium) using RT-PCR. Amplified PCR products from a representative sample of 12 isolates from eight different AG were sequenced and subjected to phylogenetic analysis and coalescent simulations to infer the ancestral lineage and determine the genetic relationship among these dsRNAs. Phylogenetic analysis of M2 dsRNA sequence data resulted in seven inferred haplotypes and there was no unique association with AG to support co-evolution of the M2 dsRNA haplotype within the fungal host. Based on coalescent analyses and the inferred genealogy, the ancestral M2 dsRNA haplotype likely evolved in R. solani AG-1-IA and has recently been acquired by isolates of Ceratobasidium. To our knowledge, this is the first report of a dsRNA occurring in isolates of binucleate Rhizoctonia that are also present in isolates of R. solani. Horizontal transmission of the M2 dsRNA between mycelia of somatically incompatible isolates of R. solani AG-3 was investigated. Eight donor isolates of R. solani AG-3 containing the M2 dsRNA were paired on potato dextrose agar with each of three different recipient isolates where the M2 dsRNA was absent. RT-PCR was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3-cm from the hyphal interaction zone. PCR-RFLP genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solani AG-3. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates ranged from 0-33% and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge, this represents the first demonstration of transmission of dsRNA between genetically different individuals of R. solani confirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solani AG-3, but that maintenance of the dsRNA in the recipient isolates was not stable following repeated subculturing on nutrient medium.
The Function and Regulation of B6 Biosynthetic Genes by Oxidative Stress Conditions
(2005-02-02) Denslow, Sheri Ann; Jose Alonso, Committee Member; Margaret E. Daub, Committee Co-Chair; Arthur Weissinger, Committee Co-Chair; Gary Payne, Committee Member
Vitamin B6 has consistently been associated in diverse organisms with conditions of cellular oxidative stress. Plants contain an alternate, and previously uncharacterized pathway for vitamin B6 biosynthesis, distinct from the well characterized pathway in Escherichia coli. The specific objectives of this work were to further characterize vitamin B6 biosynthetic genes in tobacco and Arabidopsis, assess the regulation of B6 biosynthetic genes during biotic and abiotic stress responses, and determine the antioxidant abilities of B6 vitamers. Two genes unique to the alternative pathway, PDX1 and PDX2, have been described. PDX2 has previously been shown to encode a glutaminase. Complementation studies with E. coli pdxA and pdxJ mutants are consistent with the hypothesis that PDX1 is responsible for synthesis of the pyridoxine ring. PDX1 and PDX2 genes were isolated and characterized from tobacco and Arabidopsis. Quantitative RT-PCR demonstrated that, in Arabidopsis, PDX1 (3 copies) and PDX2 are differentially regulated in response to high light, chilling, drought, and ozone. In tobacco, PDX1 and PDX2 transcript levels decreased following inoculation with the avirulent pathogen Pseudomonas syringae pv. phaseolicola and transiently increased in response to salicylic acid and methyl jasmonate. Excess vitamin B6 in tobacco leaves interfered with the development of a hypersensitive defense response and increased disease severity caused by P. syringae pv. tabaci. B6 vitamers quenched superoxide and prevented lipid peroxidation in in vitro assays. Our findings indicate that B6 vitamers have antioxidant capabilities in chemical assays and in planta, and that synthesis of vitamin B6 is regulated during plant defense responses in a manner consistent with this vitamin's activity as an antioxidant and modulator of active oxygen species in vivo.
Identification of Quantitative Trait Loci (QTL) for Gray Leaf Spot Resistance, Maturity, and Grain Yield in a Semi-tropical Recombinant Inbred Population of Maize.
