UCR

Center for Plant Cell Biology



REU 2011


REU Students and their Summer 2012 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 2012, the following twelve 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 research programs in CEPCEB laboratories.

REU Student
College/University
CEPCEB Faculty
Jessica Ball Clemson University, SC Larson Lab
Derreck Carter-House

Southwestern College, CA

Yang Lab

Kyle DeHart

University of Pittsburgh, PA

Roper Lab

Aubrie De La Cruz

Cal Poly Pomona, CA

Bailey-Serres Lab

Justin Durancik

Northern Illinois University, IL

Borkovich Lab

Christopher Galley
Chaffey College, CA Springer Lab

Caren Khachatoorian

California State University, Northridge, CA

Judelson Lab

Andrew Lyne

Kansas Wesleyan University, KS

Cutler Lab

Minhtu Nguyen

South Seattle Community College, WA

Ding Lab

Megan Riley

Moorpark College, CA

Nothnagel Lab

Benjamin Schlau

Portland State University, OR

Eulgem Lab

Erin Sternburg

California State University, Long Beach, CA

Chen Lab

 

JESSICA BALL
Clemson University, SC
Jessica Ball

In Arabidopsis thaliana, EER6 is involved in cell wall degradation, where loss-of-function mutations cause an enhanced ethylene response in the presence of ethylene, resulting in an exaggerated triple response (apical hook, short roots, and severely short hypocotyls). Currently in the Larsen lab, we are trying to understand the role of EER6 in the ethylene signaling pathway. To do so, we are creating double mutants containing an eer6 mutation and either ein (ethylene insensitivity) 2-5, ein 3-1, or ctr (constitutive triple response) 1-3, which will allow us to decipher how EER6 fits into the current model of ethylene signaling. Next, we must determine where in the plant the EER6 gene is expressed. To do so, we will perform Northern analysis to show whether it is found in the leaves, roots, stems or flowers. Next, a GUS staining assay will tell us where the promoter of the gene is active. Finally, we will use GFP localization to assess EER6 accumulation in the cell. The final piece to my part in this project is to supplement growth media with cell wall fragments and plant eer6 mutant seeds. We believe that EER6 helps regulate ethylene signaling via the cell wall fragments it produces. Therefore, by adding cell wall fragments to an eer6 mutant, we suspect that this may restore the plant to wild-type characteristics. Overall, everything we learn about the ethylene signaling pathway can help prolong fruit ripening in agricultural crops.

 

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DERRECK CARTER-HOUSE
Southwestern College, CA
Derreck Carter-House

Upon auxin interaction with auxin binding protein 1 (ABP1), ABP1 activates both ROP2 and ROP6. Through two mutually exclusive pathways, ROP2 and ROP6 create lobes and indentions respectively in plant pavement cells. ROP2 promotes F-actin accumulation via its effector protein RIC4. Increased formation of F-actin leads to out-growth, aka lobes. ROP6, via its regulator RIC1, is suspected to lead to an increased organization of microtubules (MT). The MT restrain the outward growth of the cell and creates and indention. Within seconds of detecting auxin these incredible proteins begin working antagonistically to create lobes and indentations between cells. Understanding the communication and processes that lead to lobing and indenting hold exciting potential for developmental biologists who hope to better understand polarity in developing cells. My work will focus on the rop6-2 knock out line, a knockout in the ROP6 gene. EMS mutagenesis, which allows introduction of further mutations into the rop6-2 plant line, was performed prior to my arrival. After planting the post-mutagenesized seeds in MS media or soil, I will be screening for the enhanced phenotype (less indented cells) in seedlings amongst the population. Subsequently, I will use map-based cloning to isolate mutated genes in the enhanced phenotype plants that I find.

 

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KYLE DEHART
University of Pittsburgh, PA
Kyle DeHart

