Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which are responsible for both suppression of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici and enhanced survival of 30-84 within the wheat rhizosphere in competition with other organisms. A gene (phzR) was identified just prior to the start of this work that is required for phenazine production by 30-84. PhzR was identified as a positive regulator of the phenazine biosynthetic operon. During the course of this dissertation it was discovered that PhzR belongs to the LuxR family of N-acyl- scL-homoserine lactone-responsive transcriptional regulators and that phenazine production in P. aureofaciens 30-84 is mediated by a diffusible signal molecule. The gene responsible for production of this signal (phzI) was identified. Both phzI and phzR are required for the production of phenazines in vitro. Together these two proteins (PhzR/PhzI) comprise a N-acyl- scL-homoserine lactone (AHL) response system that controls phenazine antibiotic production in P. aureofaciens 30-84. Classic AHL-mediated regulatory systems consist of two proteins, a LuxR homolog (PhzR) which transcriptionally activates target gene expression in response to AHL produced by the second protein, the LuxI homolog (PhzI). Using HPLC coupled with high resolution mass spectroscopy, the specific AHL produced via PhzI has been identified as N-hexanoyl- scL-homoserine lactone (HHL). It has been determined that PhzR activates phenazine production in conjunction with HHL produced by PhzI via transcriptional activation of the phenazine biosynthetic gene phzB. A variety of synthetic AHLs restore transcription of phzB and phenazine production in phzI mutants suggesting that phzI mutants can be used to detect the presence of exogenous AHLs. This ability was exploited to show that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere. The work presented in this dissertation is the first to show that AHL-mediated regulation, previously only examined in vitro, can operate within the natural habitat of a bacterium.

Tobacco Virgin A Mutant (VAM) exhibits apparent immunity to several potyviruses in a strain-specific manner. Its resistance was generated by UV irradiation, and is partially conditioned by the recessive gene va. This allele has been introgressed into several breeding fines such as NC745. Previously, it was observed that the inoculation of an avirulent strain of potato Y potyvirus (PVY(NN)) in both resistant genotypes, caused systemic infection in some NC745 plants only, and the virus recovered from these plants acquired an ability to easily infect both NC745 and VAM. The current study was to identify the host factors that define each one of these resistant phenotypes, and to characterize the pathogenic properties of the evolving virus. VAM cells supported a reduced rate of PVY(NN)-accumulation compared with NC745 cells, which accumulated virus progeny at, he same level as the susceptible control Burley 21 (B21). However, in both resistant tobaccos the virus cell-to-cell movement was similarly impaired. Even so, PVY(NN) was recovered sooner from NC745- than from VAM-inoculated leaves. After PVY(NN)-detection, emerging resistance breaking (RB) variants were also recovered. Surprisingly, just in VAM, the RB variants never moved out of the inoculated leaves, until they were reinoculated in the same or another uninoculated VAM plant. The inability of the emerging RB variants to exit the PVY(NN)-inoculated VAM leaves was associated with their low accumulation rate and an obstruction imposed by coinfecting avirulent genotypes. The VAM factor restricting virus accumulation was inherited independently from va and operated in an allele doses manner. This gene, named rvam2, was easily overcome by the isolated RB variants, but the underlying virus modification(s) implied a loss of fitness in B21. Thus, the systemic emergence of RB variants, starting from a quasispecies, adapted to accumulate in Rvam2 genotypes (e.g., B21), seems to require a high rate of local virus accumulation linked to a selective constraint in the virus intercellular and/or intertissular traffic. This is the first report where the combined action of two vulnerable resistance mechanisms confers a stronger plant resistance to a viral systemic infection.

