UCR

Center for Plant Cell Biology



REU 2009


REU Students and their Summer 2009 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 2009, 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 2009 research programs in CEPCEB laboratories.

An REU Poster Session was held Friday, August 21, 2009 in Keen Hall's lobby area, where students were available to discuss their projects. The Poster Session was open to the campus community.

 

REU Student
College/University
CEPCEB Faculty
Mentor
Lizz Esfeld Truman State University, MO Chen Lab Shengen Li
Shahid Jaffer

St. Olaf College, MN

Springer Lab

Pan-Ya Kim

Evelyn Pereyra

San Bernadino Valley College, CA

Yang Lab

Augusta Jamin

Tim Richardson

Riverside Community College, CA

Reddy Lab

Mariano Perales

Roxanne Sebeny

University of California, Berkeley, CA

Borkovich Lab

James Kim/Sara Wright

Rebekah Silva Riverside Community College, CA Walling Lab Melissa Smith

Julie Stutzbach

Beloit College, WI

Eulgem Lab

Mindy Salus

Alexandra Swidergal

Cornell University, NY

Raikhel Lab

Abel Rosado-Rey

Gilbert Uribe

California State University, Bakersfield

Douhan Lab

Greg Douhan

Donald Van Fossan

Riverside Community College, CA

Rao Lab

A.L.N. Rao

 

LIZZ ESFELD
Truman State University, MO
Lizz Esfeld

MicroRNA is a category of small non-protein coding RNA molecules with lengths between 21-24 nucleotides. They regulate a wide range of important gene functions either by mRNA cleavage or by translational repression. In plants, microRNAs participate in a variety processes including plant development, metabolism and stress responses. Therefore, understanding the mechanism of microRNA biogenesis and function is an important component of plant biology. It is known that plant microRNA genes are transcribed by RNA polymerase II into pri-microRNA, then pri-microRNA is processed into pre-microRNA and then into mature microRNA by DCL1, which is also greatly facilitated by HYL1 and SE. The mature microRNA will be methylated by HEN1, and can also be degraded by SDN. Finally, they are assembled into an RNA-Induced Silencing Complex (RISC) to silence the target genes. To more deeply study the microRNA metabolism and function pathway, we carried out a genetic screen from an EMS mutagenesis pool. One mutant has obvious morphological phenotypes including serrated leaf, abnormal inflorescence phyllotaxy, late flowing and bad fertility. Preliminary data shows most of the microRNA levels are reduced, indicating that this mutant is a good subject for microRNA metabolism study. We already crossed the mutant (Columbia background) with Landsberg ecotype, and got an F2 population.
           
My project for the summer in the Chen lab consists of cloning the gene responsible for this phenotype with a map-based cloning approach using this mapping population. The rough mapping is going on now. At the same time, I'm carrying out a more detailed molecular characterization of this mutant. First, I will compare the microRNA target genes expression level in the wild type and mutant plant to see whether their accumulation is increased because of decreased microRNA level. Then I will compare the pri-microRNA level in the wild type and mutant plant to gain some clues as to whether the reduction of mature micoRNA accumulation is caused by mciroRNA biogenesis, stability or degradation.

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SHAHID JAFFER
St. Olaf College, MN
Shahid Jaffer

The shoot apical meristem (SAM) is a group of undifferentiated cells at the growing plant tip that controls the formation of leaves and other lateral organs. It is essential for the regulation of all aspects of shoot architecture, including leaf initiation, morphogenesis and patterning, flower production, and axillary shoot formation. Organ boundaries are important barriers that regulate the patterning of both the SAM and lateral organs; moreover, boundaries have been proposed to function in mediating communication between these two domains. Research in the Springer lab is concerned with genes expressed in the boundary layer and the function of these genes in overall plant development.
PENNYWISE (PNY) and POUNDFOOLISH (PNF) are redundant homeodomain proteins found in the inflorescence and floral meristems of Arabidopsis thaliana.  As transcription factors, these genes regulate gene expression and play an important role in the plant’s transition from the vegetative to reproductive state.  Phenotypic characterization of pny pnf double and single mutants show that a lack of functional PNY leads to random internodal patterning and impairs the ability of the SAM to respond to chemical signals, ultimately resulting in an inhibition of floral evocation in pny pnf double mutants.

Arabidopsis LATERAL ORGAN FUSION1 (LOF1) is a gene characterized by the Springer lab that encodes MYB-domain transcription factors important in lateral organ separation and axillary meristem formation. LOF1 is thought to be critical for the maintenance of the SAM. 