The Gram-negative bacterium Pantoea stewartii subsp. stewartii is an important pathogen, which infects sweet corn and maize resulting in the disease, Stewart's wilt. It induces disease by colonizing the xylem vessels of seedlings where it aggregates into biofilms. The formation of these biofilms, a crucial component of virulence, obstruct water flow leading to wilting and plant death. When pathogens infect plants they must overcome the host defense response, which includes the production of toxic reactive oxygen species (NO, H2O2). OxyR and SoxR are both conserved bacterial transcription factors that play a role in recognizing this redox stress and mitigating it. It has been demonstrated that these oxidative stress response proteins also have a role in initial surface-cell attachment and biofilm formation. This project will continue this investigation by screening a mutant for the soxR gene(delta-soxR) to determine whether this protein also plays a role in bacterial attachment. A crystal violet staining procedure in a high throughput 96 well plate format will be used to quantify levels of attachment compared to wild-type and delta-oxyR. Furthermore, an OxyR binding domain exists upstream of rtx, a gene believed to function in bacterial adhesion based on sequence homology to other adhesion expressing genes. Using gateway recombinational cloning, we will fuse the rtx domain to a fluorescent reporter protein in the pRH009 plasmid and will then retransform it back into P. stewartii with the goal of conducting protein localization studies. The results will provide insight into P. stewartii biofilm formation which could serve useful in designing target specific chemicals that will render xylem-dwelling bacteria non-virulent.

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AUBRIE DE LA CRUZ
Cal Poly Pomona, CA
Aubrie De La Cruz

RNA binding proteins (RBPs) are of extreme significance in all organisms as they play large roles in post-transcriptional regulation mechanisms, thereby controlling gene expression. RBP functions can be very diverse. They can, for example, determine whether a mature mRNA transcript will proceed through translation, or be arrested at translation initiation, resulting in the formation of stress granules, or be tagged for degradation, resulting in the formation of processing bodies. In Arabidopsis thaliana, 1200 RBPs have been identified based on homology searches, but only the functions of a relative few are well understood. One of the goals of the Bailey-Serres lab is to take a few of these RBPs, and examine their effects on development or stress responses in Arabidopsis rbp mutants. The rbp mutant seeds will be grown under different stress conditions to help determine in which biological processes these RBPs are involved. Another objective of this lab is to measure the abundance of specific RBPs in both the wildtype and mutants. We will do this by performing Western blotting and immunopurification, using antibodies that are designed to bind these proteins. Finally, we intend to find out where RBPs accumulate in the cell by tagging them with mCherry fluorescent protein and viewing the cells using microscope imaging. With these three goals in mind, we hope to bring some of the unexplored roles of RBPs to light.

 

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JUSTIN DURANCIK
Northern Illinois University, IL
Justin Durancik

Aspergillus nidulans is a filamentous fungus and model organism for other Aspergillus species, such as A. flavus, a common pathogen of corn, peanuts, and various other crops. A. flavus and some other Aspergillus species produce a toxin known as aflatoxin. Aflatoxin is a carcinogenic secondary metabolite produced by the fungi. A. nidulans is often used to study this pathway because it used the same pathway to produce the mycotoxin sterigmatocystin (ST), a much less harmful toxin than aflatoxin. For my project, I will screen for chemicals that disrupt the biosynthetic pathway responsible for producing aflatoxin. This will be done three separate ways. First, I will analyze vegetative growth of A. nidulans in the presence of various chemicals. Second, production of norsolorinic acid (NOR) will be observed in a stcE mutant of A. nidulans. NOR is produced as a precursor in the aflatoxin biosynthetic pathway and can be easily visualized as an orange pigment. Third, I will screen for chemicals that inhibit the VeA-LaeA protein-protein interaction. This protein interaction has been previously shown to affect production of secondary metabolites, including aflatoxin, in Aspergillus species. The experiment will be done by performing a yeast 2 hybrid, so that growth of the yeast is dependent upon the interaction of these two proteins. All of the experiments mentioned above will be done in 96 well plates and use the biomek FX robot for dispensing media and chemicals. The chemicals will be obtained by the UCR chemical libraries.

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CHRISTOPHER GALLEY
Chaffey College, CA
Christopher Galley

Leaves are essential plant organs because plants use their leaves for a variety of purposes including photosynthesis, defense, or water conservation. Leaves exhibit many different shapes and sizes to accommodate their intended functions. Despite the variance in leaf structure and function, all leaves develop from the shoot apical meristem (SAM), a group of undifferentiated cells that give rise to all lateral organs. During lateral organ development, an area between the new organ and the SAM forms, known as the boundary region. The cells in this region are small in size and rarely divide compared to the surrounding cells.  The boundary region plays an important role in lateral organ development because plants carrying mutations in boundary-expressed genes have defects in organ separation (Rast and Simon, 2008). One gene that is important in organ separation in Arabidopsis is LATERAL ORGAN FUSION1 (LOF1). LOF1 encodes a MYB domain transcription factor that is expressed in the boundary region of all lateral organs. lof1 mutants exhibit fusions of axillary stems to cauline leaves, which suggests LOF1’s central function is to separate lateral organs from the SAM (Lee et al, 2009). Since transcription factors often work in protein complexes, my project in Dr. Springer's laboratory is to identify possible proteins that interact with LOF1 using a Yeast-2-Hybrid Assay. A cDNA library from Arabidopsis inflorescences will be transformed into yeast, which will then be grown on selective media. Plasmid DNA from the resulting yeast colonies will be extracted, amplified using PCR, categorized using restriction digestion, and then sequenced to identify possible genes that encode for proteins that interact with LOF1.