'The ascomycetal fungus Magnaporthe grisea causes the most damaging fungal disease of rice, blast. Resistant rice cultivars are typically dependent on the presence of one, or a few, major genes that are effective only toward particular M. grisea isolates. These isolates have avirulence genes that correspond to specific rice resistance genes, in a gene-for-gene relationship. Results presented here show the genetic identification of AVR2-MARA, a M. grisea a gene that confers avirulence toward the rice cultivar Maratelli. Two techniques were used for determining the chromosomal location of AVR2-MARA, restriction fragment length polymorphism analysis (RFLP), and bulked segregant analysis. Through RFLP analysis AVR2-MARA was mapped to Chromosome 7, between markers cos196 and cos209. Bacterial Artificial Chromosome (BAC) clones, as well as cosmids, were utilized in chromosome walking to the locus. Walks were initiated from markers on both sides of the locus, allowing the identification of sequences that were inseparable from AVR2-MARA. However, I was unable to clone the complete locus. In contrast, avr2-MARA , the virulent locus, was isolated from the virulent parental strain O-135. Sequence analysis of markers inseparable from the AVR2-MARA locus showed a higher AT content than typically observed in the M. grisea genome. Sequence analysis of a fragment inseparable from the AVR2-MARA locus also revealed a putative open-reading-frame (ORF) with significant homology to AVR-Pita. During this study I found that the segregation of avirulence on Maratelli corresponded with the segregation of avirulence towards the rice cultivars M-103 and M-201. AVR-M201, conferring avirulence toward the rice cultivar M-201, was identified several years ago (Valent et al. 1991), but never mapped. In our mapping cross, consisting of 65 progeny, AVR2-MARA and AVR-M201 are inseparable. Furthermore, we isolated two virulent mutants of AVR2-MARA via UV mutagenesis and both had also gained virulence on cultivars M-103 and M-201. This suggests that AVR2-MARA, AVR-M201 (and AVR-M103) are the same avirulence gene. Because AVR2-MARA originated in a finger millet isolate, and confers avirulence toward several different rice cultivars, it may represent a host-species-specificity factor. Such rice cultivars may be guarded from infection by finger millet isolates due to recognition of the AVR2-MARA gene product.

<p> The ascomycete fungus <i>Magnaporthe oryzae,</i> causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and <i>M. oryzae</i> is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made <i> M. oryzae</i> an ideal model system for understanding general plant-pathogen interactions as well. </p><p> The objective of this dissertation was to characterize the <i>M. oryzae</i> homologs of <i>Histoplasma capsulatum RYP</i> (<i><u> R</u>equired for <u>Y</u>east <u>P</u>hase </i>) genes that are required for transition to the parasitic phase. <i> H. capsulatum</i> is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37&deg;C, the host body temperature. Four<i>H. capsulatum RYP</i> genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. <i> RYP1</i> is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. <i>RYP2</i> and <i> RYP3</i> are homologs of <i>vosA</i> and <i>velB,</i> members of the Velvet family, best characterized in <i>Aspergillus nidulans, </i> where they coordinate morphological differentiation with secondary metabolism. <i>RYP4</i> is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the <i> M. oryzae RYP1</i> homolog, <i>RIG1</i> (<i><u> R</u>equired for <u>I</u>nfectious <u>G</u>rowth </i>), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. <i>&Delta;rig1</i> was blocked in the transition to infectious hyphal growth, similar to <i> H. capsulatum ryp1,</i> which could not transition to the yeast phase. Deletion mutants of <i>M. oryzae RYP2, RYP3,</i> and <i>RYP4 </i> homologs were similar to the wild type in somatic growth and pathogenicity indicating that although <i>RIG1</i> is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the <i>RYP</i> pathogenicity genes identified in <i>H. capsulatum. </i> </p><p> <i>&Delta;rig1</i> is the first <i>M. oryzae</i> mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.</p>

