UCR

Center for Plant Cell Biology



REU 2014


REU Students and their Summer 2014 Research Programs

Undergraduate students were invited to apply to the Center for Plant Cell Biology (CEPCEB) to pursue individual research projects in the areas of cellular and molecular biology of plants and their pathogens. In 2013, the following twelve students were selected for this ongoing 10-week residential summer program.  Please click on the following student links to see photos and read about their Summer 2014 research programs in CEPCEB laboratories.

An REU Poster Session is scheduled in the Genomics lobby at the end of the ten-week program, where students discuss their projects.

REU Student
College/University
CEPCEB Faculty

Max Baymiller

New Mexico Institute of Mining & Technology, NM

Judelson Lab

Thomas Bekman

New College of Florida, FL

Borkovich Lab

Alyssa Carpenter

East Stroudsburg University, PA

Ng Lab

Andy Chen

Hamilton College, NY

Gonehal Lab

Stephanie Cheng

Mt. San Antonio College, CA

Chen Lab

Jenniffer Flores

University of Texas at San Antonio, TX

Bailey-Serres Lab

Patrick Gallagher

The College of New Jersey, NJ

Nothnagel Lab

Katherine Guthrie

Northwest Missouri State University, MO

Walling Lab

Kebba Mbye

University of North Carolina, NC

GirkeLab

Katie Orban

Loyola Marymount University, CA

Springer Lab

Spencer Swansen

Seattle Pacific University, WA

Stajich Lab

Roland Truong

East Los Angeles College, CA

Larsen Lab

 

MAX BAYMILLER
New Mexico Institute of Mining & Technology
Max Baymiller

The oomycetes are an evolutionarily distinct group of organisms often confused with fungi because of their filamentous growth habits and frequent saprophytic tendencies. The most well-studied oomycete is the potato and tomato pathogen Phytophthora infestans, which is more commonly known as late blight. A prolific and highly destructive pathogen, P. infestans is known to have caused the infamous Irish Potato Famine and today is responsible for billions of dollars of crop losses each year. We are studying the infection strategies of P. infestans and other oomycete pathogens such as Pythium with the long-term goal of mitigating disease through a better understanding of their fundamental biology. In this summer's project, we will investigate how oomycete pathogens obtain the energy and nutrients required for growth and infection. To answer this, the expression of metabolic genes from species of Phytophthora and Pythium that infect potato will be compared. Total RNA will be extracted from infected tubers at several stages of disease, and quantitative reverse transcription-PCR used to assess the relative abundance of various metabolic gene transcripts. These data will provide insights into the differences between the metabolic strategies of the pathogens. With this improved knowledge of pathogen metabolism will come the ability to formulate more effective mitigation strategies for devastating diseases such as late blight.

 

 

THOMAS BEKMAN
New College of Florida
Thomas Bekman

Neurospora crassa is an ideal model organism for studying eukaryotic cellular processes: it is non-pathogenic, multinucleate and easily cultured. It shares many homologous genes with yeast, plants, and humans. Previously, more than 200 knockout mutants for transcription factor genes were created in a high throughput project. Our goal is to identify the role of these transcription factors by studying the phenotypic effects due to their absence. The knockouts were produced by replacing the transcription factor gene with a hygomycin resistance cassette. The mutant was then cultured on media with hygomycin. Assuming that the proper mutant was generated, proper growth would have occurred; otherwise the antibiotic (hygomycin) would have inhibited growth. We observed defects in the sexual and asexual cycle to determine their correlation with the absence of a transcription factor. The phenotypes characterized included linear growth rate of basal hyphae, height of aerial hyphae, asexual sporulation (conidiation), female fertility and general morphology. Furthermore, we are now testing various progeny of mutants that were previously only available as heterokaryons with both mutant and wild type nuclei. Heterokaryons were purified to homokaryons by crossing with wild type N. crassa and then culturing the progeny on hygomycin medium. If the knockout strain could not be purified as a homokaryon, it is defined as an inviable mutant, and thus the gene removed is essential to survival. Cataloguing of and correlation of these assays on the above phenotypic categories will help elucidate the role transcription factors play in growth and development.

