UCR

Center for Plant Cell Biology



REU 2008


REU Students and their Summer 2008 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 2008, the following eleven 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 2008 research programs in CEPCEB laboratories.

An REU Poster Session was held Friday, August 22, 2008 in Keen Hall's lobby area, where students will be available to discuss their projects. The Poster Session will be open to the campus community.

REU Student
College/University
CEPCEB Faculty
Mentor
Byron Doyle Fort Valley State University, Augusta, GA Cutler Lab Simon Alfred
Maritza Duarte

Seattle University, Seattle, WA

Reddy Lab

Ram Yadav

Benjamin Fulton

Skidmore College, Saratoga Springs, NY

Jin Lab

Chellappan Padmanabhan

Aimee Johnson

Western Washington University, Bellingham, WA

Ma Lab

Huanbin Zhou

Joseph Manson

Riverside Community College, Riverside, CA

Judelson Lab

Howard Judelson

Colin Murphree

Transylvania University, Lexington, KY

Rao Lab

Soon Choi

Michael O'Leary

University of California, Riverside, CA

Chen Lab

Theresa Dinh

Rebekah Silva

Riverside Community College, Riverside, CA

Walling Lab

Melissa Smith

Robert Washington

Cal State Polytechnic University, Pomona, CA

Raikhel Lab

Michelle Brown

Andrea Wheat

University of North Texas, Houston, TX

Borkovich Lab

Gyungsoon Park

Ali Zanial

California State University, Bakersfield, CA

Bailey-Serres Lab

Piyada Juntawong

 

BYRON DOYLE
Fort Valley State University, Augusta, GA
Bryon Doyle
Glycosylation is a process by which saccharides link to one another to produce glycans, which can be independent or attached to proteins and lipids. Glycosylated molecules demonstrate the ability to modify the duration and intensity of biological activity and have been shown to modulate drug responses.  Understanding how glycans affect specific biological processes can enhance the understanding of those biological processes.  Previous experiments examining natural variation in Arabidopsis accessions have shown that the small molecule hypostatin is a prodrug that is activated by HYR1, a UDP glycosyltransferase (UGT), to form a bioactive glucoside.  Glycosylated hypostatin modulates etiolated growth resulting in a shortened hypocotyl and an elongated root. The target of glycosylated hypostatin remains unknown. To identify the target of glycosylated hypostatin, we are employing a map based cloning approach. Hypostatin resistant mutants were obtained via a mutant screen.  The identification of the mutated gene responsible for hypostatin resistance may lead to the discovery of the target site of glycosylated hypostatin.  Currently, linkage mapping has placed the hypostatin-resistance gene on chromosome III.  Identifying the target/pathway of glycosylated hypostatin will further our understanding of Arabidopsis growth and shed light on the regulation of glycosylation in general.

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MARITZA DUARTE
Seattle University, Seattle, W
A
Maritza Duarte
The shoot apical meristem (SAM) of plants harbors a set of stem cells in the tip of the shoot apex. The above ground organs in plants are derived from these stem cells via differentiation. Genetic studies using the model plant Arabidopsis thaliana revealed the role of the homeodomain transcription factor WUSCHEL (WUS) in stem cell specification (Mayer et al., 1998). In addition, studies found that stem cells restrict their own size by secreting a peptide ligand CLV3, which interacts with CLV1-CLV2 receptor complex and negatively regulates the expression of WUS at the transcriptional level (Schoof et al., 2000). In a functional SAM, expression of WUS is confined to the organizing centre (OC) located underneath the stem cells. The disparity between the WUS expression domain and its functional requirement, led to the hypothesis that WUS acts non-cell autonomously in specifying the stem cells within the SAM. The studies thus far indicate a non-cell autonomous function for WUS. Dr. Reddy’s lab has designed antibodies against the WUS protein. With this new tool I will address the localization of the WUS protein. Therefore during my research project in Dr. Reddy’s lab I will use immunohistochemistry on sections of fixed plant tissue to visually localize the WUS protein in the SAM. In an alternate approach the WUS protein will be fused with GFP in order to visually follow in real time where the protein is localized. During this research I will learn protein localization in fixed plant tissue by using immunolocalization and live-imaging by confocal microscopy.