Sterols and isoprenoids are vital cellular constituents. In studies of their formation, metabolism, and biological effects, the separation and identification of individual isomers present formidable challenges. Approximately 30 unsaturated C27 sterols were prepared and purified as authentic standards to evaluate and develop chromatographic and spectral methods. Novel chromatographic conditions were devised for silver ion HPLC (Ag+-HPLC), a technique that provided unprecedented separations of these closely related sterols. Ag+-HPLC proved very powerful in addressing several important problems in medicine and biology relating to the biochemical effects of sterols and isoprenoids; described herein. Smith-Lemli-Opitz syndrome (SLOS) is a genetic disorder of development associated with the accumulation of unsaturated C27, sterols. My analysis of SLOS and normal blood samples unequivocally demonstrated that only the Delta5,7, Delta5,8, and Delta5,7,9(11) sterols accumulate significantly in SLOS patients. The 19-nor-Delta5,7,9 sterol, which has been reported to accumulate in SLOS blood by several research groups, was not detected in my analyses and was demonstrated to be a GC artifact arising from the thermal decomposition of cholesta-5,8-dien-3beta-ol. Alternative pathways in the late stages of cholesterol biosynthesis relating to the biological origin and metabolism of the beta5,8, beta6,8, and beta6,8(14) sterols were elucidated by incubation of tritium-labeled substrates in rat liver preparations. Also, a simple and rapid colorimetric method was developed for the clinical screening of SLOS. Ag +-HPLC played a critical role in studying a possible biochemical defect in another genetic disorder, Langer-Giedion syndrome, and in isolating isomers of (20R,22R)-dihydroxycholesterol), all-trans geranylgeranoic acid, other isoprenoids, and 4,4-dimethyl-sterols, compounds obtained from novel and efficient chemical syntheses. (20R,22R)-Dihydroxycholesterol proved to be a moderately potent activator of the nuclear orphan receptor LXRalpha, whereas all trans geranylgeranoic acid was found to be an inhibitor. The synthetic 4,4-dimethyl sterols caused a resumption of meiosis in mouse oocytes. Famesoic acid was demonstrated to be an extraordinarily potent substrate inducer of cytochrome P450BM-3.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006. / Source: Dissertation Abstracts International, Volume: 67-04, Section: B, page: 1764. Adviser: Roger W. Innes. "Title from dissertation home page (viewed June 20, 2007)."

Doctor of Philosophy / Department of Plant Pathology / William W. Bockus / Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph Septoria tritici), is an important disease of wheat worldwide capable of reducing yields by as much as 30 to 40%. In Kansas, the disease is widespread and losses in individual fields can exceed 25%. This study examined the genetic structure of Kansas populations of M. graminicola at different spatial scales (micro-plot, macro-plot, and statewide) using amplified fragment length polymorphism (AFLP) markers. Three primer pairs were used to resolve 174 polymorphic loci from 476 isolates. The results indicated high levels of genotypic variability, which is consistent with a genetically diverse initial inoculum. Genetic identities among populations representing the three spatial scales were >98%. Tests for differentiation among populations due to population subdivision revealed that on average 97.5% of the genetic variability occurred within populations with a correspondingly high migration rate of 16 to 23 individuals per generation. We observed little evidence of linkage disequilibrium, on average, only 4.6% of locus pairs were in disequilibrium. Our results indicate that Kansas populations of M. graminicola are characterized by regular recombination, are genetically diverse, and appear to be homogenous across different spatial scales. These populations are probably components of a larger pathogen pool that is distributed at least across much of Kansas and probably the central Great Plains. Because of the frequent recombination, the risk of adaptation of Kansas populations of M. graminicola to fungicide treatments or resistance genes is high and could be dispersed very quickly, whether these new pathogenic traits occur locally through mutation or by migration from other areas.

