MARESSA BELL-DEANE MT. HOLYOKE, SOUTH HADLEY, MA

While working in the Raikhel lab this summer, I will be researching the trafficking of proteins to the storage vacuole in Arabidopsis thaliana using the Landsberg ecotype. For that purpose we will perform screenings on a T-DNA mutagenized VAC2 population.
Wild type Landsberg plants have a small meristem and smooth looking siliques (seed pods) due to the function of CLAVATA3, which is secreted to the extracellular space. The VAC2 population used in the screening has a mutant form of the signaling protein CLAVATA-3 (clv3) and also has an active form of CLV3 fused with CTPP, a vacuolar-targeting signal. Neither clv3(which is inactive) nor CLV3-CTTP (which is targeted to the storage vacuole) is secreted, thus leading to a phenotype consisting of a large meristem and thick siliques in the VAC2 plants.

The VAC2 population has been mutagenized with T-DNA. If a T-DNA insertion disrupts the pathway required for targeting CLV3-CTPP to the storage vacuole, then CLV3-CTPP will be secreted from the cell, where it will be functional, thus rescuing the clv3 mutant, resulting in the wild type phenotype. Once we identify VAC2 plants with wild type phenotypes, we will perform TAIL PCR on these mutants to locate the T-DNA insertion position in the genome and we will confirm the correlation between phenotype and genotype using allelism tests, backcrosses and complementation assays.

The lab has already looked at 40 T-DNA mutant pools (approximately 40,000 seeds) out of 164 pools available (which we received from Dr. Ray A. Bressan at Purdue University) and 10 mutants have been identified.

TIFFANY CARMONA RIVERSIDE COMMUNITY COLLEGE , RIVERSIDE, CA

The research goal in Dr. Smith's lab is to understand the molecular mechanisms that regulate floral specification. To this end, we are focused on identifying transcriptional complexes that are involved in specifying floral cell fate.  Previous studies in Dr. Smith's laboratory have shown that PENNYWISE (PNY), a homeobox transcription factor, is part of a large transcriptional complex approximately 1 kD in size. The lab has also developed a purification technique to enrich for this complex from cauliflower meristem tissue.  One of my research goals is to affinity purify antibodies raised against PNY andSHOOTMERISTEMLESS (STM) then characterize these antibodies by western blot analysis and immunolocalization experiments.  After characterization of these antibodies, we will perform immunoprecipitation experiments to determine if we can immunopurify the 1 KD PNY transcriptional complex and determine the components of this complex using a proteomics approach.  We will also utilize a tandem affinity purification procedure to purify the PNY-transcriptional complex(es) and determine if STM and PNY are part of a large regulatory complex in Arabidopsis.

RHONDA EGIDY PITTSBURG STATE UNIV., PITTSBURG, KS

For my visit here in UC Riverside’s CEPCEB 9 week REU program I am gifted with the opportunity to work in Xuemei Chen’s lab under the tutelage of Theresa Dinh. The Chen lab is interested in cell fate in floral development and the biogenesis of micro RNAs.  This lab uniquely merges the cutting edge dimension of chemical genomics with that of traditional genetics. 

To identfy genes involved in miRNA biogenesis, the Chen lab first fused the reporter gene LUCIFERASE to a portion of the APETALA2 (AP2) gene, that contains a miRNA binding site.  Translation of AP2 mRNA is normally inhibited by miRNA172, so in this line, miRNA172 is predicted to cause translational repression in the
 LUC-AP2 reporter gene. The Luc-AP2 transgenic line was randomly mutagenized to produce point mutations.   The seeds from these “random” mutants were then collected into seed pools.  One of the three goals for my project is to sow many of these seed pools onto plates and image them live to determine their luciferase activity.  Those seedlings with higher and lower luciferase activities will be retained and further characterized.  They will represent putative mutations in genes that regulate miR172 production.  Secondly, screens will be completed using chemical libraries to determine “hit” compounds that cause higher or lower luciferase activity.  Luc-AP2 seeds will then be grown in 96-well plates in the presence of these various chemicals and images of the seedlings will be taken to determine luciferase activity.   Once again those found to cause higher or lower activity will be retained and further characterized.
 