I am investigating the relationship between LOF1 and PNY.  Initial data shows that the pny lof1 mutant displays none of the phenotypic characteristics shown in pny mutants, suggesting that the loss of function of LOF1 results in the “rescue” of the mutantphenotype and that PNY may negatively regulate the expression of LOF1 in wild-type plants. Determining the extent of this relationship will allow for a better understanding of the genes involved in the proper maintenance and differentiation of the SAM, as well as an increasing appreciation for the functional importance of the boundary domain in Arabidopsis.

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EVELYN PEREYRA
San Bernadino Valley College, CA
Evelyn Pereyra

The goal of our research is to study protein-protein interactions to better understand the biological process of these interactions by finding chemicals that disrupt this interaction. Our study focuses mainly on the auxin signaling pathway and the ROPs, Rho-GTPases of plants (ROPs). The auxin-signaling pathway is known to regulate many aspects of plant growth and development. The two proteins that are being studied for this interaction are SPK1, a mutant protein that confers defects in the cotyledons, trichomes, and leaves, and TMK1, a gene coding for a receptor-like transmembrane kinase.  TMK1 is also an auxin receptor.  SPK1 produces phenotypes similar to phenotypes seen when the ROP protein is manipulated. The method being used to screen these chemicals in order to quantify the interaction level between the two proteins is the yeast two-hybrid system. This technique is used to test for physical interactions between two proteins and the effects of chemicals in the CEPCEP combinatorial libraries.  TMK1 and SPK1 have already been shown to interact using the yeast-two-hybrid method. Currently, we are conducting additional high throughput screens with the help of the robot.

 

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TIM RICHARDSON
Riverside Community College, CA
Tim Richardson

The shoot apical meristem (SAM) is a collection of totipotent stem cells involved in plant organogenesis. Interconnected networks of signaling pathways influence cellular differentiation within the SAM. In Dr. Reddy’s lab, we will be focusing on the homeodomain transcription factor WUSCHEL (WUS). The WUS protein is known to bind to the promoter sites of KAN-1 and 2 and influence their expression.
Initially we will determine where WUS binds near the KAN genes in the model plant Arabidopsis thaliana. This will be achieved using selective PCR priming, bacterial cloning, and electrophoretic mobility shift assays. Once this is accomplished, the work will focus on determining the effect of acetylation of the histone proteins associated with the WUS/KAN system. We will use inhibitors of histone acetyl transferase and histone acetylase and observe the phenotypic change. Transgenic plants labeled with markers will allow us to use confocal microscopy to view these phenotype changes at the cellular level. RNA interference will be used to detect the changes in gene expression from each case. These efforts will help to unveil the complex regulatory system involved in stem cell maintenance and differentiation.

 

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ROXANNE SEBENY
University of California, Berkeley
Roxanne Sebeny

Neurospora crassa, commonly known as orange bread mold, is a model eukaryotic organism in the filamentous fungi group that is studied in Professor Borkovich’s lab.  One of the main projects in this lab is to study the heterotrimeric G-proteins (1).  Previous work in the lab has shown that a gene called ste50 might be involved in the heterotrimeric G-protein signaling cascade.  While it is already known that ste50 is part of the mitogen-activated protein kinase (MAPK) cascade in yeast (2), my work will help verify whether ste50 is part of the MAPK cascade in N. crassa, a signal transduction cascade involved in osmoregulation and stress responses (3).
As part of my project, I will be creating a recombinant DNA construct by yeast homologous recombination using polymerase chain reaction, amplifying the ste50 gene with a FLAG-tag attached to the C-terminus and fusing it with a nourseothricin marker (4).  After using PCR to obtain the correct DNA fragments, they will be inserted into Saccharomyces cereviseae, resulting in the final recombination product. From the yeast the vector will be extracted and used to transform E. coli; the bacteria will produce mass copies of the inserted vector, facilitating the process of verification. Once verified, I will electroporate the recombined DNA sequence into Neurospora conidia.  
           
Another aspect of my project consists of a phenotypic analysis of N. crassa strains, where I will analyze the morphological defects in a large set of gene-deletion mutants.  Finally, the growth habits of these mutants growing on media containing growth-altering chemicals will be studied.

References:

1. Li, L., Wright, S. J., Krystova, S., Park, G., Borkovich, K. A. 2007. Heterotrimeric G Protein Signaling in Filamentous Fungi. Annual Review of Microbiology. 61: 423-52.

2. Posas, F., Witten, E. A., Saito, H. 1998. Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway. Molecular and Cell Biology. 18: 5788- 5796.

3. Park, G., Pan, S., Borkovich, K.A. 2008. Mitogen-Activated Protein Kinase Cascade Required for Regulation of Development and Secondary Metabolism in Neurospora crassa. Eukaryotic Cell. 7: 2113-2112.

