The Agrobacterium T6SS is Activated by Host Responses and Induces Light-Dependent Host Defenses
Agrobacterium tumefaciens, a gram-negative soil bacterium, causes the well-characterized Crown Gall Disease on host plants. This bacterium is the only known organism capable of interkingdom gene transfer. The tumor-inducing Ti plasmid encodes tumor-inducing factors on the transferred DNA (T-DNA), which is delivered into the host cell via a Type IV Secretion System (T4SS) and incorporated into the host’s chromosomal DNA for the production of plant growth hormones and nutritional resources for the bacterium. In addition to the T4SS, Agrobacteria also possess a functional Type VI Secretion System (T6SS), implicated in virulence in a variety of mammalian pathogens. T6SS proteins are encoded on the 14-gene imp operon and the divergently transcribed hcp operon. A T6SS mutant strain (#20) lacking the entire imp operon exhibits attenuated virulence.
We have obtained evidence that the Agrobacterium T6SS both triggers and dampens host defense responses. We hypothesize that Efr-mediated defenses common to both WT and T6SS-deletion mutant bacteria mask T6SS-triggered defenses in WT plants, but that in the efr mutant plant, the absence of a functional EF-Tu receptor allows for T6SS-elicited responses to become apparent.
The master Arabidopsis thaliana circadian regulator gene Circadian Clock Associated 1 (CCA1) has been shown to control resistance to a number of pathogens. At the same time, many plant defense genes seem to be regulated in a diurnal manner by changes in light rather than these endogenous rhythms. This thesis investigated whether Agrobacterium-induced expression of the Arabidopsis thaliana defense response genes FRK1 and WRKY29 was under circadian and/or light control.
Our results demonstrated time-of-day-dependent induction of host defense genes as well as bacterial virulence genes. Expression of A. thaliana defense genes FRK1 and WRKY29 suggested EFR-mediated responses were under circadian regulation, while T6SS-induced defenses seemed to be under light-mediated control. In addition, bacterial T6SS gene expression was induced in response to host defense gene levels, which a functional T6SS was in turn able to dampen slightly. These results suggest that the Agrobacterium T6SS is activated by host defense responses, resulting in the induction of further light-dependent A. thaliana defense gene expression.
Characterization of Von HippelLindau (VHL) in Helobdella sp. (Austin)
The goal of the Savage lab is to identify early developmental regulators that function in annelid development. An unbiased screen was conducted to generate a list of candidate genes, of which I screened a subset. One of these, the Von Hippel-Lindau (VHL) gene product, yielded a novel and interesting expression pattern in the Helobdella embryo. VHL’s canonical role is as the substrate recognition component of an ubiquitin ligase complex, but it has more recently been identified as acting in diverse capacities, such as regulating ciliogenesis. VHL acts as a microtubule stabilizer and is implicated in the establishment of polarity through its interaction with the PAR-aPKC complex. I analyzed the VHL sequence and characterized its expression pattern in leech embryogenesis using in situ hybridization. VHL is the first blastocoel marker identified in annelids. A second blastocoel was observed in the stage 5 embryo, a previously undocumented finding. The localization of VHL gene products to the blastocoel suggests that it is regulating decisions relating to asymmetry and cell fate specification.
Strain-Specific Specialization and Functional Evolution of the Light-Harvesting and Carbon Concentrating Complexes in Prochlorococcus
Katharine H. Dusenbury
The marine cyanobacterium Prochlorococcus dominates the euphotic zone in the tropical and subtropical open oceans. Recent work from our laboratory has suggested that the heterogeneity that exists in the Prochlorococcus photosynthetic apparatus must have an important role in enabling this lineage to thrive in the varied environments from the surface to depths of 200 m (Ting et al., 2009). In particular, many Prochlorococcus ecotypes have different copy numbers (ranging from one to eight) of the pcb gene encoding the prochlorophyte chlorophyll a/b binding (Pcb) protein (Ting, et al., 2009). Pcb proteins can form antenna rings around the photosystems to maximize light-harvesting efficiency. Due to this association, it has previously been suggested that the multiplication of the pcb gene in some Prochlorococcus strains is adaptive for growth at low light levels (Garczarek, et al., 2000). However, we found evidence that the association of the Pcb proteins with the photosystems in the various Prochlorococcus strains is actually highly specialized and that the presence of particular Pcb types may be more important for ecotype differentiation and adjusting to environmental conditions than the number of pcb genes a strain has. For instance, past work has demonstrated that PcbA subunits are typically associated with PSII in all studied Prochlorococcus strains. However, the only Pcb found to constitutively associate with PSI is the PcbG protein unique to SS120. In other strains, particularly MIT9313, PcbD has been found to form an antenna around PSI, but only under iron stress. Notably, the expression of these pcb genes, as well as the genes encoding the main components of the photosystem reaction centers, is not conserved across all Prochlorococcus strains. Together, these observations support our hypothesis that the Pcb proteins in Prochlorococcus have evolved conserved, but highly specialized functions. Furthermore, the diversification of Pcb proteins within the Prochlorococcus lineage likely played a critical role in the ability of this genus to adapt to different ecological niches and dominate the open oceans.