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CAREN KHACHATOORIAN
Cal State Univ., Northridge, CA
Caren Khachatoorian

This summer I will be working on two projects involved with an organism called Phytophthora infestans. This is a member of the oomycete family that infects potato and tomato plants, causing a devastating disease known as late blight. In my first project, I will be experimenting with new promoters to evaluate their abilities in directing transgene expression. After fusing the promoters to the beta-glucuronidase (GUS) reporter gene, the constructs will be transformed into P. infestans. I will then extract proteins, quantitate total proteins, and perform a GUS assay to measure the activities of the promoters. My second project is to test an efficient silencing tool based on artificial microRNAs (miRNA), using a major secreted protein as a target. I will design the desired nucleotide sequence, amplify the sequence by polymerase chain reaction (PCR), clone it into an expression plasmid, and then insert them into P. infestans. If successful, the miRNA will block the expression of the targeted protein. I will be doing PCR, silver staining of protein gels, and western blot analysis will identify transformants that have reduced levels of expression. This project will also help us learn more about miRNAs in P. infestans.

 

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ANDREW LYNE
Kansas Wesleyan University, KS
Andrew Lyne

World’s foods are becoming less available due to population growth. Additionally, limited agricultural lands are losing more to global warming, which causes desertification or degradation of arable land. Therefore, we must find ways to overcome these problems. The only sustainable way to help these is to improve plant productivity. Major crop losses are attributed to abiotic stress, and current research focused on understanding and manipulating plant abiotic stress pathways is of importance so that new knowledge and research tools can be developed and used to improve agricultural productivity. Sean Cutler’s lab is interested in understanding and manipulating plant stress responses to help achieve these long-term goals. This project focuses on the stress hormone abscisic acid (ABA), which is one of the central molecules in plant stress physiology. ABA controls its signaling pathway that functions to help plants cope with water deficiency by regulating guard cell closure and inducing other physiological changes that assist with abiotic stress tolerance. Currently, the project is interested in identifying novel ABA signaling factors. This is being done in the lab by trying to find factors that affect ABA signaling with the goal of isolating 19 mutants related to the ABA pathway. However, the isolated 19 mutants are still not well characterized. My objective in this project is to characterize the 19 mutants thorough the phenotypes of the mutants based on seed germination, transferring the mutant seedlings to soil, water loss experiments with Arabidopsis leaves that are being grown from the mutant seeds, molecular gene expression experiments, and possibly mapping the mutations in the Arabidopsis genome related to the ABA signaling pathway. My research will contribute to identify the novel ABA signaling factors.

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MINHTU NGUYEN
So. Seattle Community College
Minhtu Nguyen

Cucumber mosaic virus (CMV) is a plant pathogenic RNA virus, which infects over 1200 species, including crops and fruits, resulting in severe economic loss worldwide. In Dr. Ding's lab, we are focusing on the mechanisms of plants for combating viral infections, particularly through RNA silencing. We attempt to elucidate how CMV replicates and propagates as well as how small RNAs mediate anti-virus defense in the model plant Arabidopsis thaliana. In previous research, using a mutated CMV, CMV-2b, in which virus silencing suppressor 2b was deleted, the lab demonstrated that vsiRNAs were produced through Dicer and amplified by RDR-dependent pathway to mediate the degradation of virus RNA in Arabidopsis, which resulted in host defense to invading virus (Wang et al., 2010). To find novel regulators controlling siRNA mediated anti-virus pathway, a different mutant of CMV, CMV-m2b, is studied. In CMV-m2b, all three start codons of the 2b gene were mutated without the loss of the overlapping reading frame of the 2a gene that codes the viral RDRP in CMV-2b (Wang et al., 2010). My work in this project includes genetic screening of Arabidopsis mutants involved in the siRNA mediated anti-virus pathway, identification of the genes underlying the mutants, and functional analysis of the novel regulators of the siRNA mediated anti-virus defense.

