Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / John C. Reese / Keeping in view the important roles of bacteria in almost every aspect of insect’s life, the current study is the first systemic and intensive work on microbes associated with Hessian fly, a serious pest of wheat crop. A whole body analysis of Hessian fly larvae, pupae, or adults suggested that a remarkable diversity of bacteria is associated with different stages of the insect life cycle. The overriding detection of genera Acinetobacter and Enterobacter throughout the life cycle of Hessian fly suggested a stable and intimate relationship with the insect host. Adult Hessian flies have the most dissimilar bacterial composition from other stages with Bacillus as the most dominant genus. Analysis of 5778 high quality sequence reads obtained from larval gut estimated 187, 142, and 262 operational taxonomic units at 3% distance level from the 1st, 2nd, and 3rd instar respectively. Pseudomonas was the most dominant genus found in the gut of all three instars. The 3rd instar larval gut had the most diverse bacterial composition including genera Stenotrophomonas, Pantoea, Enterobacter, Ensifer, and Achromobacter. The transovarial transmission of major bacterial groups provided evidence of their intimate relationship with the Hessian fly. The Hessian fly is known to manipulate wheat plants to its own advantage. This study demonstrated that the combination of a decrease in carbon compounds and an increase in nitrogen compounds in the feeding tissues of Hessian fly-infested plants results in a C/N ratio of 17:1, nearly 2.5 times less than the C/N ratio (42:1) observed in control plants. We propose that bacteria associated with Hessian fly perform nitrogen fixation in the infested wheat, which was responsible for shifting the C/N ratio. The following findings made in the current study i.e. the presence of bacteria encoding nitrogenase (nifH) genes both in Hessian fly and infested wheat, exclusive expression of nifH in infested wheat, presence of diverse bacteria (including the nitrogen fixing genera) in the Hessian fly larvae, presence of similar bacterial microbiota in Hessian fly larvae and at the feeding site tissues in the infested wheat, and reduction in survival of Hessian fly larvae due to loss of bacteria are consistent with this hypothesis. The reduction in Hessian fly longevity after the loss of Alphaproteobacteria in first instar larvae, highest proportion of Alphaproteobacteria in insects surviving after the antibiotic treatments and the nitrogen fixation ability of associated Alphaproteobacteria strongly implies that Alphaproteobacteria are critical for the survival of Hessian fly larvae. This study provides a foundation for future studies to elucidate the role of associated microbes on Hessian fly virulence and biology. A better understanding of Hessian fly-microbe interactions may lead to new strategies to control this pest.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen that causes infection in people and several vertebrate animals. One of the striking features of E. chaffeensis infection is the prolonged persistence in its vertebrate and tick hosts. The mechanism of persistent infection and the reasons for the host immune system failure to clear the infection are not well understood. One hypothesis is that differential gene expression serves as an important adaptive mechanism used by E. chaffeensis in support of its continued survival in both tick and vertebrate hosts. One way to test this hypothesis is by performing mutational analysis. However, the methods for introducing mutations in this pathogen have not yet been documented and are challenging, possibly due to its obligate, intraphagosomal growth requirement. Recently, a novel gene mutation method called ‘TargeTron Gene Knockout System’ that is based on the modified group II intron insertion strategy has been developed. This method appears to be effective in creating mutations in a wide range of gram positive and gram negative bacterial organisms. The group II intron can be programmed for insertion into virtually any desired DNA target with possibly high frequency and specificity. In this study, I focus on creating mutations in E. chaffeensis using the TargeTron gene knockout system. I prepared modified group II intron constructs retargeting for insertion into three E. chaffeensis genes: Ech_0126 (a transcriptionally silent gene), macrophage-specific expressed gene (p28-Omp 19, Ech_1143) and tick cell-specific expressed gene (p28-Omp 14, Ech_1136). In support of driving the expression of the modified group II introns in E. chaffeensis, the pathogen- specific high-expressing gene promoter (tuf) was inserted upstream to the transcription start site. In addition, a chloramphenicol acetyltransferase gene with E. chaffeensis rpsl promoter was introduced for use as a selection marker. The constructs were then evaluated by transforming into E. chaffeensis. Transformants with mutations, introduced in two of the three genes (Ech_0126 and Ech_1143), were identified by PCR and Southern blot methods. Although the mutants are detectable for up to 48 hours, establishment of stable transformants remains to be challenging. The outcomes of this project will have important implications in defining the pathogenesis of E. chaffeensis, particularly to assess the differences in the organism in tick and vertebrate hosts.

