The 7S globulins are a subclass of seed storage proteins characterized by their solubility in saline solution. These proteins are major legume storage reserves, and have been studied extensively; in the cereals, they represent a minor seed protein fraction and have been characterized to a lesser extent. Recently, a study associating a wheat 7S globulin, WP5212, with the development of type 1 diabetes in diabetes-prone rats, has renewed interest in this protein class. The present study aimed to better characterize the 7S globulins of wheat. The majority of the wheat embryo globulins were detected by anti-WP5212 polyclonal serum. These proteins varied significantly in their molecular masses and isoelectric points. Six major polypeptides were identified by mass spectrometry and/or N-terminal sequencing as belonging to the globulin 1 family. These results combined with prior studies have allowed the construction of a hypothetical model of the post-translational events contributing to the wheat 7S globulin profile in mature kernels.

Plant mitochondria possess a gene expression system in which post-transcriptional events, such as transcript end maturation and turnover mechanisms play a key role in regulating the transcriptome. In addition, during early developmental stages of embryo germination, differing transcript profiles have been seen. This research focuses on two loci in wheat mitochondria, ccmFn-rps1 and rpl5-Psirps14-cox3, to elucidate the transcription and post-transcriptional events involved in their expression. Northern analysis of the ccmFN-rps1 genes during early seed-to-seedling development reveals a 3.2 kb primary transcript and a 2.7 kb bicistronic mRNA. A 0.7 kb monocistronic rps1 mRNA is detectable up to 2d but there is no detectable monocistronic ccmFN transcript during the stages examined. Transcript ends were mapped using circular-RT-PCR and phosphatase treatment at three different developmental stages and revealed two processing sites as well as a single 3' end common to all three transcripts. The 5' ends of the processed rps1 transcripts are heterogeneous and do not always include the start codon, questioning the rps1 transcript functionality. Gene order varies between plant species due to the high recombination rate in mitochondrial genomes, as is seen for rpl5-Psirps14 in wheat and rice. In both plants, the functional rps14 gene is encoded in the nucleus and the mitochondrial rps14 copy is a pseudogene. In wheat, rpl5-Psirps14 are co-transcribed with cox3 as two RNA species of 3.5 kb and 2.7 kb at 24hr post-imbibition and exhibit developmentally-specific differences in abundance in seedlings. Two promoter regions were mapped in wheat upstream of rpl5 and both transcripts have the same 3' end. In rice 24hr and 6d however, rpl5-Psirps14 are co-transcribed as a 1.4 kb bicistronic mRNA. This presumably reflects the different regulatory signals used in different species. In addition, rpl5 has been subject to several independent gene transfers to the nucleus in the cereal lineages. For example, there is a functional copy of rpl5 in the mitochondria and the nucleus in wheat but it is absent from the mitochondria in rye and maize. In oat mitochondria, rpl5 appears to be a pseudogene and in barley, rearrangements at the 3' end and low transcript levels question its functionality. The characterization of transcription initiation sites, processing sites and 3' ends for these two loci reflect the relaxed nature and flexibility of signals exploited by plant mitochondria. This research supports the significant role of post-transcriptional events in the regulation of gene expression in plant mitochondria.

Doctor of Philosophy / Genetics Interdepartmental Program / Scot H. Hulbert / Frank F. White / Many maize lines carry Rxo1, an NB-LRR gene that confers a rapid hypersensitive response (HR) after infiltration with the rice streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) or the maize stripe pathogen Burkholderia andropogonis (Ba) carrying the effector genes avrRxo1 or avrRba1 respectively. Interestingly, when expressed as a transgene in rice, Rxo1 also confers a strong and rapid HR to Xoc strains harboring the avrRxo1 type III effector gene. To gain insights into the Rxo1 signaling network, we used a combination of functional genomics and bioinformatics, molecular genetics and reverse genetics. Microarray experiments were carried out to investigate the temporal expression profiles of nonhost and host responses to isogenic strains of Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen, and Ba with and without the Xoc type III secreted effector gene avrRxo1. Xoc AvrRxo1 induces disease resistance in maize when delivered by Xoo or Ba. We show that recognition of the two bacterial pathogens is translated into similar transcriptional outputs. Cluster analyses revealed that Xoo and Ba co-regulated genes display different kinetics and amplitudes and showed that gene clusters are associated with overrepresentation of known and putative novel DNA cis regulatory elements. One early induced gene, ZmPti1b, is a serine threonine kinase. RNAi-mediated gene silencing of a rice ortholog of ZmPti1b, OsPti1a, revealed that OsPti1a is required for Rxo1-governed resistance. Using a full length coding sequence as bait to screen a yeast-two-hybrid library, we identified 11 rice proteins that interact with RXO1. Functional analysis of two showed that Os1PVOZ, encoding a putative transcription factor, is required for Rxo1-dependent HR whereas OsATL6, a putative RING finger type E3 ubiquitin ligase gene is dispensable. Scanning of the rice genome for putative DNA binding sites suggests that Os1PVOZ is a master regulator of many signal transduction pathways, including those that mediate plant defense responses. Our investigations identified key signaling components that mediate Rxo1-specified resistance and possibly resistance mediated by other R genes.

