UCR

Center for Plant Cell Biology



REU 2006


REU Students and their Summer 2006 Research Programs

Undergraduate students were invited to apply to the Center for Plant Cell Biology (CEPCEB) to pursue individual research projects in the area of plant cell biology. In 2006, the following ten students were accepted to this ongoing 10-week residential summer program.  Please click on the following student links to see photos and read about their Summer 2006 research programs in CEPCEB laboratories.

REU Student
College/University
CEPCEB Faculty
Mentor
McKell Dilg Brigham Young University, Provo, UT Zhu Lab Paul Verslues
Caleb Jones Eastern Oregon University, La Grande, OR Borkovich Lab Liubov Litvinkova
Abby Nitschke Loyola University, Chicago, IL Yang Lab Ying Fu
Larry Page University of Missouri, Columbia, MO Ozkan (M) Lab Nate Portney
Fady Rofail Cerritos Community College, Norwalk, CA Rao Lab A.L.N. Rao
Angela Rowe Ohio State University, Columbus, OH Jin Lab Serekha Katiyar-Agarwal
Jason Schoneman California State Polytechnic University, Pomona, CA Judelson Lab Howard Judelson
Cheri Tilburg Rochester Institute of Technology, Rochester, NY Chen Lab Zhiyong Yang
John Tracey St. Norbert College, DePere, WI Eulgem Lab Mercedes Schroeder
Carrie Wang University of Colorado, Boulder, CO Smith Lab Siddhartha Kanrar

 

MCKELL DILG
BRIGHAM YOUNG UNIV., PROVO, UT
My project involves the characterization of ABA-binding proteins. ABA is a plant hormone that affects many processes in plants. These affects are mainly related to plant stresses caused by reduced water availability. Some of the processes affected are, seed drought tolerance, dormancy and protein synthesis. (Razem, et. al., 2006) On a molecular level, the pathways for ABA perception and signaling are not fully understood, namely a receptor for ABA had not been discovered. Recently, Razem, et. al., determined that an RNA binding protein FCA, which is involved in flowering, is an ABA receptor.

In order to study more in depth FCA as an ABA receptor we will attempt to construct a fluorescent ABA sensor using a portion of the FCA protein. Fluorescence can be observed as an absorption of light at a given wavelength and then subsequent emission at a lower wavelength. We will be using two specific fluorescent indicators, CFP and YFP. CFP is a cyan fluorescent protein, and YFP is a yellow fluorescent protein. The special thing about these two fluorescence proteins is that together they create what is called FRET: fluorescence resonance energy transfer. Light is first absorbed by one fluorescent molecule (CFP), and then the energy is transferred to a different molecule (YFP) and finally emitted. The larger the FRET, the closer together CFP and YFP are.

In my experiments I will construct a fusion protein where CFP and YFP are attached to opposite ends of a portion of the FCA protein. We will look for changes in CFP-YFP FRET upon ABA binding in in-vitro ways. Preliminary experiments have shown a change in FRET upon ABA binding with this fusion protein. We will attempt to first, maximize the ABA binding dependent change in FRET by modifying the linkers between the CFP-FCA-YFP fusion protein and second, further define the ABA binding site by making additional truncated versions of FCA.

Protein expression and ABA binding assays with other potential ABA binding proteins are also underway.
References: Razem, A. Fawzi, et. al. The RNA-binding protein FCA is an abscisic acid receptor. Nature, 439, 290-294 (2006).

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CALEB JONES
EASTERN OREGON UNIV., LA GRANDE, OR

The Borkovich Lab is working on a five year project with Dartmouth Medical School and UCLA to learn the function of the Neurospora crassa genome. N. crassa is a filamentous fungus that is a model organism in which plays an important role in numerous accounts of research and discoveries. N. crassa has already been completely sequenced (there are 10,620 genes), but now the goal is to learn the function of each of the genes. In order to figure this out, knockout mutants have been created.

My project this summer will be analyzing at about twenty knockout mutants and phenotyping them. To analyze them I will be growing them on different types of medium, watching their growth, and keeping a photo record, using several forms of microscopy. The genes that will be “knocked out” in these mutants are protein phosphatases. Not much is known about the phophatases in N. crassa; they have been studied more in other organisms such as Saccharomyces cerevisae. Phosphatases are dephosphorylating negative regulators in several cell pathways. They regulate physiological responses to extracellular signals. My goal for the end of the summer will be to successfully phenotype the protein phosphatase genes to see what purpose they serve in N. crassa.