References:
Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, and Laux T (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95, 805-815.
Schoof H, Lenhard M, Haecker A, Mayer KF, Jurgens G, and Laux T (2000). The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100, 635-644.

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BENJAMIN FULTON
Skidmore College, Saratoga Springs, NY
Benjamin Fulton
Small RNAs are important regulators of gene expression, including development, genome maintenance, and response to abiotic and biotic stress.  Small RNA’s are generally 20-25 nucleotides long and can be divided into two categories based on their biogenesis: small interfering RNAs (siRNAs) and microRNAs (miRNAs).  Small interfering RNAs are derived from double-stranded RNAs and microRNAs are processed from single-stranded hairpin RNA precursors.  Recently, Dr. Jin’s lab demonstrated that nat-siRNAATGB2, an endogenous siRNA, is promoted in Arabidopsis thaliana by Pseudomonas syringae, which carries the effector avrRPT2.  This nat-siRNAATGB2 appears to repress a negative regulator of the RPS2 resistance pathway and plays an important role in plant defense. Based on this previous research, the purpose of my research is to study a miRNA that is induced in response to the pathogen Pseudomonas syringae.  This specific miRNA targets the coding region of the mRNAs encoding many different genes.  We are currently investigating the roles of these genes in plant immunity to Pseudomonas syringae.  To determine the function of these genes we are using T-DNA knockout mutant lines to see the difference in pathogen resistance.  This analysis requires the use of DNA extraction to confirm the genotype of the mutants, RNA extraction and RT-PCR to determine the gene expression levels of these genes in the mutants, and pathogen assays on the mutant strains to determine their resistance to Pseudomonas syringae.

References:
Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani, Villegas AJ, Zhu JK, Staskawicz BJ, and Jin H (2006) A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci U S A 103: 18002-18007.
Katiyar-Agarwal S, Gao S, Vivian-Smith A, and Jin H (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes & Development 21: 3123-3134.

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AIMEE JOHNSON
Western Washington University, Bellingham, WA
Aimee Johnson
Plant pathogens cause disease to their hosts by secreting molecules that can then disrupt normal plant function. Pseudomonas syringae is a common bacterial pathogen that infects its host by using the type III secretion system, or T3SS, which is a needle-like apparatus for the infection of type III secreted effectors, or T3SEs. These effectors then directly interact with plant substrates and facilitate bacterial infection. HopZ is one family of T3SEs injected by P. syringae. There are three different homologs of the HopZ family — HopZ1, HopZ2, and HopZ3 — the result of a co-evolutionary arms race between pathogen infection and plant resistance. As an REU student in Wenbo Ma's lab, I will be studying hopZ1a, the most ancestral of three allelic forms of HopZ1. In soybean, hopZ1a causes a hypersensitive response whereas hopZ1b, derived from a hopZ1a-like ancestor, evades host recognition and promotes pathogen virulence. My goal for the summer is to use yeast two-hybrid screens, along with PCR and cloning experiments, to identify soybean proteins that are interacting directly with hopZ1a. Sequencing will then be performed to determine what these proteins are. Interacting proteins have already been identified for hopZ1b, and the results from my experiment will allow for the comparison of these proteins in the two allelic forms of HopZ1 and will lead to a better understanding of how hopZ1a triggers soybean defense while hopZ1b evades this recognition.