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ALYSSA CARPENTER
East Stroudsburg University
Alyssa Carpenter

Bemisia tabaci, more commonly known as whiteflies, are a common invasive insect species. The pests act as a vector passing detrimental viruses to plants through their mouthparts. The focus of this experiment was on whitefly transmission in the genus Crinivirus. Lettuce infectious yellow virus (LIYV) is a common plant pathogen and is transmitted by B. tabaci. The virions are retained in the whitefly's anterior foregut, and are able to be transmitted for days after the initial attainment. Retaining the virion in the anterior foregut showed reason for causation in the transmission of LIYV from vector to plant. Virions are attained through whitefly ingestion, are retained in the vector's anterior foregut at specific binding sites, and then spread to another plant. Through further analysis, it was discovered that a minor coat protein (CPm) of lettuce infectious yellow virus facilitated virion preservation and transmission. Therefore, there must be a factor which mediates the switch from virion retaining to virion transmission. For this experiment, pH was examined to be the mediating factor. The pH of phloem sap is typically around 8; the mouthparts of a whitefly have a typical pH of 9. Through artificial membrane feeding cages, virion retention was tested at pH 4, pH 7.4, and pH 9. In previous experiments, LIYV was retained in all but pH 7.4 through examination by reverse transcription PCR analysis. Throughout this program, I will be further investigating pH's effect on virion retention, as well as protein factors which aid in virion retention and transmission.

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ANDY CHEN
Hamilton College
Andy Chen

The model plant Arabidopsis thaliana expresses transcription factor (TF)-WUSCHEL (WUS) differentially throughout its shoot apical meristem (SAM), where the transcription factor migrates through multiple cell layers and regulates the expression of other stem-cell differentiation promoting transcription factors.  Furthermore, WUS regulates the expression of the Clavata3 (CLV3) gene, its own negative regulatory peptide, which is thought to trigger a signal cascade that represses WUS expression through a poorly understood mechanism.  Finally, new data suggests that WUS can be both a CLV3 repressor and activator; WUS concentration determines which function of regulation occurs.  To further explore this interactive system, a novel approach will be employed combining a ubiquitously expressed RNA-binding protein and an inserted RNA binding sequence in the CLV3 gene, resulting in a powerful reporter system capable of visualizing the mRNA transcript in vivo.  This technique employs the MS2 bacteriophage coat protein (MS2 CP) system, which exhibits high affinity for specific RNA stem-loop sequences (MS2 SL).  GFP-labeling and NLS-tagging of the binding protein and selective insertion of the stem-loop sequence into the CLV3 gene allows for fluorescence- and confocal-microscopy visualization of the transcript and its intracellular interactions.  Quantification of transcript dynamics further our understanding of the WUS-CLV3 cyclical feedback system and elucidate plant transcriptional control mechanisms.  Insight into the interactions of the Arabidopsis WUS-CLV3 system may yield important information about as yet unknown processes of transcriptional regulation in Arabidopsis with possible implications in other organisms.  

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STEPHANIE CHENG
Mt. San Antonio College
Stephanie Cheng

MicroRNAs (miRNAs) are short RNAs of 20-24 nucleotides found in all plants and animals, which have emerged as sequence-specific regulators of gene expression. Although miRNAs impact numerous biological processes in eukaryotes, many key players in miRNA pathways are still unknown. Therefore, to find new components work in miRNA pathways, my project in Chen lab is to identify additional factors involved in miRNA biogenesis or activity, using a forward genetics approach. We utilize a transgenic line of artificial microRNA, amiR-CTR1, as the background to screen potential candidates that work in miRNA pathways. Because the amiR-CTR1 was designed to target the CTR1 gene and employ the endogenous miRNA machinery for its biogenesis and activity, the amiR-CTR1 line mimics the ctr1 mutant. When the machinery for miRNA biogenesis or activity is disrupted, the amiR-CTR1 line cannot mimic ctr1 mutant, but exhibits the wild type phenotype.  After using EMS mutagenesis to create random mutations, only plants with a wild-type phenotype are chosen, as they would most likely have the mutation in microRNA biogenesis or activity. Besides screening for potential miRNA pathway mutants, I will also characterize the existing mutants by Northern Blot, q-PCR, and Western Blot in order to understand the mutations in miRNA biogenesis or activity in the selected mutants.   

