Nitrogen (N) is one of the most important nutrients for plant growth, and fertilizers are heavily used worldwide to provide this nutrient. However, fertilizers are costly to produce and have significant negative impacts on the environment. Microorganisms within plants, termed endophytes, were isolated from wild poplar and willow plants and shown to promote growth in several other plant species as broadly diverse as rice, tomato, and Douglas fir. N-fixation was demonstrated in wild poplar and in hybrid poplar inoculated with a consortium of the endophytes from wild poplar. In order to better understand the genes involved in N-fixation by these endophytes, I created mutant libraries by random transposon mutagenesis. I screened the libraries for inability to grow on N-free medium. The Tn insertion sites were identified by sequencing and I analyzed the results. These results as well as a comparative genomics analysis with nodule-inhabiting rhizobia may provide insight into how endophytes protect nitrogenase from inhibitory oxygen. The project will also provide mutants to be tested on plants for determining the relative importance of N-fixation in the observed growth promoting effects of endophytes. A better understanding of the processes involved in N-fixation could lead to the creation of an optimized consortia of naturally occurring, N-fixing endophytes that could reduce the need for fertilizers in the future.

While a sizable and important part of biology revolves around work with mutants, significant work must precede any such experiments. Studying of mutants allows biologists to identify gene functionality through the comparison of organisms with an ineffective or over-effective gene against those with a wild type gene. Therefore, one of the most basic and important steps in this process is to be able to properly observe the effects of a wild type gene. Our primary interest is a diazotrophic endophyte, strain WP9, isolated from wild poplar, and its ability to perform nitrogen fixation. My aim is to optimize growth of WP9 in nitrogen limited media by varying available nutrients, and in the process I have done related studies in quorum sensing and the formation of aggregates. Also, due to the presence of aggregates which interfere with spectrophotometer readings, I have sought to improve the standard procedure of measuring endophyte growth, looking beyond optical density and into alternative methods such as microscopy. In the near future, with a specific and effective growth procedure, we can grow enough bacteria to then compare it to mutants in order to identify important genes for processes such as nitrogen fixation, and hopefully improve our efforts at aiding plants through a symbiotic relationship with our endophytes.

Microbes that live within plants, called endophytes, are being heavily researched on their ability to assist host plants in processes from nitrogen accumulation to drought tolerance. Two endophyte strains named WP5 and WP9 are known to colonize Populus trees. To research the interaction of these strains and their colonization processes, both strains have been marked with fluorescent tags to enable imaging under a microscope. Through this research, experiments determine what factors affect the colonization process of WP5 and WP9 in Populus trichocarpa clone Nisqually-1. I hypothesize that the presence of one of the marked endophytes in a plant sample could affect the colonization abilities of the other endophyte. Methods for experimentation include the growth of the endophytes in selective media and varying the optical densities of these liquid cultures before inoculating the poplar clones. The plants co-cultivate with the inoculum for 24 hours, at which point they are removed, washed and placed in limited nutrient media. After removal from the inoculum as well as several days hence, root samples from the clones are taken and put onto slides to image with the fluorescent microscope. The two endophytes were labeled with different colors, one red and one green, so that they could be imaged concurrently in multiple ways. In addition to using the microscope imagery to analyze colonization, root samples are ground up and plated on selective media to quantify colony numbers. In the future, I will experiement if stressed poplar clones attract colonization by endophytes faster and in larger quantities than with unstressed individuals. We expect that plants under stressed conditions may call endophytes to colonize at a faster pace. The results of this research demonstrate plant-microbe contact and shed light on the pathways that facilitate these interactions.

With a changing climate, rising temperatures are predicted to increase frequency and severity of drought. Along with drought, there is a need to increase crop production and expanding agriculture into areas with poor quality soil is one solution. Approximately 78% of the Earth’s atmosphere consists of nitrogen; however, plants cannot use nitrogen that is available in the atmosphere, since it is in the form of dinitrogen gas. The current dominant nitrogen solution in agriculture is chemical nitrogen fertilizer, which can be harmful to aquatic ecosystems and pollute groundwater if used in large quantities. This project focuses on the uses of microbial endophytes in agriculture. Endophytes are bacteria and fungi that live within plants. Some strains of endophytes have been shown to increase drought stress tolerance and nitrogen availability. In Prof. Doty’s lab, we have tested how individual and a consortium of endophyte strains mediate drought stress alleviation in rice by subjecting the plants to a period of drought followed by a rehydration period. We anticipate the plants that were inoculated with endophytes to survive the drought period and recover during the rehydration phase. We have also tested one endophyte strain for nitrogen fixation in rice and tomato plants by inoculating the plants with the wild strain and nitrogen fixation mutant strain. These anticipated outcomes suggest that endophytes could potentially be a more sustainable solution for agricultural crops under drought and nutrient stress.