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JOSEPH MANSON
Riverside Community College,
Riverside, CA
Joseph Manson
The Judelson lab is primarily focused on the oomycete Phytophthora infestans, specifically the structure, expression, and evolution of spore genes. Using various bioinformatic programs, gene database resources, and DNA cloning techniques paired with gene sequencing, I will be researching how P. infestans spores develop, germinate, and the evolutionary relationship to the proteomes of other flagellated organisms. In order to further understand P. infestans spore development, I will evaluate predicted gene structures, design primers for polymerase chain reactions, and amplify promoters from genomic DNA. These promoters will be cloned and tagged using GUS reporters and with the use of various microscopic techniques, we will locate where genes of interest are being expressed. Furthermore, we will be sequencing the plasmids to verify proper construction as well as a transformation procedure of utilized plasmids into P. infestans. The second half of this summer’s project will be analyzing the structure and evolution of the flagellar proteome. We will be using other organisms for direct comparison such as Trypanosoma brucei, Monosiga brevicollis, Homo sapiens, Batrochochytrium dendrobatidis, and Chlamydomonas gruberi. This comparison will give us a better understanding of the evolutionary changes that have occurred in P. infestans using web-based approaches, which include alignment studies and tree-building exercises.

 

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COLIN MURPHREE
Transylvania University, Lexington, KY
Collin Murphree
Many positive-strand RNA viruses cause serious diseases of humans, animals and plants. Despite the fact that replication of RNA viruses occur in the same compartment where cellular RNAs also exist, assembled virions contain only viral progeny RNAs. Our knowledge concerning how viruses filter cellular RNA is still in its infancy.

Cucumber mosaic virus (CMV) is the type member of the genus Cucumovirus and is of substantial agricultural significance. CMV is typical of eukaryotic RNA viruses and its replication is entirely cytoplasmic. Purified virions of CMV package three genomic and a single subgenomic RNA. Some CMV strains also contain another small, single-stranded RNA species, known as a satellite RNA (Sat-RNA).

Sat-RNA is completely dependent on CMV for its replication and is efficiently encapsidated into CMV virions although it shares no significant sequence homology with CMV genomic RNA. Replication of Sat-RNA is species specific. Therefore, other members of Cucumovirus genus are incompetent to replicate the Sat-RNA of CMV and vice versa. Dr. Rao’s lab is employing a variety of molecular, cellular and biochemical approaches to address the following questions: (i) Does Sat-RNA replicate independent of its helper virus? (ii) Is packaging of Sat-RNA functionally coupled to replication? (iii) Is packaging of Sat-RNA sequence or structurally dependent? (iv) Can Sat-RNA package autonomously into virions or does it co-package with helper virus RNAs?         To find some of the answers to above mentioned questions this summer, I will be using the Agrobacterium-mediated transient expression system (agroinfiltration) developed in Dr. Rao’ lab.  The three genomic RNAs of TAV and its Sat-RNA will be subcloned into a binary vector (eg. pCASS-Rz) amenable for agroinfiltration. Following construction of the required agrotransformants, the biological activity of TAV genomic RNAs and its Sat-RNA will be tested by infiltrating desired sets of inocula into Nicotiana benthamiana plants. Northern and Western blot hybridization assays will be used respectively to monitor accumulation of progeny RNA and capsid protein. Encapsidation efficiency of Sat-RNA by transiently expressed virus replication derived capsid protein will be compared to evaluate the required forms of Sat-RNA and capsid protein. Virions assembled with transiently expressed coat protein will be compared to those of native with respect to the configuration of packaged RNAs.  Information obtained from these studies is valuable in understanding how RNA viruses replicate and package their genomes, with an eventual aim of formulating novel methods of virus disease control.