Along with having key differences in the copy number and expression of their photosynthetic apparatus genes, even closely related Prochlorococcus strains exhibit important differences in the evolution of their carbon concentrating microcompartment (or carboxysome) genes. In particular, we found that the gene encoding a carbonic anhydrase (CsoS3) present in the protein shell of the carboxysome has been evolving more rapidly in the MED4 strain than in other closely related strains. Interestingly, however, any evolutionary differences in the sequence and structure of Prochlorococcus carboxysome shell components (CsoS3, CsoS2, and CsoS1D) do not manifest themselves in significant strain-specific differences in the expression of these genes.
Through identifying specific differences in the structure, regulation, expression, and evolution of key genes/proteins involved in photosynthesis in Prochlorococcus, my work supports the overall hypothesis that Prochlorococcus isolates have evolved specialized photosynthetic strategies to best adapt to the numerous ecological niches in the open oceans. In particular, my results suggest that the specialization of the Pcb proteins, particularly PcbG and its closely related PcbD proteins, may have been integral in this process of strain-specific evolution and adaptation.
Exploring the Function of Phytosphingosine in Physcomitrella Patens
This research project is the first to utilize RNA interference to examine sphingolipid function and metabolism in the moss Physcomitrella patens. Very little is known about sphingolipids in moss, although sphingolipids in higher plants have been the subject of many recent studies. We modified sphingolipid metabolism in Physcomitrella using RNAi to silence the sole sphinganine (d18:0) hydroxylase (SH) responsible for generating the most prevalent sphingolipid LCB, phytosphingosine (t18:0). Silencing the SH gene resulted in a dramatic shift in LCB profile, the predicted result of decreased hydroxylase activity. It also resulted in a profound decrease in growth. We suspected that the reduction in t18:0 availability was the cause for the dramatic reduction in growth in the SH RNAi phenotype, although the resulting accumulation of d18:0 could also be responsible. To test this, we examined whether growth could be rescued by adding t18:0 to the growth medium of SH-RNAi plants and observing whether growth increased relative to vehicle-treated SH-RNAi plants. Indeed, we found that there was a significant increase in growth after adding t18:0. Because exogenous t18:0 stimulated growth, we hypothesize that the observed reduction in growth was a result of a lack of t18:0, and not the accumulation of d18:0.
The Agrobacterium Type 6 Secretion System Modulates Hormone-Dependent Arabidopsis Defense Pathways
Plant defenses rely upon hormone pathways for signaling and gene induction. Pathogens such as Agrobacterium tumefaciens may manipulate these pathways during the infection process. Our data suggest that host perception of the Agrobacterium Type 6 Secretion System triggers expression of these pathways. In addition, our results suggest a mechanism by which Agrobacterium may subvert host defense responses.
Are pgi and mpi Under Balancing Selection in Heliconius Butterflies?
