UCR

Center for Plant Cell Biology



REU 2011


REU Students and their Summer 2013 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 from 160 applicants for this ongoing 10-week residential summer program.  Please click on the following student links to see photos and read about their Summer 2013 research programs in CEPCEB laboratories.

An REU Poster Session was held in the Genomics lobby at the end of the ten-week program, where students discussed their projects.

REU Student
College/University
CEPCEB Faculty
Jesus Banderas Cuyamaca College, CA Eulgem Lab
Quinton Brail

Cleveland State University, OH

Bailey-Serres Lab

Janel Case

Alan Hancock College, CA

Walling Lab

Sarah (Luna) Dietrich Warren Wilson College, NC Borkovich Lab

Seth Flanders

Whitworth University, WA

Roper Lab

Matthew Holmes Haverford College, PA Reddy Lab

Lauren Kivlen

Riverside Community College, CA

Nothnagel Lab

Mikayla Kravetz

Wells College, NY

Ma Lab

Andrea Krueger

University of Wisconsin-Stout, WI

Judelson Lab

Carolyn Meyers

Victor Valley Community College, CA

Chen Lab

Galen Rask

Swarthmore College, PA

Springer Lab

Alicia Vazquez

Cal State University, Channel Islands, CA

Larsen Lab

 

JESUS BANDERAS
Cuyamaca College, CA
Jesus Banderas

Sixty thousand different molecules were screened using a high throughput screening system designed to find molecules that induce CaBP22-333 promoter-mediated reporter gene expression in transgenic Arabidopsis seedlings. 114 chemicals were identified from this process. Thirty of these chemicals have been grouped together, based on their molecular structure, and labeled as Phenylimino Methyl Phenols (PMPs). Eight of these PMPs have been chosen for further study. Two week old Arabidopsis thaliana plants, both wild type colo-0 and Arabidopsis defense mutants, will be treated with each chemical twenty four hours prior to being infected with Hyaloperonospora arabidopsidis (Hpa). By counting the spores present at seven days post inoculation, the chemical’s effectiveness at increasing the plants’ resistance to Hpa will be determined. As a second part of the experiment, tissue samples from plants will be collected twenty four hours after chemical treatment and used for RNA extraction. The integrity and yield of the RNAs will be analyzed and reverse transcription PCR will be performed. This will show the chemicals ability to induce expression of plant defense gene markers. Overall these experiments will help to understand the each chemical’s mode of action and the hierarchal level at which it interferes with the plant immune pathway. This project will hopefully yield chemicals that can be used as pesticides that will be less harmful to the environment.

 

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QUINTON BRAIL
Cleveland State Univ., OH
Quinton Brail

RNA binding proteins (RBPs) are involved in complex post transcriptional regulation pathways, such as pre-mRNA processing and mRNA translation. RBPs are classified in different families based on their RNA binding domains. In Arabidopsis the function of the majority of RBPs in not known or is under characterized. The Julia Bailey-Serres lab showed for two RBPs these RBPs function in stress survival to hypoxia and drought. This study is focused on a Puf family protein (APUM), namely APUM4. Knowledge about APUM4 and its potential role in stress resistance could shed a light on how plants adapt their post-transcriptional gene regulation mechanisms in response to stress. This information could be used to design ways to better prepare crops to withstand challenging growth conditions.

Two mutant alleles in APUM4 will be screened for stress resistance compared to wild type using growth mediums with various stressors such as high salt and sugar concentrations, exogenous hormone application, or hypoxia treatment. In addition, several double mutants in APUM4 with close APUM4 homologs (APUM1 to 3, 5 and 6) tested under the same conditions as the APUM4 single mutant alleles. APUM4 expression levels will be analyzed in at least the single mutants using qRT-PCR to determine whether the stress resistance is due to APUM4 down or up regulation in the mutants. APUM4 localization will be studied through confocal microscopy in the 35S:APUM4-mCherry-HA lines. APUM4 abundance will be analyzed in T2 transgenic 35S:APUM4-FLAG and 35S:APUM4-mCherry-HA lines through western blotting. APUM4-mRNA complexes will be isolated using an immunopurification approach.

