UCR

Center for Plant Cell Biology



REU 2010


REU Students and their Summer 2010 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 2010, 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 2010 research programs in CEPCEB laboratories.

An REU Poster Session was held Friday, August 20, 2010 in the Genomics 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
Ilse Argueta Chaffey College, CA
Springer Lab
Robert Koble
Elliott Beltran

Chaffey College, CA

Raikhel Lab

Michelle Brown

Jeremy Berg

University of Wisconsin, Madison

Ma Lab

Huanbin Zhou

Michael Cantrell

University of Idaho

Ding Lab

Zhihuan Gao

Cassandra Carrivales

Texas A&M University

Judelson Lab

Qijun Xiang

Blair Clark
Chaffey College, CA
Jin Lab

Yifan Lii

Luis Contreras

Riverside Community College

Reddy Lab

Ram Yadav

Oghenemano Evero

Chaffey College, CA

Borkovich Lab

Patrick Schacht

John Hartzheim

St. Olaf College, MN

A.L.N. Rao Lab

Devinka Bamunusinghe

Christopher Massimino

Frostburg State University, MD

Close and Roberts Labs

Wellington Muchero

Brian Perez

Riverside Community College, CA

Douhan Lab

Greg Douhan

Grace Sprehn

Tulane University, LA

Chen Lab

Rae Yumul

 

ILSE ARGUETA 
Chaffey College
Ilse Argueta

The research in the Springer Lab focuses on understanding the function of Lateral Organ Boundaries (LOB), an organ boundary gene, by two methods: Chemical Genetics and a Yeast-2-Hybrid screen. First a chemical screen will be conducted using the Lifechem chemical library, which consist of approximately 12, 000 chemicals, in an attempt to identify chemicals that will inhibit LOB overexpression in etiolated Arabidopsis thaliana seedlings. This screen will allow us to observe the changes that arise in seedlings in a LOB overexpression background, which consists of no apical hook. If we are able to find chemicals that bind and work to inhibit LOB, we will observe the formation of an apical hook. Chemical genetics has been shown to overcome redundancy, and our aim is to identify chemicals that can overcome the possible functional redundant nature of LOB. The second method will be the analysis of LOB interaction with other proteins via a Yeast-2-Hybrid screen to examine whether LOB functions as a complex or as a single protein. These assays will assist in explaining how LOB works in the boundary region between Shoot Apical Meristem (SAM) and plant lateral organs.

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ELLIOTT BELTRAN
Chaffey College, CA
Elliott Beltran

Protein trafficking within a cell is a highly dynamic process. Many of the genes involved in vesicular trafficking are essential. Given the large functional gene redundancy found in plants, discovery of new phenotypes are often too difficult for classical genetics to accomplish. The study of chemical genomics offers a novel approach that can circumvent the problems of lethality and redundancy that hinder classic genetic approaches. Chemical genomics utilizes small synthetic compounds that potentially interfere with proteins to reveal a phenotype without mutation to the organism.

Tobacco pollen serves as an excellent model for studying membrane cycling and protein trafficking. Apical growth of the pollen tube is dependent upon vigorous endocytic and exocytic trafficking. Tobacco pollen is also amenable to high throughput processing in a multi-well microtitre format. In a prior screening of over 46,000 compounds, the Raikhel Laboratory identified 360 compounds that affect tobacco pollen tube formation. These 360 compounds produced seven distinct morphological phenotypes.

The goal of this summer’s research project will be to further investigate the effects of these compounds on endocytic and exocytic pathways using laser scanning confocal microscopy with propidium iodide and the styryl dye FM4-64. These two agents will potentially help sort the compounds into groupings affecting endocytosis, exocytosis, or both pathways. The determination of pathways that are disrupted by each compound will give insight to that pathways role in development in relationship to its phenotype. In addition, the RAB2:GFP tobacco pollen marker will be assayed to determine if any of the compounds inhibit the ER-to-Golgi cycling pathway. Several compounds from this screening process have been found to also have an effect on mammalian cells. This means that many of these compounds have potential pharmaceutical as well as agricultural applications.