Heterozygote advantage, a type of balancing selection, has been studied in many different organisms, including humans. Here we tested for heterozygote advantage in two metabolic genes, mpi and pgi, in the tropical butterflies Heliconius petiverana and Heliconius rosina. The two genes were chosen based on previous evidence pointing to balancing selection. For pgi, balancing selection has been described in two high latitude butterflies, Colias and Melitaea cinxia. For mpi, a previous study in Heliconius found very high nucleotide variability and incongruence between gene trees and species trees. Taken together, these results could be indicative of balancing selection. We ran statistical tests on cDNA, to avoid the long introns, of both genes and created gene trees for each. None of the tests were statistically significant, and our gene trees were identical to the species tree. Interestingly, the Tajima’s D value was negative for each individual gene, indicating a change acting across the entire genome (possibly population expansion), rather than natural selection acting on a particular gene. There was no evidence for balancing selection acting on either gene for our species. The types of habitats Heliconius butterflies live in could explain the lack of balancing selection. However, it could also be due to environmental factors. Melitaea cinxia butterflies show heterozygote advantage in low to moderate temperatures, but homozygotes for pgi have an advantage in higher temperatures. As Heliconius butterflies are from tropical environments, the higher temperatures could account for the lack of balancing selection found.
Regional Specificity in Dopaminergic Rescue of Endogenous Arousal
The driving force behind consciousness and behavior is arousal, which can be further separated into distinct endogenous and exogenous subcategories. The biological anime dopamine (DA) has been shown to play a role in human disorders of arousal. Furthermore, previous research has linked the Drosophilia gene, DopR1, to variability in arousal. DopR1 encodes for a receptor that is homologous to the human DA1 receptor. In the present study, a mutation of in DopR1, DopRf02676 (DopR), is used. DopR is hypomorphic, and contains a UAS sequence, which when enhanced by Gal4 produces a functional version of DopR. This genetic technique can be used to target the expression of DopR into specific euroanatomical regions to examine their role in behavior. Selective expression of DopR into the ellipsoid body is seen to have effects on exogenous arousal; however, endogenous arousal and regional specificity is not yet well characterized. Here, I show that a complex pattern of neuroanatomical rescue into the mushroom bodies and fans-shaped body with DopR is sufficient to restore wild type circadian behaviors, while a subsection of these neuroanatomical regions are not. These findings suggest a complicated pattern of dopaminergic signaling between different neuroanatomical regions that holistically contribute to behavioral phenotypes.
Activating Dopaminergic Neuronal Circuits That Regulate Arousal in Drosophila Melanogaster
Construction of Chromosome Fragmentation Vectors of Localize Origins of Replication in Tetrahymena thermophila
Tetrahymena thermophila is a single-celled ciliate with two nuclei. The micronucleus, which has five pairs of chromosomes, is the germline nucleus and divides by mitosis. The macronucleus has about 45 copies each of approximately 181 chromosomes, as well as chromosome-containing ribosomal DNA, present at about 9,000 copies. The macronucleus is formed from a mitotic copy of the micronucleus during conjugation. The macronucleus divides amitotically.
DNA replication is initiated when the origin of replication complex (ORC) binds to DNA. Each macronuclear chromosome must contain an origin of replication, where the ORC would bind. However, few origins of replication have been identified in the macronucleus. Fragmentation of macronuclear chromosomes would allow for the location of origins of replication, as only fragments containing origins of replication would continue to replicate.
Chromosome 8254260 was selected for study. Chromosome fragmentation vectors must be constructed in order to break the chromosome in the desired locations for origin of replication localization. These vectors contain Tetrahymena DNA to serve as a site of homologous recombination, as well as chromosome breakage sequences to break Tetrahymena DNA and endogenously add telomeres. Three methods were used to attempt to construct vectors: overlap extension polymerase chain reaction; conventional restriction enzyme digestion and ligation; and polymerase chain reaction, restriction enzyme digestion and ligation. Once vectors are successfully produced, they can be transformed into Tetrahymena to help localize origins of replication on chromosome 8254260.
Regeneration of the Tracheosyringeal Nerve Following Transection: Central Reorganization and Effects on Song
Brain plasticity involves the concept of a brain adapting to its environment and allows for processes such as development, learning and repair of an injury. Zebra finch males learn and stabilize their songs during a critical period which ends at 90 days of age. However, injury to the tracheosyringeal nerve connecting the brain and the syrinx (the avian vocal organ) in mature birds induces a response that mediates new axon connections formed in repairing the nerve, indicating that mature zebra finches retain the ability to regenerate peripheral nerves. These newly formed projections were traced from the syrinx to nucleus nXIIts in the brain, which displayed a disrupted topographical map of syringeal muscles, despite a recovery of the template song. The number of new axon connections formed in the tracheosyringeal nerve had a negative relationship with subjects’ age, while it was directly correlated with song recovery and song consistency. This experiment did not successfully trace the source of topographical disruption within nXIIts, but it did directly relate neural regeneration to behavioral recovery.