Finally I will take part in the mapping of a hen-1 like mutant; S6.3. HEN-1 or Hua Enhancer plays multiple roles in plant development such as in the biogenesis of micro RNAs and organ identity specification in the flower.  It encodes for a novel protein that causes methylation of micro RNAs. Arabidopsis plants with hen-1 mutations exhibit pleiotropic effects, including infertility, late flowering, reduced organ and cell size, shortened internodes, and alterations in leaf number and morphology. This project will fully expose me to genetics in the forms of crossing and mapping and to the components of molecular biology: DNA, RNA, and proteins. It will also provide me with many of the necessary skills needed to successfully take part in the world of research as I one day hope to.
   
References:
Chen X, Liu J, Cheng Y, and Jia D (2002).  HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development 129: 1085-1094.
Chen X. (2005). microRNA biogenesis and function in plants.   FEBS Letters 579: 5923–593.

SHONNETTE GRANT CLAFLIN UNIVERSITY, ORANGEBURG, SC

Small RNAs (smRNAs) are major players in regulating gene expression both in plants and animals [Baulcombe, 2004].  Deep sequencing of smRNAs revealed diverse species of endogenous small RNAs from Arabidopsis [Reinhart et. al., 2002].  One of the miRNAs was shown to be involved in basal defense against Bacteria by regulating auxin signaling [Navarro et. al., 2006].  Our lab identified, another class of endogenous small RNAs derived from the overlapping region of a pair of natural antisense transcripts (nat-siRNAs) that was induced by a bacterial pathogen Pseudomonas syringae (Ps) carrying an effector avrRpt2 [Katiyar-Agarwal et. al., 2006].  My work involves examining the effect of pathogen on RNA silencing pathway genes in A. thaliana.  RNA silencing in Arabidopsis is accomplished by 4-DCLs, 10-AGOs and 6-RDRs.  We are interested to examine the expression levels of these pathway genes by using RT-PCR and Northern blotting.

Techniques Involved:
Plant material preparation
Media preparation
Pathogen culture preparation and inoculation
Samples collection at various time points
RNA extraction
RT-PCR
Northern blot hybridization and Data analysis

References:
Baulcombe, D. 2004. RNA silencing in plants. Nature 431: 356-363.
Reinhart, B.J., E.G. Weinstein, M.W. Rhoades, B. Bartel, and D.P. Bartel. 2002. MicroRNAs in plants. Genes Dev. 16: 1616-1626.
Navarro, L., P. Dunoyer, F. Jay, B. Arnold, N. Dharmasiri, M. Estelle, O. Voinnet, and J.D. Jones. 2006. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312: 436-439.
Katiyar-Agarwal, S., R. Morgan, D. Dahlbeck, O. Borsani, A.J. Villegas, J.K. Zhu, B.J. Staskawicz, and H. Jin. 2006. A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci U S A 103: 18002-18007.

TOM HIRSCHAUER UNIVERSITY OF DAYTON, DAYTON, OH

Phytophthora infestans is a fungus-like microbial eukaryote that causes late blight in potato.  In addition to causing the "Irish Potato Famine" in the mid-1800's, the disease is also a major problem that currently limits the production of potato crops worldwide.  Since asexual spores are actively involved in the dispersal of P. infestans and its infection of the plant, it is important to understand how P. infestans produces its spores, and how they might germinate.  If genes and proteins important to spore function are identified, inhibitors might be developed that would be safe and effective fungicides. The Judelson lab is using current genetic tools and genomic resources to analyze and identify which genes are most upregulated during asexual and sexual sporogenesis.

My project this summer will be to learn more about the genes that are turned on in spores by studying the structure and the expression patterns of the protein kinases.  Studying the structure of the kinases involves annotating genes from the Broad Institute database that contains the entire P. infestans genome.  By using programs that find open reading frames with codon biases deviating from the norm, and comparing these sequences to genes from related species such as Phytophthora sojae and Phytophthora ramorum, accurate gene models can be created.  Sequence comparisons are performed using BLAST (Basic Local Alignment Search Tool), and can be searched against other genome databases.  In addition to computational techniques, I will also be purifying RNA from different developmental stages and performing reverse transcription-polymerase chain reaction (RT-PCR) assays to measure the concentration of kinase gene transcripts.  This should validate the microarray expression data, and also obtain data for kinases not represented on the arrays.  Another part of my experiments will be the analysis of "control" genes, which are needed to equalize RNA levels between different preparations.