Doctor of Philosophy / Department of Biochemistry / Michal Zolkiewski / ClpB is a bacterial heat-shock protein that disaggregates and reactivates strongly aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two ATP-binding AAA+ modules, a linker coiled-coil domain, and a highly mobile N-terminal domain. It forms ring-shaped hexamers in a nucleotide-dependent manner. The unique aggregation reversing chaperone activity of ClpB involves ATP-dependent translocation of substrates through the central channel in the ClpB ring. The initial events of aggregate recognition and the events preceding the translocation step are poorly understood. In addition to the full-length ClpB95, a truncated isoform ClpB80, that is missing the whole N-terminal domain, is also produced in vivo. Various aspects of the structure and function of ClpB were addressed in this work. The thermodynamic stability of ClpB in its monomeric and oligomeric forms, as well as the nucleotide-induced conformational changes in ClpB were investigated by fluorescence spectroscopy. Equilibrium urea-induced unfolding showed that two structural domains-the small domain of the C-terminal AAA+ module and the coiled-coil domain-were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation or interactions during formation of the ClpB rings. Several locations of Trp-fluorescence probes were also found to respond to nucleotide binding. The biological role of the two naturally-occurring ClpB isoforms was also investigated. We discovered that ClpB achieves optimum chaperone activity by synergistic cooperation of the two isoforms that form hetero-oligomers. We found that ClpB95/ClpB80 hetero-oligomers form preferentially at low protein concentration with higher affinity than homo-oligomers of ClpB95. Moreover, hetero-oligomers bind to aggregated substrates with a similar efficiency as homo-oligomers of ClpB95, do not show enhanced ATPase activity over that of the homo-oligomers, but display a strongly stimulated chaperone activity during the reactivation of aggregated proteins. We propose that extraction of single polypeptides from aggregates and their delivery to the ClpB channel for translocation is the rate-limiting step in aggregate reactivation and that step is supported by the mobility of the N-terminal domain of ClpB. We conclude that the enhancement of the chaperone activity of the hetero-oligomers is linked to an enhancement of mobility of the N-terminal domains.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Phloem-feeding insects like aphids feed on a variety of crop plants and limit plant productivity. In addition they are vectors for important plant viruses. Efforts to enhance plant resistance to aphids have been hampered by lack of sufficient understanding of mechanisms of plant defense against aphids. I have utilized a plant-aphid system consisting of the model plant Arabidopsis thaliana and the generalist aphid, Myzus persicae Sulzer (green peach aphid [GPA]), to study plant response to aphids. These studies have demonstrated an important role of premature leaf senescence in controlling aphid growth in Arabidopsis. Molecular and physiological studies suggest that the Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT 4) gene modulates the GPA feeding-induced senescence process. Furthermore, in comparison to the wild type plants, GPA growth was higher on pad4 mutant plants, suggesting an important role for PAD4 in plant defense against GPA. In contrast, constitutive expression of PAD4 in transgenic Arabidopsis enhanced basal resistance against GPA. Unlike its involvement in plant defense against pathogens, the role of PAD4 in Arabidopsis resistance to GPA is independent of its involvement in phytoalexin biosynthesis and of its interaction with EDS1, a PAD4-interacting protein. Instead, the heightened resistance to GPA in these PAD4 constitutively expressing plants was associated with the rapid activation of leaf senescence. The association of premature leaf senescence in basal defense against GPA is supported by our observation that in comparison to the wild type plant, GPA growth was restricted on the Arabidopsis hypersenescence mutants, ssi2 and cpr5. Gene expression studies suggested some overlap between plant responses to pathogens and aphids, for example, activation of genes associated with the salicylic acid (SA) signaling pathway. However, the characterization of aphid performance on Arabidopsis SA biosynthesis and signaling mutants have ruled out the involvement of SA signaling in controlling aphid growth.