Huddling and Nest Building Do Not Completely Ameliorate the Cold Stress of Typical Housing in Mice
Huddling and nest building are two methods of behavioral thermoregulation commonly utilized by mice under cold stress. Lab mice are typically housed at an ambient temperature (Ta) of 20°C, approximately 10°C below their thermoneutral zone of 30°C. We tested the hypothesis that the thermoregulatory benefits of huddling and nest building would lower heart rate (HR), blood pressure (BP) and metabolic rate (MR) of mice housed at 20°C to levels typical of mice housed at 30°C. Outbred mice were implanted with EKG/BP/core body temperature (Tb) telemeters, housed in metabolic cages and, using a repeated-measures crossover design, exposed to one of four conditions: 1) singly housed at 20°C, 2) group housed at 20°C, 3) singly housed with nesting material at 20°C, 4) singly housed at 30°C. There was a main effect of temperature in all measured parameters and no interactions due to group housing or nesting. In a second study, the same telemeter implanted mice were singly housed at 20°C before being assigned to either group housing or nesting at 20°C for a 10 day acclimation period. The mice were then singly housed at 30°C for one week. Again, there was a main effect of temperature in HR, BP, and daily food consumption, but no interaction due to group housing or nesting. In a final study, six groups of mice were allowed to acclimate to one of three conditions at either 20°C or 30°C: singly housed, group housed, or with nesting material. Group housing and nesting at 20°C significantly decreased plasma T3 levels and UCP1 expression in brown fat below that of singly housed mice at 20°C. These findings indicate that huddling and nest building lessen but do not completely ameliorate cold stress at 20°C.
The Role of Sex Hormones in Rapamycin-Induced Diabetes in Mice
While rapamycin is known to increase longevity in aging mice, it has also been shown to induce insulin resistance and glucose intolerance. We initially tested the hypothesis that long-term rapamycin dosage induces diabetes in mice. Outbred male and female mice were fed a rapamycin-containing diet for approximately 1 year, and analyzed for markers of diabetes. Only male mice were found to exhibit diabetic symptoms, including elevated fasting blood glucose (256±12 vs. control-fed: 87±5mg/dL) and detectable urine glucose (14,600±1,680mg/dL). Female mice remained euglycemic (91±3 vs. control-fed: 83±2mg/dL) with no detectable urine glucose. We then hypothesized that testosterone (T) was facilitating the progression of and 17β-estradiol (E2) was protecting against rapamycin-induced diabetes. A subset of males and females from the initial group was gonadectomized and monitored with weekly glucose tolerance tests for changes in diabetic status. Over 11 weeks post-castration, male mice showed a decreased rate of decline in glucose tolerance (∆AUC=3114±2288 vs. sham: 9,094±5053mg.min/dL). Female mice progressively developed glucose intolerance (∆AUC=8265±2329 vs. sham: -900±1153mg.min/dL) during the 10-week period post-ovariectomy . This was partially reversed by E2 administration over 4 weeks (∆AUC=-7491±3542 vs. vehicle: -2141±1430mg.min/dL). Analysis of plasma samples indicated that glucose intolerance was not distinctly associated with fasting insulin concentration: among rapamycin-fed mice, ovariectomized vehicle-implanted females, ovariectomized E2-implanted females, and sham-operated males showed comparable levels (1.0±0.09, 1.08±0.36, and 1.08±0.22ng/mL respectively). Western blots indicated no significant association between Akt phosphorylation and glucose intolerance. These results implicate T in the progression of and E2 in the protection against rapamycin-induced diabetes, through pathways distinct from regulation of insulin production and Akt signaling.