Perenniporia phloiophila (Aphyllophorales: Polyporaceae) colonizes the bark of live oak (Quercus virginiana Mill.) and is known only in the southeastern United States in this host. Cultural characteristics and mating systems of P. phloiophila and P. medulla-panis, vegetative incompatibility of P. phloiophila and temperature relationships and decay capacities of vegetative isolates of P. phloiophila, P. ohiensis and P. fraxinophila were investigated. Cultural studies indicate macroscopic and microscopic differences between the four species. Antagonistic hyphal interactions developed between different vegetative isolates. Self crosses were compatible. Optimum temperature ranges and maximum growth temperature differed for all species. Mating test results of both species indicate their heterothallic tetrapolar nature. Woods differed significantly in percent weight loss (PWL) caused by each Perenniporia species. No significant difference occurred between different isolates of the same species tested on the same wood. PWL was greatest on oak wood for all fungal species tested.

Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Both qualitative and quantitative resistance mechanisms are important contributors to disease resistance in rice. To identify useful sources of durable resistance for Korean breeding programs, the distribution of rice blast isolates compatible to widely used resistance (R) genes was analyzed. Interactions of 3,747 Korean rice Magnaporthe oryzae isolates with eight monogenic lines, each harboring a major blast R gene, were tested. Lines with R gene Pi-9 and Pi-5 were susceptible to the fewest M. oryzae isolates, and therefore, this gene might be applied for blast resistance in breeding programs in Korea. Six major blast resistance genes were susceptible to more than 60 % of the population, suggesting limited utility of these genes in breeding programs. Quantitative trait loci (QTL)-based resistance is predicted to provide durable and broad spectrum resistance to rice diseases. A candidate gene approach was applied to a population of 164 recombinant inbred lines to identify sources of quantitative resistance. Resistance gene analogs and defense response genes were mapped on the rice chromosomes, and analyzed for their association with blast and bacterial blight resistance QTL. A total of 21 putative QTL for blast resistance were identified on chromosomes 1, 4, 5, 6, 8, 9 and 12. Four putative QTL for bacterial blight resistance were identified on chromosome 3, 5 and 10. Thirteen RGA markers were associated with 11 different QTL on chromosome 1, 5, 8, and 9. The role of one disease resistance QTL associated gene, Os02g39330, encoding a chitinase was investigated for contributing to basal defense responses. RNAi silencing was used to evaluate contributions of the gene for the resistance to Rhizoctonia solani and M. oryzae. Five transgenic lines harboring the silencing construct and which differed in the level of expression of Os02g39330 were screened for responses to R. solani and M. oryzae. The chitinase gene expression levels were inversely correlated with sheath blight disease severity, suggesting a role for this defense gene in resistance to R. solani. Rice blast disease was not affected by silencing Os02g39330. Both qualitative and QTL-based resistances provide valuable sources of disease resistance, and a combination of R gene Pi-9 and QTL harboring the Os02g39330 chitinase may help to stabilize resistance.