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JENNIFFER FLORES
Univ. of Texas at San Antonio
Jenniffer Flores

To regulate growth and development, in addition to biotic and abiotic stress, plants monitor and integrate environmental signals to regulate metabolism and energy homeostasis. Flooding and complete submergence of plants is an environmental stress that contributes to large agricultural losses. During flooding events, decrease in cellular oxygen (hypoxia) and reduce light levels affect energy production within the plants. To survive flooding or submergence stress, some plants, like Arabidopsis thaliana enter a quiescent state in which their growth is inhibited to restrict energy usage and allow for the plant to survive until the flooding events subsides. In this research project, TARGET OF RAPAMYCIN (TOR) signaling, a process that positively regulates growth, will be investigated in various Arabidopsis Thaliana mutant lines with various T-DNA insertions that lead to over and under-expression of TOR. These lines will be investigated using techniques such as qPCR and Western immunoblot to determine if TOR will also be performed to evaluate the downstream processes related to TOR.  It is important to fully understand the functionality TOR expression and TOR signaling is affected. Hypoxia and submergence tolerance assays will also be performed to determine if survival is affected in the TOR mutant lines, and if TOR signaling contributes to low oxygen responses. Gaining a greater understanding of the role of TOR in low oxygen stress can aid in the production of more tolerant crops, which can lead to increased crop yield and prevent crop shortages due to environmental variation. 

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PATRICK GALLGHER
The College of New Jersey
Patrick Gallagher

Plant cell walls and plasma membranes contain arabinogalactan-proteins (AGPs), a class of glycoproteins which are thought to function in cell division, growth, and other aspects of plant development. The AGPs of the moss Physcomitrella patens contain the unusual 3-O-methyl-L-rhamnosyl (3-O-Me-Rha) residue, which occurs in cell walls of relictual plants up to and including the four phyla of gymnosperms but does not occur in angiosperms, the most derived plants. These methylated sugar residues are of interest because their extra carbon atom could be relevant toward more efficient production of biofuels from cell wall biomass. This project has the goal of identifying which gene in Physcomitrella patens encodes the methyltransferase enzyme that generates 3-O-Me-Rha. Using bioinformatics, five likely gene candidates in moss have been selected, and these genes are being transformed into transgenic tobacco plants. The immediate aim for this summer is to test the transgenic tobacco tissue for the presence of 3-O-Me-Rha. Wild-type tobacco produces AGPs containing Rha residues but no 3-O-Me-Rha residues. The hypothesis is that if one of the five candidate genes does encode the rhamnosyl-3-O-methyltransferase in the moss, then the transgenic tobacco expressing this moss gene will be able to synthesize AGPs containing 3-O-Me-Rha.

 

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KATHERINE GUTHRIE
Northwest Missouri State Univ.
Katherine Guthrie

After wounding of tomato leaves, Leucine aminopeptidase A (Lap A) accumulates in the chloroplast stroma. It creates a signal that moves to the nucleus to up-regulate the production of proteinase inhibitors and polyphenol oxidases and down-regulate another set of genes. This mode of regulation (chloroplast to nucleus) is called retrograde signaling. Recently, transcription factors were found to move from the chloroplast to the nucleus to control defense genes. These proteins have nuclear localization signals (NLS) and transit peptides suggesting dual localization. This project will identify transcription factors that may be involved in LAP-A's retrograde signaling. In tomato, WRKY transcription factor 5 (WRKY5) has a NLS and transit peptide and its residence in the plastid stroma was confirmed by proteomics. Given the role of the Arabidopsis homolog of SIWRKY5 in defense signaling, SIWRKY5 is likely a mobile signal. The goal is to monitor the movement of both LAP-A and WRKY5 after wounding. To this end, the Lap A and WRKY5 cDNAs will be amplified using gene specific primers and PCR and inserted into Gateway vectors, pEarleyGate 101 and 102 to make yellow and cyan fluorescent fusion proteins, respectively. These vectors will be delivered into leaves using agroinfiltration. The location of the fusion proteins after wounding will be determined using confocal microscopy. If the protein moves, its role in retrograde signaling of these proteins will be further investigated. This knowledge will be important in the creation of insect resistant tomato crops and prevention of crop loss.

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KEBBA MBYE
University of North Carolina
Kebba Mbye

Next-generation sequencing technologies allow present high-throughput genome profiling approaches to generate massive amounts of data that are frequently difficult and time-consuming to decode. Gene sets are groups of genes that share common chromosomal location, biological function(s), or regulation processes. Gene set enrichment tests are used to determine over- and/or under-representented pathways and biological processes in a gene set of interest. These processes help facilitate the interpretation of gene sets and enrichment of genes, however, it is often problematic and time inefficient to combine the results from multiple enrichment tests. Through the development of a R-based analysis environment for performing multiple enrichment tests across any number of genes, this project aims to develop a single program that will execute these functions in an efficient, reproducible, and user-friendly manner.