Chronic Rapamycin Treatment Confers Partially Reversible Diabetes Mellitus
Chronic rapamycin treatment, initially thought to promote longevity in mice, is now associated with impaired glucose homeostasis and insulin resistance. It was therefore hypothesized that chronic rapamycin treatment would cause reversible diabetes in mice. Outbred mice were treated with oral rapamycin for a period of one year and were assessed with glucose, insulin and pyruvate tolerance tests (GTT, ITT, PTT). Area under the curve analysis (AUC) of GTTs showed glucose intolerance in rapamycin treated males (AUC 10419Â±1839 vs. 2975Â±318 min-mg/dL) and rapamycin treated females (AUC 4744Â±867 vs. 2936Â±261). In a PTT, rapamycin treated males (AUC 7614 Â±1044 vs. 3288 Â± 575) showed pyruvate intolerance, suggesting excess gluconeogenesis. The ITT showed normal insulin sensitivity in both rapamycin treated males and females suggesting diabetes complications are a result of damaged islet cells, however an analysis of pathology showed no significant islet degradation. When rapamycin was removed from the diet, fasting blood glucose fell from 294Â±28 mg/dL to 135Â±11mg/dL (ad lib cohorts: 96Â±6 mg/dL) and had increased glucose tolerance (AUC 4877Â±742) in males. Females also exhibited increased glucose tolerance (AUC 2571Â±322) after removal of rapamycin treatment. These data suggest impairment of islet cell function in rapamycin treated mice that is partially reversible.
Investigating the Presence of Female Preference for Species-Typical Syntax in Male Zebra Finch Song
Zebra finch males sing in a syntactically linear manner. Although each male sings a unique combination of syllables, the pattern of singing is the same across the species. What might drive species typical syntax? One possibility is sexual selection. Female preferences may guide the development of zebra finch song. This project assessed the role of syntax linearity in female preference, and whether female presence during adolescence drives a male to develop a syntactically species-typical song. Females viewed a video presentation that was artificially generated. Short clips of a male singing were combined with either an atypical (variable) or typical (linear) syntax, assembled from prerecorded male syllables. Females’ responses to the songs were video recorded, and their behavior was subsequently scored. All females showed a significant attention preference for either typical, atypical, or both syntax types, showing they attended to videos of males singing. However, there were no overall preferences for variable or linear syntax, although some individual females had significant preferences for one of the stimulus types.
In addition, four males were raised in isolation chambers without fathers, so that they did not have the opportunity to copy from a male tutor during song learning and development. Two males were raised with their sisters and two males were raised alone. Once the males’ songs had crystallized, these songs were recorded and analyzed for syntax structure. Syntax structure was calculated as sequence stereotypy and stop variability. Males raised with their sisters had normal stereotypy and stop variability. Males without females did not have typical stereotypy and variability values. One male had significantly higher stereotypy and lower stop variability, indicating a highly stereotyped song with little variation. The other male had significantly lower stereotypy, with average stop variability.
Females appear to play an important role in the development of male song. Males raised with their naive sisters developed species-typical syntax. In contrast, males raised alone developed either more variable or more linear syntax than typical zebra finch song. However, the behavioral assay testing adult, experienced females did not reveal a consistent preference for typical, linear male syntax over all subjects. It may be that this behavioral assay was not sensitive to female preference, and that the small sample size limited the results. Nevertheless, the paradoxical nature of these results raise interesting questions about the interplay of male and female influences on song development.
Effects of Long Chain Base Modification on Growth and Metabolism in Physcomitrella Patens
This thesis examined the effects of long chain base modification on growth and metabolism in Physcomitrella. We utilized RNAi to silence hydroxylase and a desaturase in Physcomitrella. In both of these transformants, a dramatic reduction in growth was observed. Comparison of cell length to total growth area showed that reduced cell size was strongly correlated with a reduction in the growth area of the plantlets. Transmission electron microscopy showed that both SH and DS transformants exhibit apparent plasmolysis. LCB profiling of the SH transformant showed a decrease in from the phytosphingosine (t18:0) and an increase in sphinganine (d18:0) variant, as was expected from silencing hydroxylase activity. Surprisingly, LCB profiling of the DS transformant showed an increase in desaturated LCBs. We hypothesize that this may be due to activity of another desaturase compensating for the silencing of the first desaturase. RT-PCR analysis was used to examine changes in the sphingolipid metabolic pathway. Most notably, we observed a change in expression consistent with a shift from glycosyl inositolphosphoceramide formation to glucosylceramide formation in the SH-RNAi and, to a lesser degree, DS-RNAi transformants.