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JANEL CASE
Alan Hancock College, CA
Janel Case

Plants have many plant defense hormones and one of which is salicylic acid (SA). Upon attack by pathogen or insects, plants respond with an increase in salicylic acid. An increase in SA response may lead to the triggering systemic acquired resistance (SAR) in the plant immune response. SAR is a sustained defense response to pathogen or insect at remote sites after local infection of plants. SAR increases the plant’s defenses against future infection and aids in the recovery process. SAR can be passed on genetically from one generation to the next generation. Using Arabidopsis plants, the goal is to find out if whiteflies, a common agricultural pest and an inducer of SA-regulated defenses can pass on SAR from previous generation to the next resulting in transgenerational SAR responses. Arabidopsis wild-type Columbia plants will be infested with whiteflies and seeds will be collected from these plants. Concurrently, wild-type Columbia control plants without whitefly infestation will be carried out.   With the seeds collected, the first generation of plants will be assessed for transgenerational SAR responses by two procedures. Plants will be grown and challenged with PstDC3000 bacteria and measured for the presence of transgeneration SAR between control non-infested plants and whitefly infested plants. The first generation of plants acquired from seeds of non-infested and whitefly infested plants will be challenged with whiteflies again. The ability of the whitefly to infest this first generation of plants between non-infested and whitefly infested plants will be compared. If transgenerational SAR is triggered by whiteflies and passed on to following generations, there would be an increase in resistances with successive generations. This knowledge can be utilized in help farmers create more whitefly resistant plants and prevent loss of crop due to whiteflies.

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SARAH (LUNA) DIETRICH
Warren Wilson College, NC
Sarah Dietrich

Neurospora crassa is an ideal model organism for studying human biology and cellular processes. It is a non-pathogenic eukaryote that is easy to culture and is more closely related to mammals than plants or yeasts. We are studying roughly 200 transcription factor mutants previously generated by a high throughput knockout project. In this study, we are specifically looking for defects in the sexual cycle and major asexual cycle. We are performing phenotypic characterizations of all uncharacterized transcription factor mutants available, testing for defects in linear growth of hyphae, aerial growth of hyphae, conidiation, female fertility and general morphology. Additionally, we are picking and testing homokaryon progeny of mutants which were previously only available as heterokaryons. For these strains we will phenotype newly obtained homokaryons and report those which are unable to be purified to homokaryons as inviable mutants, indicating the knocked out transcription factor is an essential gene. In this way we can determine the roles of every transcription factor within Neurospora's genome.

 

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SETH FLANDERS
Whitworth University, WA
Seth Flanders

Pantoea stewartii subsp. stewartii. P.stewartii causes wilting and death of its corn host by primarily colonizing and obstructing the xylem tissue. As the bacteria become established in the plant they produce large amounts of an exopolysaccharide matrix called stewartan. The bacteria form biofilms and it is these matrix-encased biofilms that allow successful colonization of the plant. Plants have many defense mechanisms to ward off and kill pathogens, including production of reactive oxygen species (ROS), such as hydrogen peroxide. ROS are also present in plant tissue as byproducts of normal plant development, such as xylem lignification. We hypothesize that one mechanism that P.stewartii utilizes to protect itself from these deleterious ROS is through the production of a protective EPS layer. Furthermore, we have evidence that EPS production is regulated, in part, by OxyR. OxyR is a transcription factor that regulates gene expression in response to hydrogen peroxide. We hypothesize that P. stewartii regulates expression of stewartan EPS production, in part, through sensing external oxidative stress via OxyR. We are testing this through the use of a fluorescent reporter (GFP) construct to monitor expression of EPS biosynthetic genes in response to ROS in the presence or absence of OxyR. We are also creating a knockout mutant in a known regulator of EPS production to determine the hierarchy of regulation that leads to the production of the protective EPS matrix and biofilm formation. This will aid future scientific endeavors in understanding bacterial survival mechanisms and gene regulation during plant-pathogen interactions.

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MATTHEW HOLMES
Haverford College, PA
Matthew Holmes

The stability of cell identity within the plant stem cell niche is a result of hormone and protein interactions within the shoot apical meristem. WUSCHEL, a symplastic transcription factor protein, forms a gradient within this region that may play a significant role in establishing cell identities. WUSCHEL is synthesized within the rib meristem – a region consisting of the shoot apical meristem’s central corpus. An interesting feature of WUSCHEL is its feedback loop with another protein, CLAVATA3. These two proteins play a role in meristem structure regulation. WUSCHEL also forms a gradient that tapers off towards the SAM epidermis. This gradient may, in part, determine cell identity within the meristem. Why does WUSCHEL form this gradient, though? Previous research has indicated that, in Arabdopsis thaliana, WUSCHEL is modified and regulated by the signaling pathway of cytokinin – a hormone produced in the central zone of the apical meristem. I am seeking to understand how the cytokinin pathway interacts and alters WUSCHEL protein stability, and affects the gradient. I will be using an array of biochemical techniques (such as using dexamethasone induced two component systems to change cytokinin distribution) within the context of genetic approaches to study various Arabidopsis thaliana constructs. Primarily, I will be observing these constructs with fluorescent confocal microscopy to observe cytokinin level impacts on WUSCHEL movement.