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JEREMY BERG
Univ. of WI, Madison
Jeremy Berg

Type III secretion systems (T3SSs) are potent weapons used by many bacterial pathogens to overcome their host’s defense systems. Proteins known as type III secreted effectors (T3SEs) are injected into the cytoplasm of host cells via this needle-like structure, where they promote bacterial virulence. Pseudomonas syringae is a model plant pathogen, which secretes a T3SE known as HopZ1. Previous work in Wenbo Ma’s lab has identified two HopZ1 alleles of interest: HopZ1a and HopZ1b [add reference]. P. syringae carrying the HopZ1a allele elicit a robust defense response – hypersensitive response (HR) in soybean, Nicotiana benthamiana, and Arabidopsis thaliana, while P. syringae carrying the HopZ1b allele fail to elicit HR in soybean and A. thaliana.

These results, in conjunction with sequence analysis, indicate that HopZ1a and HopZ1b are evolutionarily divergent and differentially recognized by plant defense systems. We hypothesized that this differential recognition may be due to allele-specific substrate binding by HopZ1a and HopZ1b in the host. Using yeast two-hybrid screens, soybean proteins that interact with HopZ1a, but not HopZ1b were identified. Our preliminary data suggest that one of the proteins identified from this screen, known as ZINP2 (HopZ INteracting Protein 2), is involved in the pathway that triggers HR in response to HopZ1a in soybean.

I will use a yeast two-hybrid screen to identify proteins that interact with ZINP2, and thus may be involved in downstream signaling to initiate the HR in response to HopZ1a. The relevant genes identified by this screen will then be isolated from the yeast and sequenced to determine their identity. Since the function of ZINP2 is currently unknown, identification of the ZINP2-interacting proteins may help define the function of ZINP2 in the plant cell, as well as determine if and how the interaction between HopZ1a and ZINP2 leads to an HR. Ultimately, this work will shed light on how HopZ1a and HopZ1b differ in their effects on plant defense, and will help paint a broader picture of the ongoing arms race between plant pathogens and their hosts.

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MICHAEL CANTRELL
University of Idaho
Michael Cantrell

Viral Immunity can be established through either the innate or adaptive immune response. Shou-Wei Ding’s lab has proven that RNA silencing serves as an innate immunity against RNA viruses. His colleagues are currently studying the innate immune response to viruses through forward and reverse genetic screens. To better understand this immune response in C. elegans, a self-replicating portion of the flock house virus (FHV) called RNA1 has been tagged with GFP and introduced into the genome. RNA1’s replication or its accumulation is normally blocked by antiviral mechanisms in C. elegans, but the accumulation of RNA1 can be observed in the transgenic worm when the innate immunity machinery has been inhibited. This model has been used for identification of antiviral immunity genes functioning in multiple antiviral pathways. My project this summer in the Ding lab will be to use this model to carry out a reverse genetic screen in order to find additional genes functioning in these antiviral pathways. This screen will be performed using a library of E. coli expressing the double stranded RNAs (dsRNAs) which target roughly 86% of the estimated 19,000 predicted genes in C. elegans. In addition I will be involved in mapping mutants which have been found through a forward genetic screen as well as in characterizing candidate genes picked up from my primary reverse genetic screen.

 

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CASSANDRA CARRIZALES
Texas A&M University
CASSANDRA CARRIZALES

The current research performed in the Judelson lab focuses primarily on the fungus-like eukaryotic pathogen Phytophthora infestans, the casual agent of potato and tomato late blight. Through investigation of specific molecules in this pathogen, specifically those controlling asexual development, we aim at understanding this pathogen’s molecular mechanisms and regulatory network. My research focuses on Myb transcription factors, a group of sequence specific transcriptional regulators found in various pathways of animals, plants and other eukaryotic species (1). Because these transcription factors are expressed at specific developmental stages in P. infestans, the current genes of my interest are the sporulation-specific Myb2R4 and Myb2R3 proteins and their roles in the growth and development are under investigation through RNA interference based-gene silencing and over-expression experiments (2). RT-PCR and western blotting techniques are being performed to determine the silencing or over-expression levels; and the phenotypes of the isolates with altered gene expressing levels are being assessed. This project will hopefully give insight into how the Myb2R4 and Myb2R3 proteins are involved in the asexual development of sporulation.

References:

1. Judelson, H. S., Blanco, F. A., 2005. The Spores of Phytophthora: Weapons of the Plant Destroyer. Nature Reviews: Microbiology. 3: 43-57.

2. Qijun, X., Judelson, H. S., 2010. Myb transcription factors in the oomycete Phytophthora with novel diversified DNA-binding domains and developmental stage-specific expression. Elsevier: Science Direct; Gene 453 (2010): 1-8.