Spatial and Temporal Population Genetic Structure in Disjunct Populations of the Arctic Plant Sagina nodosa, in Isle Royale, Michigan
Sagina nodosa (knotted pearlwort) grows circumboreally in the sub-artic zone. Disjunct populations grow in the Isle Royale archipelago, Michigan. These populations occur at the southernmost edge of the species’ range, making the population of interest in relation to thermal tolerance and climate change adaptation. The results of our analysis will add to our limited understanding of genetic responses to climate change. Given Isle Royale’s protected status as a National Park, any changes in the population are likely due to measured shifts in weather patterns. We conduct microsatellite analyses of temporal and spatial separation in S. nodosa from Isle Royale. Here, we analyze a total of 179 individuals collected in two time periods: 1997-2000 and 2011, as well as from 4 distinct islands in each time period. We find evidence of strong hierarchical structure between islands and high inbreeding within island. We believe that S. nodosa pollination and seed dispersal methods prevent significant gene flow from occurring across the water channels between islands. We additionally report strong evidence of genetic change over the past decade, indicating that climate induced environmental stress may be affecting populations of S. nodosa in Isle Royale.
Demonstrating Chromosome Fragmentation and Telomere Addition in Tetrahymena thermophila
The ciliate, Tetrahymena thermophila, exhibits nuclear dimorphism. The germline micronucleus contains five pairs of chromosomes, and divides mitotically during asexual reproduction. The somatic macronucleus, derived from a copy of the micronucleus during conjugation, contains ~45 copies of 180 chromosomes and ~9000 copies of the rDNA chromosome. The macronucleus divides amitotically during asexual reproduction, which can lead to an unequal distribution of chromosomes to the daughter nuclei. However, cell lethality across generations due to chromosome loss is not observed, and thus, the presence of a copy number control mechanism that preserves the correct copy number of each macronuclear chromosome is indicated.
Artificial fragmentation of the macronuclear chromosomes using plasmid vectors containing a Chromosome Breakage Sequence (Cbs) should allow for the detection of a copy number control region. The vector inserts into the genome by homologous crossing over, and fragmentation occurs due to Cbs processing and telomere addition to the fragmented ends of the chromosome. Previous students have obtained and characterized transformants, although telomere addition to the fragmented ends of the chromosome has not been conclusively shown. In this thesis, PCR analysis demonstrated telomere addition to the fragmented end of the recovered chromosome in nine different transformants, and Southern hybridizations to further show telomere addition initiated. Furthermore, the construction of additional fragmentation vectors was attempted.
Hspb7 Expression in Both the Embryo and Yolk Syncytial Layer Contributes to Zebrafish Valvulogenesis
Congenital heart defects occur in 1-2% of the roughly 4,000,000 annual live births in the United States; understanding the causes of these defects is critical to their prevention. Unlike other organs, the heart begins functioning before fully completing development, making it sensitive to feedback from contractility and hemodynamic forces exerted by shear stress or hydrostatic forces of flowing blood. Zebrafish are a useful model for the human heart at three weeks of development, a critical period at which heart morphogenesis begins.
Previous research has identified a role for small heat shock protein (sHsp) hspb7 in heart development. Morpholino knockdown of hspb7 during the 1-4 cell stage randomizes heart laterality (Lahvic, 2010). Moreover, hspb7 morphants suffer from reduced ventricle size, over-proliferation of valve tissue, and failed or reduced migration of cardiac progenitor cells (Springel, 2012). Interestingly, the migration defect is also observed when hspb7 morpholino is injected 3-4 hours post-fertilization (hpf). At this stage, morpholino is restricted to the extra-embryonic yolk syncytial layer. This observation raises two questions 1. What is the identity of the migration-promoting YSL signal? and 2. What effects does this signal exert on later events in heart development?
Work on the first question remains ongoing, but to address the second question, we tested whether valve formation is cell autonomous (i.e. independent) of hspb7-regulated signals in the yolk syncytial layer (YSL)—however, the evidence presented here suggests that both YSL and embryonic hspb7 contribute to valve formation. In addition, we also tested hspb7 cDNA for alternative splice isoforms as part of an ongoing effort to understand the failure of previous attempts to rescue the phenotype of hspb7 injection. Our data provide no evidence for hspb7 splice isoforms in zebrafish based on sequencing of 3’ cDNA ends.