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ABBY NITSCHKE
LOYOLA UNIV., CHICAGO, IL
Plant cell shape is directly related to cell function. In Arabidopsis, the leaf epidermal cells (pavement cells) take on a jigsaw puzzle like appearance, which requires coordination between neighboring cells to form lobes and neck regions. In Dr. Yang’s lab, we study the mechanisms governing cell shape formation, using Arabidopsis pavement cells as a model system. Previous studies have already determined that cell shape formation is controlled by a ROP GTPase network involving several ROP GTPase downstream target proteins including RIC1. RIC1 is a novel microtubule associate protein (MAP). The over expression of RIC1 suppresses cell lobe formation and expansion. In order to understand this mechanism, we need to identify other proteins that interact with RIC1. Our approach is to search for genetic mutations that affect the RIC1 over expression phenotype. As my project for the summer, I will be screening for RIC1 suppressors in mutant lines that have already been created using chemical ( EMS ) induced mutagenesis. The phenotype of the epidermal cells will be used to identify possible useful mutations. Taking advantage of the unusual shape of epidermal cells from plants overexpressing RIC1, I will be able to determine whether or not a particular mutation can counteract the effect of RIC1 overexpression. I will also create new mutants using T-DNA tagging under a ric1 knockout background as well as a RIC1 overexpression (low level) background. These lines will be used to screen both enhancers and suppressors in the future. Continuing investigation on these enhancers and suppressors will lead to a thorough understanding of the mechanisms behind the cell morphogenesis.

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LARRY PAGE
UNIV. OF MISSOURI, COLUMBIA
Atomic Force microscopy (AFM) is a powerful technique for visualization of biological systems up to a resolution of 10 nanometers. Some of the advantages of this microscope are that the sample does not have to be fixed in order to view it, it can accurately view soft biological membranes, and it has a high enough resolution to distinguish some of the surface topography of cells and virus particles. Using this microscope, I will be visualizing Cow Pea Mosaic Viruses (CPMV) attached to Iron Oxide Nanoparticles (IONs). CPMV-ION hybrids offer a possible mechanism for new types of dyes to be used in therapeutics. It is possible to mutate virions to target specific types of cells (i.e. cancerous cells) and the IONs will allow for visualization using magnetic resonance imaging. IONs offer an advantage over typical cancer detection methods because they allow the visualization of much smaller tumors. This is important because detecting cancers before they are large enough to spread throughout the body makes them much easier to treat and remove. I will be creating the CPMV-ION hybrids, visualizing them using AFM, and analyzing their interaction with living human breast carcinoma and non tumerogenic epithelial cells.

 

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FADY ROFAIL
CERRITOS COMM. COLLEGE , NORWALK, CA
Positive-strand RNA viruses cause major diseases in humans, insects, animals and plants. Almost all viruses are disseminated to new hosts only in fully assembled form. Therefore it is imperative to understand how these viruses are assembled and mature. I work under the supervision of Dr. A. L. N. Rao, on a research project focusssing on how eukaryotic viruses package their RNA genomes into virions. Dr. Rao’s lab uses two single-strand, positive sense RNA viruses as model systems: a plant infecting multicomponent brome mosaic virus (BMV) and flock house virus (FHV) an insect virus with broad host range including plants. Dr. Rao’s lab developed a T-DNA based Agrobacterium-mediated transient expression system (agroinfiltration) that not only facilitates efficient transient expression of one or many combinations of a desired set of viral RNAs without having to replicate but also effectively uncouples replication from packaging.

The major focus of my project is to examine how FHV is transported between plant cells to achieve systemic movement. The genome of FHV is bipartite: RNA 1 (F1) codes for a non structural protein that is required for viral replication and another non structural protein B2 expressed as a subgenomic RNA3 (sgRNA3). Protein B2 has been identified as a suppressor of RNA silencing. RNA2 (F2) encodes the structural capsid protein gene which results in co-packaging of RNA1 and 2 into a single virion. The sgRNA3 is not encapsidated into virions. Although FHV replicates efficiently in plant cells, it can not spread from the point of initial entry to neighboring cells and to distal part of the plant due to the lack of genes that are involved in this process (referred to as movement protein). This defect however can be circumvented, by complementing with movement proteins of either tobacco mosaic virus (TMV) or red clover necrotic mosaic virus (RCNMV). In the absence of complementing movement proteins the spread of FHV remain subliminal. In addition to movement protein, some viral systems also require additional proteins such as capsid protein.