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BLAIR CLARK
Chaffey College, CA
Blair Clark

Katiyar-Agarwal, et al. (2006), found an endogenous natural antisense small interfering RNA (nat-siRNA) that is induced by a Pseudomonas synringae strain which has a certain effector gene called avrRpt2. The nat-siRNA is created from the overlap of two genes: a Rab2-like small GTP-bind protein gene and a pentatricopeptide (PPRL) repeat protein-like gene. The PPRL gene is a negative regulator of the RPS2 disease resistant gene (R gene). When effectors from a pathogen trigger the R gene, a hypersensitive response occurs in which infected cells will undergo cell death in order to protect the healthy cells (Hammond-Kosack K, 1996). We will be running a chemical genomics screen in order to discover new components of this nat-siRNA biogenesis and functional pathway. This screen will help identify small molecules that target important components of this pathway.

In our chemical screen, we are using transgenic Arabidopsis thaliana plants that have contain the avrRpt2 effector gene. The gene has also been linked to a dexamethasone (DEX)-inducible promoter. The plant also contains a luciferase (LUC) reporter gene that is linked to the nat-siRNA target site. Using 96-well culture plates, we grow seedlings in chemicals from the LATCA library and the Spectrum library. Once the seedlings have grown, if the chemical did not inhibit growth, we will spray plants with DEX and luciferin, causing the plants to express LUC, or glow. The plants will continue to glow until the nat-siRNA silences the LUC reporter gene and it will no longer glow and the plant will undergo a hypersensitive response. However, if a chemical interferes with this nat-siRNA pathway, the plant will continue to express LUC. Along with this LUC expression, there will be no hypersensitive response. These chemicals that interfered with the pathway will be tested again using a transgenic line of Arabidopsis that carries 35S-potato virus X:GFP and 35S-GFP. With no chemicals, the plants do not express GFP, or fluoresce, because of post-transcriptional gene silencing (PTGS). Once a chemical is added that affects the PTGS, the plant will express GFP. We are trying to find these chemicals that target proteins that play a role in the nat-siRNA mediated silencing pathway and small RNA (PTGS) pathway.

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LUIS CONTRERAS
Riverside Community College, CA
Luis Contreras

Pluripotent stem cells are located in the central zone (CZ) of specialized structures, the shoot apical meristems (SAMs). The stem-cell progeny undergo differentiation in adjacent peripheral zone (PZ) leading to organogenesis. Despite a continuous transition of stem-cell progeny to differentiation pathways, a stable set of stem-cell are always maintained. WUSCHEL, a homeodomain transcription factor expressed in cells of the Rib-meristem (RM) located beneath the CZ has been shown to provide cues to cells of the CZ to specify them as stem-cells. WUS not only specifies stem-cells but also activates CLV3 within the CZ which in turn activates a receptor kinase network to repress WUS expression. The current hypothesis states that WUS activates a non-cell autonomous signal that communicates with the CZ. In our lab we are exploring the hypothesis that WUS protein itself could move into the CZ to activate CLV3 expression. In order to test this we are generating WUS:GFP fusion proteins involving the WUS protein and several different truncated forms so that the protein could be visualized by using Laser Scanning Confocal Microscopy (LSCM).

 

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OGHENEMANO EVERO
Chaffey College, CA
Oghenemano Evero

I will be working in Dr. Katherine Borkovich’s lab, which has studied G protein signaling in the filamentous fungus Neurospora crassa for nearly 20 years. G protein signaling is mediated by three subunits: Gα, Gβ and Gγ. The pathway is activated by a ligand binding to the G protein coupled receptor (GPCR) which leads to the exchange of guanosine diphosphate (GDP) on Gα for guanosine triphosphate (GTP) which the leads to the dissociation of Gα from Gβγ dimer. The Gα and Gβγ dimer activate downstream effectors, until the GTP bound to Gα is hydrolyzed, thereby losing one phosphate to produce GDP. It was recently discovered that this pathway is also activated by RIC8 -a cytosolic protein, not present in plants but important in fungi, many of which are plant pathogens.

As an REU student, I will be conducting chemical screening using the UCR chemical libraries of about 70,000-100,000 compounds to find a compound that interferes with the interaction between RIC8 and Gα protein in the G protein pathway of filamentous fungi. This will be done with yeast 2 hybrid system (Y2H) which uses a genetically engineered yeast strain that lives or dies based on the interaction between two proteins. To speed up the process of the screening, I will be programming a robot to load the media into the 96 - well plates and dispense the chemicals to those plates. Using the robot, I will be able to screen at least half of the compounds found in the chemical library. If and when an active compound is found, it will be tested on Neurospora to study Gα-dependent functions of RIC8 and also to study RIC8 in a time-dependent manner.