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MEGAN RILEY
Moorpark College, CA
Megan Riley

The Nothnagel lab is researching arabinogalactan proteins (AGPs) in Physcomitrella patens with a specific focus on methylated sugar components, which are not present in angiosperm AGPs. Previous experiments in the Nothnagel lab have provided statistically significant evidence that the knock out of a P. patens protein (K01) results in a reduced 3-O-methyl-rhamnose to rhamnose ratio. This suggests that the K01 protein may work as a methyl transferase. I am researching the function of the K01 protein, as little is known about it. I am purifying and characterizing a large batch of AGPs to determine if all AGPs are equally effected by the knockout or if only a subset shows a reduced 3-O-methyl-rhamnose to rhamnose ratio. To do so, after homogenization and differential centrifugation of P. patens gametophytes, the soluble AGP fraction is precipitated using Yariv phenyl glycoside and NaCl. The AGPs are extracted from the Yariv phenyl glycoside using DMSO and acetone. After obtaining purified AGPs, they are separated by charge using ion exchange chromotography. The different AGPs will then be characterized using gas-chromatography-mass spectrometry to determine if all AGPs in the K01 strain of P. patens are affected or a specific portion show a reduced mehtylated rhamnose to rhamnose ratio. If the K01 protein is identified as a methyl transferase, it may be used to identify other possible methyl transferases, to research the function of methylation in carbohydrates, and in biofuel applications.

 

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BENJAMIN SCHLAU
Portland State University, OR
Benjamin Schlau

During research in Dr. Eulgem’s lab on the RPP7-mediated plant immune response to an isolate of the oomycete Hyaloperonospora parasitica, post-doc Tokuji Tsuchiya accidently discovered that Arabidopsis thaliana with rpp7 knockout mutations exhibited inhibited root morphology compared to wildtype when grown on NaCl-treated growth media. Further testing confirmed his original observations. The data may be the first evidence that an immune recognition protein is involved in salt-stress response pathways. However, previous studies demonstrate that certain proteins known to only be involved in salt-stress response are specific to NaCl. I am running the wildtype v. mutant salinity root response experiment again, but with KCl to investigate whether this is true of RPP7. The research may lay the foundation for future development of salt tolerant crops, which could improve global food security as irrigation of agricultural lands increases soil salinity due to erosion of canals and aquifers. Before one can empirically study gene functions through knockout mutations, identification of homozygous mutants is necessary as first generation lab mutants are heterozygous, and mutations are recessive. While waiting for rpp7 mutants to grow in saline media, I am screening the progeny of selfed mutant Arabidopsis for homozygous individuals by Polymerase Chain Reaction in addition to learning techniques for precise quantification of gene expression under abiotic stress through application of Reverse Transcription-PCR. When time permits, I have been applying my own background in ecophysiology by comparing levels of RPP7-dependent salt-stress response for further investigations that could account for survivorship and fecundity, crowding, and root competition.

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ERIN STERNBURG
Cal State Univ. Long Beach, CA
Erin Sternburg

Micro-RNAs (miRNA) are RNA strands approximately 20-24 nucleotides in length that act as regulators in many eukaryotic processes. miRNAs exhibit complementary base pairing to mRNAs, allowing them to bind and repress translation. This repression has been identified to work via mRNA degradation or inhibition of transcription. The mechanism involving mRNA degradation has been well studied and is understood, but little is known about the mRNA inhibition pathway. The Chen lab studies this inhibition pathway in Arabidopsis, and a candidate protein, AMP1, has been identified. My work this summer includes identifying protein-protein interactions involving AMP1 using a yeast two hybrid screen. In this screen, AMP1 will serve as the “bait” protein, and an Arabidopsis c-DNA library will serve to identify potential “prey” proteins. I will also be examining possible interactions with AMP1 found from a previous yeast two-hybrid screen in Arabidopsis via immunoprecipitation and Western blotting. My work this summer will help to identify proteins involved in the inhibition pathway which will help elucidate this cellular pathway.


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University of California, Riverside
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Center for Plant Cell Biology
Botany & Plant Sciences Department
2150 Batchelor Hall

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

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