4. Raymond, C. K., Pownder, T. A., Sexson, S. L. 1999. General Method for Plasmid Construction Using Homologous Recombination. Biotechniques. 26: 134-141.

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REBEKAH SILVA
Riverside Community College, Riverside
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 has 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.  

Since gene silencing can be an overall organism stressor that may inhibit related genes or induce compensatory responses, it is necessary to complement the LapA-SI transgenic lines.  One way to complement the LapA-SI transgenic lines is through a chemical genetics screen.  Using this technique, small molecule(s) can be found that are effective inhibitors of LAP-A in vitro.  Through a chemical genetics screen, the desired phenotype can be acquired in vivo without concern regarding potential transgenic side-effects confounding the experiment.  Other advantages of chemical genetics include the specificity and reversibility of the inhibition, control over the timing and concentration of the inhibition, and the adaptability of the assay to multiple plants and plant species. 

Additional screening will be performed on any “hits” to assess the specificity of the inhibition using neutral LAP (LAP-N) from tomato and LAP2 from Arabidopsis.  Future study will use only ligands specific to LAP-A in vivo to identify which “hits” mimic the phenotype of LapA-SI transgenic lines.  Additional work can involve monitoring tomato plant response and recovery following in vivo exogenous ligand treatment during other stresses or in other tissues to elucidate LAP-A’s specific role in other aspects of tomato response and development.  These data, combined with the chemical genetics screen, will further elucidate the specific regulatory role of LAP-A and possibly the peptidase target or targets in tomato. 

References:

Fowler JH, Narvaez-Vasquez, J, Aromdee DN, Pautot V, Holzer FM, and Walling LL  (2009)  Leucine Aminopeptidase Regulates Defense and Wound Signaling in Tomato Downstream of Jasmonic Acid.  Plant Cell.  21, 1239-1251. 

Stockwell, BR (2000)  Chemical Genetics: Ligand-Based Discovery of Gene Function.  Nature Reviews Genetics.  1, 116-125. 

Walling LL  (2000)  The Myriad Plant Responses to Herbivores.  J Plant Growth Regul.  19, 195-216.

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JULIE STUTZBACH
Beloit College, WI
Julie Stutzbach

Plants have developed a complex immune system used to recognize and fight pathogenic organisms.  However, at times plants fail to recognize these pathogens, which can make them susceptible to disease.  Dr. Euglem’s lab examines the interaction of the pathogenic oomycete Hyaloperonospora arabidopsidis and the model plant Arabidopsis thaliana.  The goal of my project is to use chemical genomics to discover potential candidates for chemicals that help Arabidopsis to fight Peronospora infections by inducing its defense pathway.  I will study compounds that have already been indentified as potential chemicals involved in resistance to Peronospora such as compound 442, 2-(5-bromo-2-hydroxyphenyl)-thiazolidine-4-carboxylic acid These experiments should determine the compound’s optimal effective concentration- the lowest level that will activate a gene cascade that leads to resistance.  Additionally, I will attempt to dissect the defense pathway activated by these compounds by examining their impact on various mutants.  This should help determine where in the pathway that the compound acts, which may distinguish it from other compounds already known to be involved in the defense pathway of Arabidopsis.  In addition, I will screen chemical libraries to find other candidates for compounds that induce a response to the oomycete.  This project could have implications for the development of pesticides that don’t kill the pathogen itself but actually emulate nature by enhancing the plant’s own natural immune system.

 

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ALEXANDRA SWIDERGAL
Cornell University, NY
Alexandra Swidergal

A crucial component in normal cellular function is the highly regulated and conserved protein trafficking system that transports essential proteins throughout the cell. The Raikhel Lab has identified a mutation in the Arabidopsis ribosomal complex structure that causes errors in translational control and protein sorting regulation. The mutations in rpl4 family members modify the interactions between the C-terminal (CTPP) propeptides that lead to the vacuole and the ribosomal exit tunnel which regulates the incipient folding process.  It was previously shown that a similar r-protein knockdown family produced a pleiotropic phenotype resulting from reduced ribosome biogenesis (Degenhardt et al., 2008). Those phenotypes were postulated to be linked to maintenance of auxin homeostasis. Preliminary results in our mutant family show similar phenotypic features to those described in previous papers. The objective of this experiment is to establish a connection between protein trafficking and auxin response. I will be focusing on the auxin response in our rpl4 mutants. 