Pseudomonas aureofaciens 30-84, a biological control bacterium for the soil-borne disease take-all of wheat, is a model system for biological control of root-infecting fungal pathogens. Strain 30-84 inhibits the causal agent of take-all, Gaeumannomyces graminis var. tritici, primarily through the production of phenazine antibiotics, which are important for survival of the bacterium in the rhizosphere. Prior to this work, phenazine production was shown to be regulated by an N-acyl-homoserine lactone (AHL) response system encoded by phzI and phzR. This work identified a second regulatory system involved in the phenazine regulatory cascade. The two-component regulatory system involving the GacS/GacA proteins regulates the production of phenazines, extracellular protease, hydrogen cyanide and fluorescent siderophores. GacS/GacA regulates the production of phenazines at multiple levels. They control the production of the AHL signal required for expression of the phenazine biosynthetic operon by tightly regulating transcription of phzI. This was the first report of a linkage between a two-component regulatory system and an AHL response system. GacS/GacA also control phenazine production through a second mechanism. Preliminary evidence suggests translational regulation of one or more genes involved in the phenazine regulatory cascade through transcriptional control of a regulatory RNA (rsmB RNA) required to neutralize the negative effects of the translational repressor RsmA. Another aspect of this work was the analysis of the formation and rhizosphere competence of spontaneous gacS and gacA mutants of strain 30-84. These are commonly isolated from laboratory cultures of all biocontrol bacteria and could pose a threat to the efficacy of biological control if they arise in the rhizosphere and displace the phenazine-producing wild type strain 30-84. This work indicated that the mutants did arise on wheat roots and did displace strain 30-84 on roots in sterile soil. However, the mutants did not displace strain 30-84 on roots in natural soil. In fact, the wild type strain 30-84 appeared to compete more favorably with indigenous microorganisms in the presence of a subpopulation of GacS/GacA mutants. Therefore, the results presented here indicate that a subpopulation of gacS and gacA mutants is a normal and beneficial part of the P. aureofaciens community in the rhizosphere.

Biological nitrogen fixation (BNF) is the nitrogenase-catalyzed conversion of dinitrogen to ammonia by a select group of Bacteria and Archaea called diazotrophs. In turn, plants and other microbes assimilate ammonia during the synthesis of nucleic acids, proteins and other biomolecules. BNF is of special interest in agriculture where it replenishes soil nitrogen lost during repetitive farming. Basic knowledge of BNF might eventually lead to less dependence on expensive and polluting chemical fertilizers. For the studies presented here, two model diazotrophs, the free-living Azotobacter vinelandii , and the alfafa symbiont, Sinorhizobium meliloti, were used to investigate mechanisms controlling nitrogen fixation and nitrogen metabolism. In A. vinelandii, ammonia inhibits nitrogenase expression by limiting activity of the two-component activator, NifA; this involves the negatively acting sensor protein, NifL. Groundwork indicated that a global nitrogen-sensing system, present in many bacteria might control NifA activity since glnD mutants were unable to fix nitrogen. In other organisms, nitrogen limitation signals GlnD-mediated uridylylation of PII-like signal transduction proteins, which signals activation of a suite of genes involved in nitrogen source utilization. The goals of the current study were to characterize the operon encoding a PII-like protein in A. vinelandii, named GlnK, and determine its influence on NifA and nitrogen metabolism. The results indicated that glnK is an essential gene and that uridylylation of GlnK is required for activation of glutamine synthetase and NifA. Also presented here is evidence that GlnK interacts with NifL to stimulate its inhibitory properties. These results are consistent with a model in which uridylylation of GlnK in response to nitrogen limitation signals relief of NifL inhibition. In the last section of this dissertation, glnD of Sinorhizobium meliloti was cloned and sequenced because a PII-like protein had been previously implicated in control of nodule development and symbiosis. Unfortunately, S. meliloti glnD mutants could not be isolated unless glnD and flanking genes were provided in trans, indicating that the glnD operon is indispensable. These studies provide new insight into the global mechanisms controlling nitrogen fixation and metabolism and suggest that GlnD and PII-like proteins may regulate other targets, some of which are essential.

In April 1989, lettuce (Lactuca sativa L.) plants showing severe root rot symptoms were received for diagnosis from a commercial hydroponic facility in Southern California. A species of Phytophthora was consistently isolated from necrotic roots. Pathogenicity trials were conducted under hydroponic conditions in a greenhouse at root temperatures of 18 and 28 C. Root necrosis, along with stem decay and plant death, occurred within 5-7 days after inoculation at both temperatures. Reisolation of the fungus from roots and stems of inoculated, but not from roots of non-inoculated plants, confirmed pathogenicity. The fungus was identified as Phytophthora cryptogea Pethybr. and Lafferty on the basis of cultural and morphological characteristics. However, crosses with A1 and A2 mating types of Phytophthora cryptogea and other species of Phytophthora were not successful. This is the first report of Phytophthora as a root pathogen of cultivated lettuce.