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LAUREN KIVLEN
Riverside Community College, CA
Lauren Kivlen

Arabinogalactan-proteins (AGPs) are a class of heavily glycosylated macromolecules found within the plant cell wall and plasma membrane. These proteins function in several aspects of plant growth and development such as the control of cell proliferation, expansion and programmed cell death. AGPs produced by the moss Physcomitrella patens structurally resemble those found in higher plants, however, they contain unusual, methylated, 3-O-methyl-L- rhamnosyl (3-O-Me-Rha) residues. While the production of 3-O-Me-Rha is conserved in mosses, lycophytes, pteridophytes and gymnosperms, it is not found in angiosperm AGPs or polysaccharides. Moreover, the presence of an additional oxidizable carbon in methylated sugars such as 3-O-Me-Rha makes it relevant to the production of biofuels. The current goal of the Nothnagel lab is to identify the gene that encodes the methyltransferase responsible for the generation of 3-O-Me-Rha. Identification of this gene may lead to the characterization of other methyltransferases and their possible utility towards the production of biofuels. Currently, the assessment of tissues gathered from tobacco plants that have been engineered to express the Physcomitrella KO9 candidate methyltransferase gene is underway. Young leaves from these plants were previously examined and appeared to exhibit a low abundance of 3-O-Me-Rha. The focus of my research is to measure the expression of 3-O-Me-Rha in the older leaves, stems, flowers and roots of these plants using gas chromatography-mass spectrometry. My objectives are to confirm the presence of 3-O-Me-Rha in these transgenic plants and determine its abundance in these tissues as compared to young leaves.

 

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Mikayla Kravetz
Wells College, NY
Mikayla Kravetz

A bacterial effector is a protein that a pathogenic bacterium injects into a host cell in order to cause disease. The HopZ1 effectors are important virulence factors for the plant pathogenic bacterium Pseudomonas syringae, which has a variety of plant hosts. HopZ1 effectors are hypothesized to be acetyl-transferases, disrupting the activity of various targets in the host cell via a “ping-pong” mechanism of picking up an acetyl group through autoacetylation and then transferring it to a target molecule. HopZ1 has two functional forms. The co-evolutionary arms race between pathogen and host has resulted in plants recognizing the more ancestral HopZ1a effector, leading to an immune response called hyperactive resistance (HR) wherein cell death occurs in the infected tissue. However, the HopZ1b effector, form of HopZ1 that appears to have evolved more recently, does not trigger this response. The researchers in Dr. Ma’s lab are trying to determine which of the differences between the sequences of the two proteins are most significant to the biological functions of the effectors in causing disease and suppressing plant immune response.

For this project, I will be generating single-point mutations in different potential autoacetylation sites of HopZ1a gene, changing lysine, serine and threonine to their corresponding amino acid in HopZ1b. These single point mutants will be used together with other chimeric constructs, combinations of intact regions of each protein, to test the potential biological function of these amino acids and sequences to HopZ1 function.

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ANDREA KRUEGER
University of Wisconson-Stout, WI
Andrea Krueger

Phytophthora infestans is a plant pathogen which causes late blight mainly in potato and tomato. The Judelson laboratory is studying different aspects of development in this pathogen. Transcription factors like homeo domain and MADS-box proteins are responsible for the expression of targeted developmental genes in many species. I have used bioinformatics analysis to identify possible homeo proteins in P. infestans and will silence them via RNA interference-based constructs. The process includes primer design, PCR amplification, gel electrophoresis, and molecular cloning in plasmids leading to bacterial and oomycete transformation. The effect of silencing will then be observed at all life stages of the pathogen to evaluate the developmental functions of the proteins. These stages include hyphae, spores, zoospores, germinated cysts, and cleaving sporangia. This study will lead to further understanding of the roles of these proteins in growth, development, and pathogenesis.