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KATIE ORBAN
Loyola Marymount University
Katie Orban

One of the challenges humanity currently faces is an exponentially increasing population and a projected food shortage. One mechanism to increase crop yields in order to feed this growing population may be to develop crops that can be grown at high density. This may be achieved by manipulating crops to have a more vertical architecture, by decreasing leaf angle. LATERAL ORGAN BOUNDARIES (LOB) is a plant-specific transcription factor in Arabidopsis thaliana that functions to define organ boundaries (areas between organs) by limiting the accumulation of brassinosteroids (BR), a growth-promoting hormone. LOB also regulates phototropic responses to blue light (the main wavelength used for photosynthesis). Pisum sativum (pea) is an important agronomic plant and is closely related to many other agronomically important legume crops. In pea, the LOB ortholog Apulvinic (Apu) functions to specify the pulvinus, a motor organ located at the base of the leaf that controls nighttime folding of leaves. Because LOB defines organ boundaries by affecting BR levels, it is possible that Apu functions in the same way. Similarly, if the blue light response function of LOB is conserved in pea, then altered Apu expression may result in peas that have altered phototropism and possibly leaf angle, which could allow for planting at an increased density.  This function may also be conserved in related legumes such as soybean. To test the hypothesis that Apu regulates pulvinus formation and leaf angle by affecting BR levels, wild-type and apu-1 mutant peas will be grown under two conditions: treated with the BR biosynthesis inhibitor propiconazole and a control treatment. The pulvinus region will be examined across genotypes and growth conditions via microscopy, and phenotypes will be documented. Analysis of the resulting data may determine the specific role of BR in pulvinus development in pea.

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SPENCER SWANSEN
Seattle Pacific University
Spencer Swansen

A common method of competing for resources involves inhibiting the growth of closely located competitors. This study is a continued exploration of inhibitory growth interactions between fungi. The chytrid fungus Homolaphlyctis polyrhiza has been shown to inhibit growth in the filamentous fungi and model organism Neurospora crassa. This interaction has not been observed between N. crassa

 and other chytrid fungi. The range of fungi affected in this way by H. polyrhiza has been previously explored along with traits of resistance to cold, heat and protease existing in the compound exuded by H. polyrhiza. The genome of H. polyrhiza was previously sequenced and this information was used to identify potential gene products involved in the production of this unique secondary metabolite.  Our work this summer involves further research on the range of fungi inhibited by H. polyrhiza and the properties of the exudate, in addition to continued sequence analysis. I personally maintained different fungal cultures, which I examined with different microscopes to detail inhibition, and performed web based sequence analysis to help in the process of searching for genetic secondary metabolite information.

 

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ROLAND TRUONG
East Los Angeles College
Roland Truong

 Aluminum (Al) has been shown to trigger a DNA damage detection pathway involving ATR, ALT2 and SOG1. These factors have been found to shut down plant growth in the presence of Al and promote repair of the plant. The loss of function of any of these factors results in Altolerance, which means that even though Al is being internalized,  plant growth fails to arrest. While more investigation is necessary to understand what is the true nature of the Al-dependent DNA damage, the detection mechanisms for the apparent damage will continue to be defined by research done in the Larsen Lab. I will investigate how plant cells are programmed to repair the perceived Al-dependent DNA damage. Preliminary evidence suggests that Al inhibits repair by homologous recombination (HR). By utilizing transgenic Arabidopsis containing a GUS reporter construct, visualization of HR can be assessed. Experimentation includes determining the proper concentration of bleocin to grow Arabidopsis in, treating those seeds with increasing Al concentration, and histochemical staining with X-gluc. We hypothesize that bleocin induced damage normally repaired through HR, should show a reduction in GUS accumulation in step with increasing concentrations of Al, indicating an Al-dependent inhibition of repair via HR. 

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University of California, Riverside
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Riverside, CA 92521
Tel: (951) 827-1012

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Center for Plant Cell Biology
Botany & Plant Sciences Department
2150 Batchelor Hall

Tel: (951) 827-7177
Fax: (951) 827-5155
E-mail: genomics@ucr.edu

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