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JOHN HARTZHEIM
St. Olaf College, MN
John Hartzheim

Positive-strand RNA viruses are pathogenic to many organisms, including plants, insects, and humans. These viruses reproduce via the injection of the viral genome, in the form of positive sense RNA, into a host cell. Flock house virus (FHV) is an important model for the study of positive-strand RNA viruses due to its relatively small genome. Positive-strand RNA viruses replicate progeny genomes in vesicles formed in the host cell’s membranous features, such as the mitochondria, lysosomes, or the endoplasmic reticulum. In FHV infection, protein A, which serves as a viral RNA-dependent RNA polymerase, has been shown to localize to the mitochondria in Saccharomyces cerevisiae and D. melanogaster cells. Additionally, analysis of protein A indicates the N-terminal domain inserts into the mitochondrial membrane, and that such an insertion possibly initiates the formation of the mitochondrial membrane invaginations in which viral genome replication occurs.

Flock House Virus demonstrates a remarkable ability to infect organisms across a variety of kingdoms. In Dr. Rao’s lab, I will be studying the role of the capsid precursor protein during FHV viral genome replication in plant cells. FHV RNA will be expressed transiently in Nicotiana benthamiana using Agrobacterium cultures transformed with viral genes, a procedure known as agroinfiltration. This will allow the expression of coat protein (CP) singularly or in combination other viral components. Agrobacterium cultures are injected into N. benthamiana plants in accordance with the desired inoculation effect. Northern and Western blot hybridization assays will be used respectively to monitor accumulation of RNA and CP, in terms of days post inoculation (dpi). Tissue samples will also be labeled via immunofluorescence and examined using confocal microscopy to confirm CP localization on the mitochondria. This study will support research on the Brome Mosaic Virus, in which CP localizes on the ER. Further work will use electron microscopy to examine CP expression in association with structural alterations in mitochondrial membranes.

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CHRISTOPHER MASSIMINO
Frostburg State University, MD
Christopher Massimino

The UCR Cowpea Genome Research Group has developed a high density single nucleotide polymorphism (SNP) genetic linkage map (Muchero et al. 2009) and coupled this to a 10X-coverage physical map of the cowpea genome. These resources together with knowledge of syntenic relationships between the genomes of cowpea and fully sequenced reference legumes, soybean and Medicago truncatula, have been utilized to genetically and physically map several interesting traits. One of the most visually striking of these is root mucilage production. Root mucilage is of importance to plant-microbe interactions in cowpea and other legumes (Knee et al. 2001). A specific region of the cowpea genome bearing a major gene controlling root mucilage has been identified which contains several candidate genes that may explain this trait.

The goal of the summer project is to test one or more of these candidate genes. The methods that will be utilized include cowpea and pea seedling culture, stereomicroscopy, cowpea and pea DNA extraction, oligonucleotide design and polymerase chain reaction (PCR), DNA fragment purification using gel electrophoresis, DNA sequence analysis, genetic mapping, comparative genomics, and the basics of relational databasing. Root mucilage will be analyzed for carbohydrate content via HPLC. Phenotypes of cowpea germplasm accessions will be compared to their existing SNP haplotypes spanning the region of interest to categorize haplotypes containing different alleles for the mucilage trait. The sequences of different alleles of candidate genes will be determined.

The overarching goal of the Close lab with the Cowpea Research Group and collaborators is pioneering advances in genetic analysis through genomic sequencing and genotyping technologies that can be used in high-throughput approaches to facilitate genomics-assisted breeding in cowpea and other legumes (Varshney et al. 2009).

References:

1. Muchero W, Diop NN, Bhat PR, Fenton RD, Wanamaker S, Pottorff M, Hearne S, Cisse N, Fatokun C, Ehlers JD, Roberts PA, Close TJ. 2009. A consensus genetic map of cowpea [Vigna unguiculata (L) Walp.] and synteny based on EST-derived SNPs. Proc Natl Acad Sci (USA) 106:18159-18164.