The overall objective of this study was to investigate the insecticidal and synergistic properties of an ethylacetate extract of Piper nigrum L. (Piperaceae) seeds used alone and in conjunction with pyrethrin, a botanical insecticide extracted from the flowers of Chrysanthemum cinerariaefolium Benth. & Hook. (Asteraceae). It was concluded that P. nigrum extract is a highly promising candidate for a novel botanical synergist for pyrethrin and could potentially be used as a replacement for piperonyl butoxide in certain pyrethrum formulations. The upregulation of mRNA transcripts encoding cytochrome P450 detoxification enzymes by a P. nigrum extract may indicate possible target proteins to enhance the toxicity of insecticides and synergists. The seven common genes differentially expressed in two or three of the sublethal treatments with pyrethrin, P. nigrum extract or pyrethrin plus P. nigrum extract merit further study of their functions relating to detoxification processes and defense responses. Future work should include a detailed, tissue-specific characterization of the genes of interest identified in this study and an investigation of the activity of the P. nigrum extract against the CYP enzymes associated with the Cyp genes that were differentially expressed in response to a P. nigrum extract. (Abstract shortened by UMI.)

A novel alteration in secondary metabolism of maize transformed with wheat oxalate oxidase gene (OXO) was determined using HPLC and microscopy. Phenolic concentration was significantly increased, but DIMBOA synthesis was down-regulated. The high levels of soluble phenolic acids, in particular free ferulic acid, most likely contributed to the insect resistance in the OXO maize. To facilitate future cowpea transformation with the OXO, a stable in vitro regeneration system was established in blackeye cowpea via shoot organogenesis. The optimal initiation medium comprised MS salts, B 5 vitamins, 2 mg 1-1 BAP, 3% sucrose and 0.8% agar at pH 5.8. The highest initiation frequency and shoot number were obtained from the shoot apices of 3-5 days old seedlings. For shoot elongation, 0.5-5.0 mg 1-1 GA3 was required. Rooting medium was MS salts supplemented with B5 vitamins, 3% sucrose and 0.8% agar.

Acetobacter diazotrophicus is a true endophyte of sugarcane and is often found in plants grown in agricultural areas of low nitrogen fertilizer input. Results from our laboratory, using mutant strains of A. diazotrophicus unable to fix nitrogen, have shown that there are two beneficial effects of A. diazotrophicus on sugarcane: one dependent on nitrogen fixation, and the other independent of nitrogen fixation. A plant growth promoting substance like indole-3-acetic acid (IAA) may represent the latter effect that accounts for improved plant growth. My first project was to characterize the genes responsible for nitrogen fixation, and determine their regulation. In summary, I have isolated, sequenced, and analyzed the major 31.5 kb nif gene cluster, including both nif and associated genes of A. diazotrophicus. This cluster of 33 genes represents the largest and most complete assembly of contiguous nif/fix and associated genes characterized in any diazotrophic bacterial species. My second project has been to determine whether nitrogen fixation and/or IAA production are important for the ability of A. diazotrophicus to stimulate plant growth. In order to determine the role of IAA directly, mutants of A. diazotrophicus producing reduced amounts of IAA were generated by Tn5 mutagenesis. Among IAA - candidates, one excreting less than 6% of IAA compared to the parent strain was further characterized. The mutation was mapped to genes involved in cytochrome c biogenesis (ccm genes-c&barbelow;ytochrome c&barbelow; m&barbelow;aturation genes). A Nif -/Iaa- double mutant and Nif- mutant were constructed by inserting a chloramphenicol cassette into nifD region. Plant inoculation experiments using mutant strains also demonstrated that A. diazotrophicus could stimulate plant growth regardless of N availability, as evidenced by the significant growth difference between plants inoculated with wild type and uninoculated plants. Under N-limiting conditions plants inoculated with wild type had greater height and biomass than plants inoculated with Nif- or Nif -/Iaa- mutants, suggesting nitrogen fixation by A. diazotrophicus stimulates sugarcane growth. Plants inoculated with Iaa- mutants were always comparable to uninoculated plants regardless of N availability, indicating that IAA biosynthesis is a major bacterial factor influencing sugarcane growth, particularly under N-sufficient conditions.