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MICHAEL O'LEARY
Riverside Community College,
Riverside, CA
Michael O'Leary
Gene expression can be, but is not necessarily, regulated at two levels – the transcriptional level by siRNA-mediated transcriptional gene silencing (referred to as TGS), and at the post-transcriptional level by microRNA-mediated post-transcriptional gene silencing (referred to as PTGS).  My two research projects this summer are to 1) use a forward chemical screen to identify players involved in microRNA-mediated PTGS and/or microRNA biogenesis and 2) utilize map-based cloning to identify a mutant involved in TGS that was previously isolated by a post-doc in Dr. Chen’s lab. Dr. Chen has previously generated a 35S::luc-AP2 reporter line.  This reporter construct is driven by a double 35S promoter that generates siRNAs leading to DNA methylation of the reporter gene.  This reporter also contains a miRNA binding site (for miRNA172) of the AP2 gene, allowing us to potentially discover factors involved in microRNA-mediated PTGS. In the chemical screen I will use libraries available on campus to screen for players involved in TGS and microRNA-mediated PTGS.  The plants will be grown in 96-well plates to the two leaf stage, at which point each well will be treated with a different chemical and then assayed for increased luciferase activity.  Compounds that result in increased activity will undergo a secondary screen as well as DNA-methylation assays to confirm that the chemicals affect the TGS pathway.
The goal of the map-based cloning project is to pinpoint the position of a putative mutant defective in microRNA-mediated PTGS and clone the gene.  This mutant was isolated from an EMS screen (EMS is a chemical agent that produces random point mutations) and has increased luciferase activity, as well as a PIN-like phenotype at the cotyledon stage.  The mutant was backcrossed to Ler and the F2 progeny from this cross will be used for the map-based cloning.  Preliminary mapping indicates that this gene may be located at the bottom of chromosome three.

 

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REBEKAH SILVA
Riverside Community College, Riverside, CA
Rebekah Silva

The Walling Lab is currently studying the signal transduction pathways in tomato (Solanum lycpersicum) that are triggered when the plants experience biotic and abiotic stress.  Much of the focus is on the octadecanoid pathway activated by mechanical wounding and chewing insects which produce the potent elicitor jasmonic acid (JA).  Recently, the Walling lab hs found a role for leucine aminopeptidase-A (LAP-A) in the late branch of the octadecanoid pathway, which regulates genes involved in insect deterrence.  Transgenic lines developed in the Walling lab (35S:LapA silenced (LapA-SI) and 35S: LapA over-expressed (LapA-OX)) have shown that in the absence of LAP-A, the tomato plant’s ability to respond to herbivory significantly decreases in comparison to the wildtype (WT) control.  In addition to the extensive plant damage, late gene RNA accumulation in the LapA-SI lines was considerably diminished.  LapA-OX lines have also shown that increased levels of LAP-A decrease the amount of plant tissue damage caused by chewing insects, relative to WT tomato plants.  These data suggest that LAP-A is a regulator of the late gene wound response.  Many other signaling molecules have also been implicated in regulating the wound response.  One such example is hydrogen peroxide (H2O2).  Levels of H2O2 have been shown to increase in response to plant stress, and treatment with H2O2 is also known to induce expression of late response genes in unwounded plants.  While the mechanism of action is unknown, both H2O2 and LAP-A only appear to function in the late wound response pathway.  Therefore, LAP-A may regulate H2O2, which in turn regulates gene expression.  In order to determine if LAP-A is a regulator of H2O2, LapA-SI, WT, and LapA-OX lines of tomato plants will be treated with H2O2 to determine if H2O2 can complement the absence of LAP-A in late wound response.   

References:
Fowler JH, Aromdee DN, Pautot V, Holzer FM, Beckage N, and Walling LL  (2007)  Leucine aminopeptidase regulates wound and defense signaling in tomato (Solanum lycopersicum).  (submitted). 
Orozco-Cardenas ML, Narvaez-Vasquez J, and Ryan CA  (2001)  Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate.  Plant Cell. 13, 179-191.
Walling LL  (2000)  The Myriad Plant Responses to Herbivores.  J Plant Growth Regul.  19, 195-216.