Doctor of Philosophy / Department of Plant Pathology / Justin D. Faris / Bikram S. Gill / The Q gene is largely responsible for the widespread cultivation of durum and common wheat because it confers the square spike phenotype and the free-threshing character. It also pleiotropically influences many other domestication related traits such as glume shape, glume tenacity, rachis fragility, spike length, plant height, and spike emergence time. The objectives of this research were to confirm or reject the hypothesis that a candidate AP2-like gene is Q, confirm the dosage and pleiotropic effects attributed to Q, and begin defining the differences between the Q and q alleles. The identity of the Q gene was verified by analysis of knockout mutants and found to have a high degree of similarity to members of the AP2 family of transcription factors. Southern analysis of multiple Triticum taxa containing either Q or q indicated that the Q locus is not composed of duplicated q alleles. Ectopic expression analysis allowed the observation of both silencing and over-expression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were directly associated with the level of transgene expression due to the dosage effects of Q. Comparisons of Q and q indicated structural differences as well as variation in the level of transcription. One amino acid difference and several base changes within the promoter were identified as possible critical differences between Q and q. Very little genetic variability was found within the sequenced Q alleles suggesting it arose only once and that q is the more primitive allele.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. Rowland / Severe acute respiratory syndrome (SARS) is caused by an enveloped, positive-stranded RNA virus, the SARS coronavirus (SARS-CoV). Coronaviruses along with the arteriviruses are placed in the order, Nidovirales. Even though nidovirus replication is restricted to the cytoplasm, the nucleocapsid protein (N) of several coronaviruses and arteriviruses, localize to the nucleolus during infection. Confocal microscopy of N protein localization in Vero cells infected with the SARS-CoV or transfected with the SARS-CoV N gene failed to show presence of N in the nucleoplasm or nucleolus. Recombinant N remained cytoplasmic after the addition of leptomycin B (LMB), a drug that inhibits nuclear export. SARS-CoV N possesses a unique lysine-rich domain, located between amino acids 369-389, which possesses several nuclear localization signal (NLS) and nucleolar localization signal (NoLS) motifs. A chimeric protein composed of the 369-389 peptide substituted for the NLS of equine infectious anemia virus (EIAV) Rev protein (ERev) showed no nuclear localization activity. Three negatively charged amino acids, located at positions 372, 377 and 379 in SARS-CoV N were hypothesized to play a role in the loss of nuclear targeting. Substitution of aspartic acid-372 with alanine restored nuclear localization to the chimeric protein. A full-length recombinant SARS-N protein with the alanine-372 substitution localized to the nucleus. Therefore, the presence of an aspartic acid at position 372 is sufficient to retain N in the cytoplasm The mechanistic basis for how aspartic acid-372 interrupts nuclear transport is unknown, but may lie in the electrostatic repulsion with negatively charged amino acids located within the NLS binding pocket of importin-alpha.

Doctor of Philosophy / Department of Entomology / Gerald R. Reeck / John C. Reese / Salivary secretions are a key component of aphid-plant interactions. Aphids’ salivary proteins interact with plant tissues, gaining access to phloem sap and eliciting responses which may benefit the insect. In an effort to isolate and identify key components in salivary secretions, we created a salivary gland cDNA library. Several thousand randomly selected cDNA clones were sequenced. We grouped these sequences into 1769 sets of essentially identical sequences, or clusters. About 22% of the clusters matched clearly to (non-aphid) proteins of known function. Among our cDNAs, we have identified putative oxido-reductases and hydrolases that may be involved in the insect's attack on plant tissue. C002 represents an abundant transcript among the genes expressed in the salivary glands. This cDNA encodes a novel protein that fails to match to proteins outside of aphids and is of unknown function. In situ hybridization and immunohistochemistry localized C002 in the same sub-set of cells within the principal salivary gland. C002 protein was detected in fava beans that were exposed to aphids, verifying that C002 protein is a secreted protein. Injection of siC002-RNA caused depletion of C002 transcript levels dramatically over a 3 day period after injection. With a lag of 1 – 2 days, the siC002-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections of adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands.