Determination of the relationship between auxin and our ribosomal mutant will be conducted though evaluation of the differences between rpl4 and Col WT in the localization of auxins, the transport of auxins, and the plant’s ability to sense auxin. To determine the location of auxin, GUS:RPL4 promoter will be utilized to evaluate whether expression is regulated by auxins in time control experiments. The analysis of crosses in mutant backgrounds will identify auxin transport carrier problems in DR5:GUS, PINS, and AUX1:GFP.  By analyzing the phenotypic characteristics in relation to gravitropism, cellular elongation, and IAA/NAA induction, sensory ability will be analyzed. Analysis of results will give greater understanding of the protein trafficking system and its relationship to auxin.

References:

Degenhardt, Rory F., Bonham-Smith, Peta C. (2008) Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development. Plant Physiology. 147, 128-142.

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GILBERTO URIBE
California State University, Bakersfield, CA
Gilberto Uribe

Parasitism is a ubiquitous life history strategy where organisms obtain resources from their hosts. Parasites can be categorized into those that are considered generalists or specialists, and it is thought that parasites that are highly host specific have had a long history of coevolutionary interaction with their hosts. Therefore, having an overview of patterns of species distribution and population genetic data from both the host and the parasite should provide evidence that may aid in elucidating reproductive and coevolutionary patterns, and levels of specificity among populations of hosts and parasites.

In Dr. Douhan’s lab, the host-parasite system to be studied will focus on various species of mycorrhizal fungi in the Boletales and parasites in the genus Hypomyces, in which some of the parasites appear to be generalist pathogens whereas other species appear to be more host specific. However, previous work in Dr. Douhan’s lab has demonstrated that undescribed cryptic species exist in Hypomyces and that some known morphological species of Hypomyces exist in California but have never been reported. Therefore, little is known about the distribution of ‘species’ in this system. Therefore, this study will focus on comparing several populations of hosts and parasites from southern California and one population from northern California. It is expected that the fungal populations from southern California will be more closely related to each other than to the northern population due to the extensive geographic barrier that would make genetic exchanges highly unlikely. Also, due to the specificity with which some parasites attack hosts, and the generality of others, it is also expected that there will be a higher degree of genetic variation among generalist individuals when compared to those demonstrating high host specificity.

To accomplish this, we will use molecular techniques to identify the hosts and parasites and use amplified fragment length polymorphism (AFLP) to investigate the population genetic structure of the parasites.

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DONALD VAN FOSSAN
Riversity Community College, CA
Donald Van Fossan

Research involving single stranded, positive sense RNA viruses in plants plays a key regulatory role in better understanding the other pathogenic viruses significant to humans and other animals such as human immunodeficiency virus (HIV) and poliomyelitis or polio. Single stranded, positive sense RNA viruses or +ssRNA viruses are unique because their nucleic acid sequence is 5’ to 3’ and, therefore, shares the same basic sequence of mRNA. This allows the viral RNA to be immediately translated into proteins without having to go through the process of transcription, essentially, because it is complementary to mRNA. However, whether or not being able to translate proteins from +ssRNA without going through transcription is beneficial to the virus or not is still unknown. Dr. Rao’s lab is interested in the study of a specific +ssRNA plant virus, Brome Mosaic Virus or BMV from the family Bromoviridae, which is icosahedral in symmetry.

Research on BMV is significant, primarily, because many pathogenic viruses with icosahedral symmetry share a common characteristic of other eukaryotic RNA viruses with the same symmetry, which is their ability to assemble infectious virions by the capsid protein; this process involves specific protein-protein interactions. We are interested in two specific protein-protein interactions, first, the coat protein-coat protein interaction (CP-CP), which is essentially the process of capsid protein formation producing icosahedral virions. Secondly, we are interested in replicase-coat protein interactions, which we know is imperative in viral genome packaging specificity.

In order to monitor the specific CP-CP and replicase-CP interactions I will employ a novel procedure termed Biomolecular Fluorescence Complementation (BiFC) analysis. This procedure is significant in monitoring protein interactions at the sub-cellular level and can be employed in various cell types. The BiFC analysis will be employed when two proteins bound to non-fluorescent components of a fluorescent protein fuse and interact allowing the protein to fluoresce. In this novel procedure Yellow Fluorescent Protein (YFP) will be used.

My role is to engineer the fusion of these fluorescent components to the N-and C-terminal ends of the BMV CP and test for the BiFC analysis. I will obtain this by producing four vectors from plasmids and inserting YFP and CP genomes into the plasmids until 4 diverse vectors are formed. Then, I will employ an Agrobacterium-mediated transient expression (agroinfiltration) to study BMV viral RNA packaging in the plant Nicotiana bethamiana. Protein-protein interactions can then be observed by using confocal laser scanning microscopy. Ideally, we wish to observe where in the host plant cell the BMV genome packaging occurs. This will allow us to further understand not only BMV but also other pathogenic viruses harmful to humans and animals.


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Center for Plant Cell Biology
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E-mail: genomics@ucr.edu

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