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CAROLYN MEYERS
Victor Valley Comm. College, CA
Carolyn Meyers

The AMP1 gene is very important in plant growth and development. Recent study in our lab identifies AMP1 as a new player in the plant miRNA pathway that facilitates miRNA-mediated translation inhibition of target mRNAs. However, the specific role of the AMP1 protein in translation inhibition remains unclear. Therefore, my project in the Chen lab is to find proteins that interact with AMP1. Since AMP1 is an integral ER membrane protein, we apply a membrane-based protein complementary assay in
yeast called the split ubiquitin system to look for its interacting proteins. In this
system, we fuse C-terminal ubiquitin to AMP1 and co-express it with Arabidopsis library proteins fused with N-terminal ubiquitin. The N-terminal and C-terminal ubiquitin can find each other when AMP1 interacts with one of the library proteins. The re-assembled ubiquitin can be recognized and cleaved by an ubiquitin specific binding protease, then lead to the release of an artificial transcription factor. The released transcription factor goes into the nucleus where it starts to transcribe the reporter genes. We aim to screen through the Arabidopsis cDNA library in yeast to find the interacting partners of AMP1 and further confirm their interaction in plants by BiFC and Co-IP assays.

Dualsystems Biotech AG. (2010). DUALmembrane starter kits User Manual. 1-58.

Igor Staglijar, Chantal Korostensky, Nils Johnsson, and Stephan Te Heesen. (1998). A genetic system based on split- ubiquitin for the analysis of interactions between membrane proteins in vivo. Genetics. 95. 5187-5192.

Shengben Li, Lin Liu,Xiaohong Zhuang, Yu Yu, Xigang Liu, Xia Cui, Lijuan Ji, Zhiqiang Pan, Xiaofeng Cao, Beixin Mo, Fuchun Zhang, Natasha Raikhel, Liwen Jiang, and Xuemei Chen. (2013). MicroRNAs Inhibit the Translation of Target mRNAs on the Endoplasmic Reticulum in Arabadopsis. Cell. 153. 562-574.

 

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GALEN RASK
Swarthmore College, PA
Galen Rask

The human population is expected to reach 9 billion individuals by the year 2050. In order to feed the increasing population, the amount of food produced by crops such as rice (Oryza sativa) must increase. In rice, the plant hormone brassinosteroid (BR), controls plant height and leaf angle. BR-deficient rice plants have a dwarfed phenotype with decreased fertility, but also have a more upright stature than wild type. Prior research has noted that more upright leaf growth enables sunlight to penetrate to the lower leaves, increasing photosynthetic efficiency, as well as enabling crops to be planted more densely, increasing yield from a given area (Divi et al., 2009). The Springer lab has created a transgenic rice line by using an Arabidopsis promoter (pATLOB) that has been found to drive expression in the lamina joint of the rice leaf, fused to the Arabidopsis BAS1, a gene that inactivates BRs. This is expected to result in plants with more erect leaves and no other changes in plant architecture. Currently, we are in the process of creating true breeding lines. I will be working to characterize the severity of leaf angle alterations and will document other possible phenotypic effects in the transgenic plants. I am taking measurements of plants and performing DNA extraction and PCR in order to determine if the DNA insertion has become homozygous and a true breeding line has been created. I will also compare the measurements of each lineage to ensure that the T-DNA insertion has not had a negative impact upon plant growth.

 References:

 Divi, Uday K., and Priti Krishna. "Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance." New Biotechnology (2009), 26: 131-136.

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ALICIA VAZQUEZ
Cal State Univ., Channel Isl., CA
Alicia Vazquez

The overall purpose of this research project is to establish a strong foundation from the data collected to aid in a better understanding of the biochemical Auxin biosynthesis pathway in Arabidopsis thaliana. Arabidopsis Col-0 wild type will be exposed to our chemical in different amounts, the model concentration and reaction Arabidopsis Col-0 wild type has to our chemical will allow us to determine the function our chemical has in the Auxin biosynthesis pathway. In addition to including our chemical to the media, one set was exposed to silver while the other was exposed to ethylene. The purpose of exposing the Arabidopsis Col-0 wild type to both silver and ethylene was to see if our chemical is best active in the presence or absence of ethylene. It is predicted that in the presence of our chemical, the roots of the Arabidopsis Col-0 wild type will grow upwards rather than in the normal downwards direction. Root growth will be monitored by planting the seeds in the media in a vertical fashion.


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E-mail: genomics@ucr.edu

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