(2005-04-12) Jines, Michael P.; Jim Holland, Committee Member; Gary Payne, Committee Member; Major Goodman, Committee Chair
Identification of QTL can aide in future breeding objectives by allowing breeders either to improve a line through targeted introgressions or assist in forward breeding strategies. Such analyses may be particularly helpful in integrating exotic germplasm into a breeding program. The percentage of tropical maize germplasm grown in U.S. farmers' fields is almost nonexistent. Tropical germplasm in maize (Zea mays L.) is a valuable resource to decrease the dependence upon a limited genetic base currently used to produce commercial hybrids, extend selection limits for grain yield, and to provide an insurance function against emerging biotic and abiotic stresses. Results of research presented in this dissertation support these recommendations. Experiments were conducted to evaluate 143 S4:5 recombinant inbred lines (RILs) resulting from a cross between NC300, an all-tropical, temperate adapted line, and B104, a stiff stalk line. The 143 RILs were topcrossed to the Lancaster tester FR615xFR697 and randomly subdivided into two sets. The two sets were evaluated for resistance to GLS disease and yielding ability in three and eight North Carolina environments, respectively. Spatial trends were examined in the GLS trials. Significant (P ≤0.01) trend effects were fitted in five of the six set-by-environment combinations, which led to improved analyses within and across environments for both sets. Ninety-three and eighty-two percent of the RILs in topcrosses (RILT) were significantly (P = 0.05) more resistant to GLS when compared to the mean of the commercial checks for set 1 and 2, respectively. Twenty-one RILs from both sets did not differ significantly (P = 0.05) for grain yield when compared to the mean of the commercial checks. RIL 2070 yielded significantly (P = 0.05) higher when compared to one commercial check, HC33.TR7322. RIL 1991 was rated the most resistant entry in set 1 and also did not differ from the mean of the commercial checks for grain yield. The RILs were genotyped at 94 simple sequence repeat loci, and a linkage map was constructed that included nine chromosomes. Composite interval mapping was used to map QTL for GLS resistance, days to fifty-percent pollen shed (DTP), and grain yield. QTL associated with GLS rating were identified at individual environments on chromosomes 1, 4, and 8. One QTL located on chromosome 4 was associated with GLS resistance from the combined analysis across environments and explained 24.5% of the phenotypic variation on an entry-mean basis. A multi-locus model was constructed that involved four marker main effects and a significant epistatic interaction that together accounted for 35.0% of the phenotypic variation. Associations between GLS and maturity have been observed previously. The genetic correlation between GLS rating and DTP in this population was 0.46. A genomic region significantly associated with DTP corresponded to a region associated with GLS resistance on chromosome 8. Further, linked GLS-rating and DTP QTLs occurred together on chromosomes 1, 4, and 8. QTL associated with grain yield were identified on chromosomes 1, 3, 7, and 9. In the combined analysis across the 2003 environments the NC300 allele had an increasing effect of 0.15 t ha-1 for the QTL on chromosome 1 and explained 11.0% of the phenotypic variation on an entry-mean basis. The QTL detected on chromosome 3 was from the combined analysis across all environments. The B104 allele had an increasing effect of 0.20 t ha-1 on chromosome 3 and explained 31.0% of the phenotypic variation. B104 also had alleles associated with an increasing effect for other grain yield QTLs identified on chromosomes 7 and 9. These experiments collectively demonstrated the potential that tropical maize germplasm has to improve grain yield and resistance to GLS via conventional or marker-assisted selection strategies.
Insight into Filamentous Fungal Secretion and Evolution through Genomic Analysis
(2005-12-08) Diener, Stephen Ericson; Gary Payne, Committee Member; Ignazio Carbone, Committee Member; Ralph Dean, Committee Chair; Jeffrey Thorne, Committee Member
Filamentous fungi are of broad economic importance due to their roles in industry, medicine and agriculture. There are several filamentous fungi, such as Trichoderma reesei, which have been harnessed as protein factories due to their immense secretion capacity. Unfortunately, their full potential cannot be exploited due to an incomplete understanding of the pathways and genes involved in the fungal secretion system. Through the development of bioinformatic tools and the use of genomic technologies including expressed sequence tags and bacterial artificial chromosomes, the genome of T. reesei has been partially characterized and a number of genes involved in the secretion system have been identified. Pathogenic fungi, such as Magnaporthe grisea, are of great economic importance due to their devastating effect on agriculture. M. grisea is responsible for the loss of incredible amounts of rice crop yearly and has recently had its genome completely sequenced and annotated. The genome sequence has revealed the set of transposable elements in M. grisea which have then been analyzed using gene genealogies and the coalescent. The genealogies and coalescent have revealed that all elements analyzed showed a rapid expansion at some point in the past. This can be explained as a genomic event leading to the acceptance of transposable element activity most likely caused by the loss of genomic defense mechanisms. As a pathogen, the ability to evolve quickly in the face of plant defense mechanisms is essential. Transposable element activity can provide means for rapid evolution and this acceptance may represent a shift of these elements from genomic parasitism to mutualism.