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ROBERT WASHINGTON
Cal State Polytechnic University, Pomona, CA
Robert Washington
Plant endocytic pathways have only recently been under investigation. Up until the last decade, the scientific community believed that the cell wall and the plant cell’s turgor pressure prevented endocytosis from occurring.  The use of dyes, labeled sterols, and reporter-fusion markers revealed that plants do undergo endocytosis. To understand this highly active process, a chemical genomics approach was utilized. This method employs small molecules to disrupt protein function. This allows the researcher to eliminate the problems of redundancy of large gene families and the lethality of essential genes. In an initial chemical genomics screen of a 2,000 compound library, the Raikhel laboratory has discovered a novel compound that affects endocytosis in plants. The Raikhel laboratory now has expanded the screen to include an additional 59,000 compounds contained in the Center for Plant Cell Biology (CEPCEB) library. The performed primary and secondary screens usetobacco (Nicotiana tabacum) pollen as a model system of active endocytosis. Tobacco pollen requires minimal care, and is easily imaged for high-throughput format via confocal microscopy techniques. The primary screen assays for aberrant phenotype and/or inhibition of germination of the pollen tube. Derived from positive primary screening results, the secondary screen will assay pollen tube phenotype and the localization of a ROP GTPase-associated protein marker that localizes at the apical tip in pollen tube growth. The compounds exhibiting mis-localization of the protein marker phenotype will then be assayed on Arabidopsis thaliana seedlings.This summer I will be participating in the data analysis of 36,000 images from the primary screen, perform the actual screening on the last 12,000 compounds to be tested, and assist in the secondary screening of approximately 400 compounds.

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ANDREA WHEAT
University of North Texas, Houston, TX
Andrea Wheat
The Borkovich lab is currently working with Neurospora crassa, a filamentous fungus and model organism. The genome for N. crassa has previously been sequenced and I will be working with 16 phosphatase mutants previously made in the lab by the throughput knockout procedure. Phosphatases aid in the regulation of dephosphorylation within the cell cycle. Each mutant has one of the phosphatase genes “knocked-out” by homologous recombination. A hygromycin resistance gene replaced the target gene and the mutants were then grown on hygromycin, leaving behind only true mutants. Now the mission is to use phenotypic analysis and determine the function of each of the phosphatase genes. I will be using microscopy to analyze vegetative growth, cell integrity, and sexual development.

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ALI ZANIAL
California State University, Bakersfield, CA
Ali Zanial
The Bailey-Serres’ lab is mainly concerned with the regulation of gene expression of an mRNA encoding a specific protein. These mRNAs are undergoing translation, but those who are not involved in translation, are sequestered into distinct mRNA/protein complexes that either preserve or destroy the transcripts. The lab’s long-term goal is to fully understand how the cell differentiates between mRNAs under stress conditions, more specifically under hypoxia (low oxygen). This goal involves a variety of cellular proteins that either bind mRNAs or are vital translational machinery compenents. My project this summer will examine the regulation of mRNA translation by focusing specifically on the role of plant ribosomes and ribosomal proteins in Arabidopsis thaliana. Ribosomes are essential cellular organelles that are composed of two subunits (large and small subunit) and consist of four rRNAs and 80 proteins. The purpose of my project is to develop key important tool which will be utilized to determine the role and the function of a set of four proteins, called “P-proteins,” of the large ribosomal subunit. The JBS lab is interested in studying the “P-proteins” for several reasons: 1) The majority of proteins are highly basic, whereas the “P-proteins” are highly acidic. 2) They form a flexible complex that assist in the translocation step of the polypeptide formation. 3) All four are phosphorylated. 4) All four are evolutionary distinct and form complexes. My project will utilize transgenic Arabidopsis lines that have been produced that contain an epitope-tag version of P1 and utilize the molecular technique of Co-immunoprecipitation of polyribosomes that posses a FLAG-tageed ribosomal protein with a-FLAG agarose beads to determine protein interactions. My second project, involves generating DNA constructs that will enable the production of more transgenic Arabidopsis lines that will produce an epitope-tagged versions of the P2a, P2b, and P3 proteins. Finally, my third project will involve in aiding a graduate student in performing experiments to evaluate mechanisms of translational regulation.

More Information

General Campus Information

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

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Center Information

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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