For chemigation of nonsoluble pesticides, small oil-pesticides droplets (dmax tend to wash-off from foliage while large droplets tend to stick. Large droplets (dmax are buoyant, tend to rise in the irrigation pipeline and exit at the beginning of the pipeline; thus, uniformity and efficacy are poor. For this research, a new chemigation system was proposed. The system removes water from the irrigation pipeline, injects the oil-pesticide into the water stream, increases dispersion velocity in successively smaller tubing diameters, and finally injects the dispersion back into the irrigation pipeline. The higher velocity flow with high turbulent shear forces breaks the oil-pesticide into desired size droplets. Droplet break-up research was reviewed, and a model developed to predict maximum droplet size and size distribution. A maximum relative error of 40% was observed when dmax predicted by the model was compared against literature data. Equations to predict friction factor in helically coiled pipes and effective viscosity of oil-in-water dispersions were evaluated. The friction factor predicted by the Ito equation was in good agreement with the experimental data. Effective viscosity of soybean oil- and kerosene-in-water dispersions was predicted satisfactorily by the Richardson equation with k₄ = 2.5. Finally, center pivot field experiments were conducted using the new and conventional chemigation systems. For the conventional system, the soybean oil uniformity coefficient along the lateral was 61%, and oil applied over the last tenth of irrigated area was 9% of the initial concentration. For the new system, the uniformity coefficient was 73% and 98% for dmax of 875 mum and 98 mum, respectively; oil applied over the last tenth of the area was 27% and 90% of the initial concentration. Field data were compared with those predicted from a pipeline transport model for nonsoluble pesticides. Agreement between the model and the field data was excellent for both experiments using the new chemigation system. Based on the field results and simulation analyses, droplets < 150 μm should be desirable to keep the discharge uniformity coefficient over 97%, for 0.92 ≤ ρ(d)/ρ(c) ≤ 1.04.

The biological and molecular properties of Sinaloa tomato leaf curl virus (STLCV) were investigated to test the hypothesis that STLCV is a previously uncharacterized whitefly-transmitted geminivirus from North America. STLCV causes leaf curling and yellowing in tomato plants. STLCV was transmissible to N. benthamiana by sap inoculation, and to Solanaceous and Malvaceous species by the whitefly vector. STLCV has transmission characteristics like other persistent viruses, and was not transovarially passaged. PCR fragments containing the large intergenic region (IR) of the STLCV A and B components and coat protein gene (AR1) were cloned from STLCV-infected tomato, and their DNA sequences obtained. Regions 174 nt in length containing diagnostic sequences present in the IR of geminiviruses, and a putative ORF AR1 of 756 nt were identified. A and B component IR sequences were 97.9% identical, suggesting a homogeneous, bipartite viral quasi-species. Pairwise alignment (Wilbur-Lipman) of STLCV AR1 and those of subgroups I, II, and III geminiviruses indicated 22-81% similarity, whereas STLCV AR1 was 36-61% similar to subgroup III viruses, collectively, suggesting STLCV is a unique viral quasi-species (>90% = same virus). Multiple sequence alignment (Clustal) and parsimony analysis (PAUP) of IR or AR1 sequences supported placement of STLCV with Western Hemisphere subgroup III viruses. Both A and B types of the whitefly vector transmitted tomato yellow leaf curl (TYLCV-Th) and chino del tomate (CdTV) geminiviruses, and transmission frequencies increased with greater AAPs. TYLCV-Th was transmitted by both vectors at a higher frequency than was CdTV. The B type, indigenous to the Eastern Hemisphere, transmitted the Old World TYLCV-Th (87%) more effectively than the New World A type vector (63%). The Western Hemisphere CdTV, was transmitted more often by the A type whitefly (50%), also from the New World, than by B type (27%). PCR detection of geminiviruses in single whiteflies indicated virus ingestion occurred after a 0.5 h AAP. Detection frequencies increased in both whiteflies given longer AAPs (0.5-72 h), irrespective of virus tested. PCR primers were designed that effectively discriminate between Old and New World geminiviruses, and between monopartite and bipartite genomic organizations.