Modified Nanodiamonds for Detoxification.
(2010-10-27) Gibson, Natalie; Tzy-Jiun Luo, Committee Chair; Donald Brenner, Committee Chair; Yaroslava Yingling, Committee Member; Gary Payne, Committee Member
Physiological and Molecular Analysis of Nitric Oxide Synthase During Bacterial Infection of Pea (Pisum sativum L.)
(2005-05-21) Wong, Mui-Yun; Eric Davis, Committee Co-Chair; Margaret Daub, Committee Member; Gary Payne, Committee Member; Jeng-Sheng Huang, Committee Co-Chair
Nitric oxide (NO) and reactive oxygen species are two key components in the induction of the hypersensitive response (HR) during plant defense against pathogen infection. In animal cells, the production of NO is catalyzed by nitric oxide synthase (NOS). Although NOS activity has been documented in plants, the process of NO synthesis in plants is not well understood. Isolation of the NOS protein and/or cloning of the corresponding gene will greatly facilitate the understanding of NO synthesis and its role in plant defense. The objectives of this research were to analyze the physiological and biochemical properties of a NOS-like protein (peaNOS) of pea (Pisum sativum L.), to purify and characterize peaNOS, and to clone the gene(s) encoding peaNOS and relate its expression to NOS activity in pea-bacteria interactions. The application of abiotic agents that induce systemic defense in plants [copper chloride, Actiguard®, Triton-X100 and salicylic acid (SA)] to pea leaves did not induce NOS activity and verified reports that NO and NOS function upstream of SA in the signaling pathway of defense responses. Maximum (two-fold) NOS activity was detected three hours before the onset of HR in pea leaves infiltrated with incompatible bacteria (Ralstonia solanacearum), which is consistent with the effect of NO in the activation of HR after interaction with H2O2. The compatible bacteria (Pseudomonas syringae pv pisi) induced NOS activity significantly, suggesting that NO generation may also be a general response to biotic stress in plants. Antibodies raised against mammalian NOS did not have apparent specificity and utility for isolating peaNOS and should be used with caution in non-mammalian systems. The peaNOS protein was most efficiently extracted under alkaline conditions (pH 8.5 and 9.0) as compared to the neutral conditions (pH 7.4-7.5) in animal systems. Precipitation of the peaNOS protein with various concentrations of ammonium sulfate, sodium citrate and sodium chloride caused rapid loss of NOS activity. The peaNOS protein did not bind to 2',5'-ADP-Sepharose and calmodulin (CaM)-agarose indicating that the protein lacks binding sites for NADPH and CaM. Cloning of a peaNOS gene based on mammalian NOS was unsuccessful suggesting that the structure of peaNOS gene may be significantly different from mammalian NOS. Analysis of the Arabidopsis thaliana genome database identified two gene sequences related to animal NOS, i.e., accessions At4g09680 (similar to NOS of Rattus norvegicus) and At3g47450 (similar to NOS of Helix pomatia). Gene At4g09680 is probably not expressed since attempts to clone cDNA of this gene using reverse transcription-polymerase chain reaction (RT-PCR) consistently failed, even when RNA of Arabidopsis was used as a template. A potential expressed peaNOS gene was successfully cloned using RNA template of pea HR tissues in RT-PCR. The 784-bp peaNOS cDNA sequence had 50% nucleotide identity to the At3g47450 coding sequence and had no other significant match in the database. The correlation of the gene expression of P protein of glycine decarboxylase complex (GDC) of pea (peaP) and NOS activity during HR in pea was not demonstrated here but peaP gene was highly expressed concomitant with NOS activity during disease development. The NOS-like protein involved in NO production during HR in pea appears to be more related to At3g47450 sequence, and is possibly encoded partially by the cloned 784-bp pea cDNA.