2. Knee, EM, Gong, FC, Gao, M, Teplitski, M, Jones, AR, Foxworthy, A, Mort, AJ, Bauer, WD. 2001. Root Mucilage from Pea and Its Utilization by Rhizosphere Bacteria as a Sole Carbon Source. Phytopathology. 14:775-784. Varshney, RK, Close, TJ, Singh, NK, Hoisington, DA, Cook, DR. 2009. Orphan legume crops enter the genomics era!. Curr Opin Plant Biol. 12:202-210.

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BRIAN PEREZ
Riverside Community College, CA
Brian Perez

Species of Hypomyces are fungi that parasitize many common mushrooms found in natural ecosystems. With respect to Hypomyces species that colonize members of the Boletales, some appear to be highly host specific while others are considered generalist parasites that infect many genera. Due to the observed host specificity among certain Hypomyces species with certain bolete hosts, coevolution between some of the hosts and parasites is thought to be a driving force in shaping the genetic structure among populations of both the parasites and hosts. Therefore, one of the long term goals of this research project is to study these relationships. Previous work has focused on understanding the genetic structure of some parasite populations but no studies have been focused on the hosts.

To initiate studies on the host organisms, PCR based genetic markers specific to the host are needed. Obtaining these genetic markers is necessary to help genetically characterize the hosts and they must also be species specific so that contaminating DNA does not also get amplified. Xerocomus dryophilus is one of the most commonly found parasitized hosts and is the target host for this project. These fungi are ectomycorrhizal (EM) and form mutualistic relationships with coast live oak (Quercus agrifolia). To isolate and characterize host specific markers, microsatellite regions of genomic DNA will be targeted because microsatellites are short tandem repeats of nucleotides found in the genome and are usually highly polymorphic and usually suitable for population genetic studies.

To obtain this goal, DNA from the inner stipe of unparasitized and newly emergent fruiting bodies of X. dryophilus will be isolated. The genomic DNA will be digested with restriction enzymes and size selected by gel purification to obtain DNA fragments of approximately 500-1200 bp. Specific adapters will be ligated to the ends, PCR amplified, and will then be hybridized to various biotinylated microsatellite specific oligonucleotides to search for microsatellite containing fragments. Magnetized streptividin beads will be used to enrich the fragments since they have a high binding affinity to biotin and can be captured using a magnet and re-amplified using the specific adaptor primers. Microsatellite enriched fragments will then be cloned, sequenced, and primers will be developed that flank the regions. Representative host DNA samples will then be characterized for the level of variation within the microsatellite loci. The molecular markers characterized from this project will be essential for future studies investigating host population biology as well as initiating studies in host-parasite coevolution.

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GRACE SPREHN
Tulane University, CA
Grace Sprehn

The flowers of Arabidopsis thaliana consist of four organs, regulated by class A, B, C, and E genes. The organs are structured into four whorls: one of sepals, then petals, stamens, and carpels. Sepals are specified by A and E genes, petals by A, B, and E together, stamens from interactions between classes A, C, and E, and carpels require C and E. AGAMOUS (AG), a C gene, acts in specifying stamens and carpels, and in flower determinacy (1). Class C (or ag) mutants lack reproductive organs and fail to stop producing sepals and petals, producing new flowers in the original flower. This demonstrates AG’s role in flower determinacy (2). The ag weak allele, ag-10, produces fertile plants with only few flowers with additional sepals or petals. The ag-10 mutant is a good choice for sensitized screening because it facilitates the identification of new mutations owing to the mild determinacy defects in ag-10 mutants.

Previous work in the Chen lab involved the mutagenesis of ag-10 seeds, and I am using map-based cloning to identify genes affected in mutants that enhance the weak ag-10 phenotype. The F1 of a plant homozygous for a mutation and the wild type plant of a different ecotype are allowed to self. F2 plants homozygous for the mutation are then identified. Near the position of the mutation the plants will be homozygous for the first ecotype, in this case Landsberg. Far from the mutation, the frequency of Columbia (another ecotype) and Landsberg will be approximately equal. The goal of mapping is to define a small region containing the mutation. Several markers on each chromosome are tested, and areas of low recombination frequency are explored in more detail. Once the position of the mutation is defined closely, the area can be sequenced to identify the mutated gene. Ultimately, the genes will be characterized molecularly, genetically, and biochemically to explain their function in floral determinacy.

References:

1. Theiben, G (2001) Development of floral organ identity: stories from the MADS house. Curr Opin Biol, 4, 75-85.

2. Prunet N, Morel P, Negrutiu I, and Trehin C (2009) Time to stop: flower meristem termination. Plant Physiology, 150, 1764-1772.


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