Master of Science / Department of Biochemistry / Rachel Zufferey / Glycerolipids are the most abundant lipids and are important constituents of various virulence factors in the protozoan parasite Leishmania. The dihydroxyacetone phosphate acyltransferase LmDAT catalyzes the first step of the ether, and possibly ester glycerolipid biosynthetic pathway. A L. major null mutant of LmDAT grew slowly, died rapidly during the stationary phase of growth, and more importantly, was attenuated in virulence in mice. The goal of this study was to determine the molecular basis responsible for the attenuated virulence. Western blot analysis revealed that the ∆lmdat/∆lmdat null mutant synthesized altered versions of the virulence factor lipophosphoglycans that were not released in the media, suggesting that its lipid anchor structure was altered. The ∆lmdat/∆lmdat strain differentiated into virulent metacyclics, but with lower efficiency compared to the wild type. Using the autophagosomal marker ATG8-GFP, the ∆lmdat/∆lmdat line produced twice as many autophagosomes as the wild type, suggesting that it is either defective in degradation of autophagosomes or that autophagy is simply induced. In conclusion, the attenuated virulence of ∆lmdat/∆lmdat may be explained by i) its inability to synthesize and release normal forms of lipophosphoglycan, ii) its inability to fully differentiate into virulent metacyclics, and iii) altered autophagy.

Master of Science / Department of Biology / Jyoti Shah / Plant defense responses are constitutively activated in the Arabidopsis thaliana ssi2 mutant plant. In addition, the ssi2 mutant allele confers a dwarf phenotype. The SSI2 gene encodes a stearoyl-ACP-desaturase, which converts stearic acid (18:0) to oleic acid (18:1), suggesting a role for lipids in plant defense. Microarray analysis identified several genes which encode putative acyl hydrolases/lipases that are expressed at elevated levels in the leaves of ssi2, in comparison to the wild type plant. One gene in particular, At5g14180, was expressed at 60-fold greater level in ssi2 than in the wild type plant. To study the involvement of At5g14180 in plant defense and lipid metabolism, two transgenic lines containing T-DNA insertions within the At5g14180 gene were identified. These two T-DNA insertional alleles of the At5g14180 gene attenuate the ssi2-conferred heightened resistance to a virulent strain of Pseudomonas syringae pv. maculicola in the ssi2 At5g14180 double mutant plant. Furthermore, pathogen growth was enhanced in the At5g14180 single mutant plants, as compared to the wild type plant. Profiling of lipid composition in leaf tissue identified changes in the lipid composition between the At5g14180 mutant and wild type plants, suggesting that the At5g14180 encoded protein may impact lipid metabolism in Arabidopsis leaves.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice (Oryza sativa L.) production worldwide is constrained by rice blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Rice blast has become a model system for the study of fungal plant diseases based on its global relevance to agriculture and on our ability to apply molecular genetic and genomic analyses to both the pathogen and the plant. We have applied molecular and cellular analyses to understand critical processes in the M. oryzae disease cycle. The dark melanin pigment produced by the fungus is critical for the function of its specialized appressorial cell, which punches the leaf surface by generating the highest pressure known in any biological system, estimated at 80 times the atmospheric pressure. Without melanin, the fungus can neither generate this pressure nor puncture the plant surface and disease does not occur. M. oryzae genome sequencing identified a cluster of melanin biosynthesis genes that included an attractive candidate for the transcription factor that regulates melanin biosynthesis in appressoria. We report the structural and functional characterization of this putative transcription factor, although its role remains elusive. Host cellular responses after appressorial penetration are equally important in determining if disease will occur. We have characterized the cellular response of one rice variety to a compatible fungal strain (causes disease), an incompatible strain (fails to cause disease due to specific triggering of rice defenses) and a non-host strain (causes disease in barley but not in rice). Distinctive fungal and rice cellular responses correlated with the outcome of each particular pathogen-strain rice interaction. We report contrasting responses in two rice leaf sheath assays that are amenable to live cell microscopy, as well as a novel cellular response of crystalline aggregations deposited inside the host cell under appressoria on the leaf surface. Our studies have important implications for future analyses of pathogenicity and host specificity in rice blast disease.