My research will focus in addressing the following questions: (1) Does spread of FHV require both movement protein and capsid protein? (2) To what extent does FHV coat protein contribute to cell-to-cell spread? (3) Does FHV spread in virion or non-virion form? To find answers to these questions I will use a FHV RNA2 derived defective interfering RNA (DI-634) as an experimental vehicle in combination with green fluorescent protein (GFP) as a tracer molecule. DI-634 replicates in the presence of F1 and is also encapsidated by the FHV capsid protein. After subcloning GFP ORF into DI-634, the resulting DI-634/GFP will be inserted into pCass4 binary vector amenable for agrotransforamtion. Following agrotransformation, DI-634/GFP will be co-infiltrated with F1 and F2 to Nicotiana benthamiana leaves. Infiltrated leaves will be harvested at regular time intervals and are subjected to confocal laser scanning microscopy for monitoring temporal expression and localization of GFP. For studying encapsiadtion, virion RNA profile will be compared to that of total RNA by Northern hybridization using riboprobes specific for FHV RNAs. The inherent nature of the research project will expose me to various recombinant DNA techniques such as PCR, cloning, gel electrophoresis, agrotransformation, infiltration, virus purification, RNA isolation, confocal laser scanning microscopy, Northern and Western blot hybridizations.

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ANGELA ROWE
OHIO STATE UNIV., COLUMBUS, OH
Pseudomonas syringae is a gram negative bacterial pathogen which attacks a wide range of commercial crops. The purpose of my research this summer will be to characterize the function of several candidate signaling genes in plant disease resistance and defense responses. We will use both loss-of-function and gain-of-function for this study. A PCR based screening method will be used to isolate homozygous Arabidopsis mutants for an intracellular nucleotide binding/ leucine rich repeat (TIR-NBS-LRR) gene and RRM protein. The western blot technique will be used to screen for the amount of expression of the desired protein in our over expression transgenic plants. These will be inoculated with P. syringae via agro bacterium infiltration. Pathogen growth assays and hypersensitive response assays will be performed to determine the effect of these proteins.

 

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JASON SCHONEMAN
CALIFORNIA STATE POLYTECHNIC STATE UNIV, POMONA, CA
Phytophthora infestans is a highly destructive plant pathogen on potato. The resulting destruction creates an annual total of billions of dollars in damage to the crop worldwide. Around the mid-nineteenth century, a pandemic of this late blight disease on potatoes resulted in the death and displacement of the population of Ireland. Recently, localized, severe epidemics have occurred in Europe and Asia due to specific mating types of the pathogen on potato being sent to these locales from P. infestans’ presumed place of origin—Mexico. Accordingly, the Judelson lab is using current genetic tools and genomic resources to analyze and identify which genes are upregalated the most during asexual and sexual sporogenesis. Discovery of specific genes or gene groups regulating these processes may prove essential to developing successful control strategies against this tenaciously successful organism.

Asexual spores are extremely important in the pathogenesis of P. infestans. They are actively involved in the infection of the plant and the dispersal of inoculum. Asexual spores consist of sporangia and the zoospores formed within the sporangia. In P. infestans, sporangia can detach from the hyphal body and act as a dispersal agent. At cooler temperatures six or more biflagellate zoospores can exit the sporangia and further travel to find their host. Before the zoospore can infect the host, it has to encyst and germinate. Essentially, all the steps from hyphae to germination of encysted zoospores may involve the expression of important sporogenesis genes.

The Judelson lab has assembled an EST dataset consisting of genes upregulated greater than ten times during asexual sporulation. They produced this dataset from microarray (Affymetry Genechip) analysis studies. I will use the EST dataset to construct correlating promoter regions in closely related Phytophthora species with bioinformatics tools. Using the BLAST (Basic Local Alignment Search Tool) of an annotated database, I will find gene and promoter homologs in these closely related Phytophthora species. Also, I will BLAST the EST clones on a raw sequence dataset for P. infestans (an annotated database for this species is not yet developed). This will allow me to move or “walk” in the sequence database to find promoter regions. After compiling the promoter homologs of the closely related Phytophthora species, I hope to find conserved blocks within these promoters using programs such as CLUSTAL, GIBBS MOTIF, SAMPLER, and others. Using Polymerase Chain Reaction, the promoters will be amplified and then cloned into the pOGUS reporter plasmid. I will then transform the plasmid into P. infestans.