The protein Transient Receptor Potential Ankyrin type 1 (TRPA1) is an ion channel found in nociceptive (pain-sensing) sensory neurons. TRPA1 is activated by several noxious compounds, including those found in mustard plants, garlic, smoke, and tear gas, among others. It is responsible for the sensation of irritation and pain that these compounds cause, as well as some related chronic pain disorders. A better understanding of TRPA1 could lead to novel therapeutics against chronic pain. For this reason, the mechanism by which TRPA1 activates is an active area of research. TRPA1 contains a coiled-coil domain at its C-terminal end and several ankyrin repeat domains (ARDs) at its N-terminal end. These are both very common repeating protein motifs; ARDs in particular often modulate protein-protein interactions. Many channels in the Transient Receptor Potential (TRP) family, which includes TRPA1, contain these domains, but their role in channel activation is not fully understood. However, a recently solved atomic structure of TRPA1 provided some key insights. The structure showed that TRPA1’s coiled-coil domain is tightly enveloped by its ARDs, and they appear to interact with each other. No other TRP channel with a known structure exhibits this unique structural arrangement. My project aims to better characterize the interaction between the ARDs and coiled-coil. I am studying a mutation in the ARDs of the human TRPA1 channel, which is at its interface with the coiled-coil. The mutation, K591E, changes a key lysine amino acid residue, which is positively charged, to a negatively charged glutamate. It is found in rattlesnake TRPA1, which unlike the human protein is activated by temperatures above 27 °C. Current results show that the K591E mutant is active at room temperature, even without any other compounds. A stronger understanding of TRPA1’s activation mechanism will be vital to the development of effective next-generation pain therapies.

Arsenic, a known carcinogen, has been linked to numerous cancers, and its toxicity and persistence in the environment at low levels impact both human health and ecosystem integrity. Due to natural and anthropogenic factors, the concentration of arsenic has elevated in many areas. Phytoremediation is a cost-effective, eco-friendly technology that uses the natural ability of plants to remediate pollutants, such as arsenic, from contaminated soils. Endophytes, mutualistic microbes colonizing the internal tissues of plants, have been shown to increase host-plant tolerance to a wide range of environmental stressors, and we hypothesize that arsenic-tolerant endophytes may improve the tolerance of their host plants to arsenic. The arsenic-tolerant isolate PD12R, identified as Rahnella aquatilis, has been shown to grow at a higher concentration in the presence of arsenic, than it does without. We hypothesis that this unexpected result may have to do with its ability to form a protective biofilm. This biofilm is made up of a matrix of extracellular polymeric substances (EPS) which bacteria use as a protection against environmental stressors. EPS may be protecting PD12R from arsenic either by slowing diffusion to the cells, or by chemically chelating the arsenic in the EPS matrix. This has an important application in plants because it may indirectly help reduce free arsenic in planta by promoting plant tolerance, thereby enhancing the process of phytoremediation. We are currently testing PD12R, and two other isolates, to determine whether biofilm production is correlated with arsenic concentration in the media, and whether arsenic is bound by the EPS matrix by using a 96-well plate set up to test optical density and colony forming units. The results will then be compared to the EPS data which will be quantified using a crystal violet assay.

Early childhood experiences determine future success in well-being, school, work, and in the community. Adversity creates an accumulated number of risks that are potentially damaging and could contribute to greater vulnerabilities for negative health and social outcomes, including the likelihood of entering the juvenile justice system. Recidivism of offending from the juvenile into the adult years is higher for people who start offending at an early age. My research project focused on examining the relationship between childhood adversity and early behavioral indicators of risks for entering the juvenile justice system and the role of cultural connection and social support as protective factors against the negative effects of adversity across different ethnic groups. Data was collected using baseline and Wave 9 data from the Fragile Families and Child Well-being Study (N=4,898). The measured variables were parental adversity, cultural connections, and social support to investigate the relationship between childhood adversity and externalizing behaviors. Findings provide evidence towards the potentially increased risk and vulnerabilities of children exposed to multiple adversities in childhood towards later involvement in the juvenile justice system. This study showed that there is a positive relationship between childhood adversity and externalizing behaviors, where greater adversity was significantly associated with worse externalizing behavior (p<.01). Social support was a significant protective factor for all ethnic groups. Additionally, results show that cultural connection and social support are promising resources that could mitigate adversity’s impact on behavioral risks and vulnerabilities of young children. Examining protective factors in the context of early exposure to adversity helps to identify potential targets for preventive interventions.

The family of Transient Receptor Potential (TRP) proteins contains ion channels with a wide array of functions, including invertebrate phototransduction, responding to painful stimuli, responding to temperature changes, and many others. Of particular interest is the polymodal receptor TRPV1, which responds to many stimuli, including capsaicin, heat, pH, etc. TRPV1 is known to play a role in the sensation of both pain and heat; however, the structural dynamics that underlie TRPV1’s ability to transduce these signals are still incompletely understood. Therefore, an understanding of the activation, regulation, and structure of TRPV1 are of clear importance. To address these questions, I have sought to use Xenopus laevis oocytes as an expression platform to perform studies on the structure and function of TRPV1. Since TRPV1 is not naturally expressed in Xenopus oocytes, the genetic information needed for the cell to build the protein was provided for the oocyte via microinjection of RNA. After an incubation period of several days, Western blot techniques were applied to analyze the presence and strength of expression of TRPV1. To better understand specific structural changes made when in the activated conformation, the fluorescent, non-canonical amino acid L-ANAP was integrated into TRPV1 using amber stop codon suppression and an engineered tRNA synthetase. Integrating the non-canonical amino acid ANAP enables structural and functional analysis of the membrane protein via fluorescence microscopy. The results of applying such methods to understand the function of TRPV1 will provide insight into the use of TRPV1 for therapeutic purposes, most prominently the reduction of the sensation of pain.