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CHERI TILBURG
ROCHESTER INST. OF TECHN., NY
First, a little background: MicroRNA (miRNA) and small interfering RNA (siRNA) have recently been identified as important in specific gene regulation in plants and animals. These small RNA molecules are highly conserved, between 20-24 nucleotides in length and are thought to be involved in early development, cell proliferation and cell death, apoptosis and fat metabolism, and cell differentiation. Both miRNA and siRNA bind to mRNA and lead to the destruction, modification, or inhibition of the specifically targeted base pair sequence of an mRNA. In plants, methylation at the 2' OH on 3' terminal nucleotide protects from enzymes that target the 3' OH at the 3' terminus. The Chen lab has previously proven that the methylation is done by the HEN1 protein, which works as a methyltransferase in vitro. HEN1 acts with other unknown proteins, and the next step is using the yeast two-hybrid system to find associated proteins.

The yeast two-hybrid system was developed using the GAL4 gene in E.coli to study protein interactions in all organisms. First we will attach the known gene of protein, X, to the binding domain (BD) of GAL4, creating the "bait". Next, we will attach each gene of the Arabidopsisthaliana cDNA library to an activating domain (AD), which creates the "hunters". When both of these plasmids are transformed into yeast the proteins in the cDNA library that interact with HEN1 will bring the BD and AD close together thus activating the GAL4 reporter gene. The GAL4 gene encodes for B-galactosidase activity, and when a lift filter assay is performed the colonies that are blue show the positive result of a protein's interaction with HEN1. The goal of this summer's REU project is to use the yeast two-hybrid system to identify the proteins that interact with HEN1 to methylate the siRNA in plants. For more information on the yeast two-hybrid system please visit http://www.biochem.arizona.edu/classes/bioc568/two-hybrid_system.htm

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JOHN TRACEY
ST. NORBERT COLLEGE, DEPERE, WI
Throughout the summer, I will be working in Dr. Thomas Eulgem’s lab under the supervision of graduate student Mercedes Schroeder. Dr. Eulgem’s lab focuses mainly on genetic regulation of the plant defense response. Plants are sessile and therefore have evolved complex immune systems and strategies of dealing with pathogens. Plants have what are known as incompatible and compatible defense reactions. My project involves looking at these response reactions in the model plant Arabidopsis thaliana to the fungus-like oomycete Hyaloperonospera parasitica (Peronospera). The main goal of the project is to use enhancer trapping methods to analyze the sequences of crucial enhancer and promoter elements used in regulation of the defense response.

We are screening over 11,000 Arabidopsis trap lines. The enhancer traps consist of a reporter gene fused to a minimal promoter, which were then inserted randomly into the genome. If the trap inserts near an enhancer, the reporter gene will be turned on. The reporter gene in the enhancer traps is known as the GUS gene and encodes for the enzyme ­β-glucoronidase. Since we are interested in the traps that are inserted near genes involved in the immune response, we “GUS-stain” the leaves and look for the staining of tissue near the Peronospera infection, along hyphae or next to a germinating spore. Individual leaves are examined using light microscopy and then tissue staining is verified with confocal microscopy. Once candidates are discovered, DNA will be isolated and amplified using TAIL-PCR (Thermal Asymmetric Interlaced), which amplifies DNA of unknown sequence upstream and downstream of the T-DNA insertion of the enhancer trap. We will then BLAST amplified sequences against the Arabidopsis genome and analyze them. Finally, a 5’deletion analysis will be initiated in order to examine the location and sequence of the enhancers used to regulate defense genes. A long term application of the research involves modifying these enhancers to help improve disease resistance in crops.

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CARRIE WANG
UNIV. OF COLORADO, BOULDER, CO
In flowering plants, the transition from the vegetative to reproductive stage constitutes a major developmental phase change. However, the cellular and molecular mechanisms for floral specification are poorly understood. Our laboratory focuses on the mechanisms in the shoot apical meristem (SAM) that initiate flowers in response to floral inductive cues. In Arabidopsis thaliana, two paralogous BEL1-like (BELL) homeodomain transcription factors, PENNYWISE (PNY) and POUNDFOOLISH (PNF) are required for floral specification during reproductive growth. Double mutants of pny-pnf are not capable of transitioning from vegetative to reproductive development. However, the SAM still displays the physical and molecular modifications which occur after receiving floral inductive signals.

To understand the biochemical functions of PNY/PNF, I am developing a procedure to purify the PNY-transcriptional complex from isolated inflorescence SAMs. The goal is to identify the molecular mass of the complex and the components that constitute the complex. Cauliflower, a close relative of Arabidopsis, will be utilized as the source for inflorescence meristems due to its availability and ease of isolation. Techniques such as ion exchange, gel filtration, SDS-PAGE, and immuno-precipitation will be used in order to purify the PNY-transcriptional complex. Future research will focus on determining the function of these proteins during flowering, and the mechanisms of inflorescence development.


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