DANIEL LIN UNIV. OF CALIFORNIA, BERKELEY, CA

The shoot tip of a plant is the location of the shoot apical meristem (SAM), part of which is a stem cell reservoir. The tip of the SAM is known as the central zone (CZ), and is where stem cells are located. Outside the CZ is the peripheral zone (PZ). Once progeny of CZ cells enter the PZ, they enter differentiation pathways and become the precursors of various organs. When CLAVATA genes are mutated, the CZ sees excess stem cell accumulation and CZ expansion. Mutation of the gene WUSCHEL results in deficient stem cell production. It has been determined that CLAVATA and WUSCHEL constitute a feedback network that functions in maintaining CZ size and stem cell production. The exact nature of how these genes interact is unknown, and it is likely that there are other players in this feedback network.

Through microarray analysis of stem and non-stem cells, novel Arabidopsis thaliana genes enriched in stem cells have been identified by the Reddy Lab. My project will be to create transgenic plants in which conditional misexpression of these genes is possible. This is done using a two-component transcription activation system. This system utilizes a transcription factor, LhG4, fused to a glucocorticoid receptor, GR. In the presence of Dexamethasone (DEX), GR-LhG4 migrates from the cytoplasm to the nucleus and binds to 6xOP, a multimerized LhG4 binding sequence, and activates transcription of the corresponding downstream gene. The project will involve placing novel stem cell genes downstream of 6xOP, and then transforming the 6xOP::novel gene construct into a 35S::GR-LhG4 ; pCLV3::GFP-ER background. This will allow for gene expression to be induced by DEX. Furthermore, because pCLV3::GFP-ER is only expressed in CZ cells, the effect of misexpression on CZ organization can be observed by special live imaging methods. This will help in the identification of genes involved in the CLAVATA-WUSCHEL feedback network.

UGOEZE NWOKEDI EL CAMINO COLLEGE, TORRANCE, CA

Bioinformatics is a developing field of science that draws from the different disciplines of biology, computer science, and information technology to create databases to store and analyze biological information. The information could be specific sequences of regulatory DNA elements or amino acid sequences of proteins. The database also contains a description of the type of molecule and also the organism it was isolated from.

My project this summer involves working with the genome of the model plant Arabidopsis thaliana to discover regulatory elements by string enrichment analysis (SEA). First, the frequencies of the regulatory elements in the entire genome are found and serve as background noise levels. Then, these frequencies are compared to those found in a particular gene or group of genes. A regulator database will be constructed that contains all possible 6-8mer DNA motifs along with their frequency of occurring in each promoter sequence. Then, the promoter sequences with enriched motifs will be isolated with a hypergeometric test that assigns discrete values to the probability of the number of successes in identifying the same motif from a string of elements.

I will also learn to search databases for gene or promoter sequences and how to map the genome to define the function of unknown genes. Also, I will write computer scripts for large-scale data analysis using the R program for statistical computing and use different graphical procedures to depict and analyze data.

Bioinformatics is important because with information in databases and different computational approaches, a better understanding of cellular processes could be attained because a better idea of the function of a gene and possibly gene families are known. This offers scientists a wealth of information that can be resourced in their research. In the long run, this knowledge could be very important in pioneering advances in diagnosis, treatment, and prevention of genetic diseases.

References:
Horan, K., Girke, T. Genome-Scale Discovery of Regulatory Elements by String Enrichment Analysis (SEA).http://www.ncbi.nlm.nih.gov/About/primer/bioinformatics.html

ALEX PAYA OHIO WESLEYAN, DELAWARE, OH

Our summer research project, a part of the IGERT graduate study program, is focused mainly on re-examining chemicals that were screened from the UCR chemical Database and shown to inhibit a protein-protein interaction in Arabidopsis. The protein of interest, ROP2, is part of a large family of ROPs or Rho proteins in plants. These plant specific proteins are part of a larger family of proteins called G-proteins or GTPases. G-Proteins act as transducers of extracellular signals, which function by toggling between an inactive GDP-bound and active GTP-bound conformation. ROP proteins have been reported to be involved in polarized pollen tube growth, root hair development, negative regulation of abscisic acid flow, and a host of other important physiological processes in plants. For example, mutations in the ROP1protein that cause it to be constitutively active (CA) have been observed to cause depolarized pollen tube growth. A series of different reporters are being used to test the ability of synthetic chemicals to inhibit the interaction between ROP and REN (ROP enhancer). My part of the research involves a yeast-2-hybrid screen using yeast that contains the cloned CArop2 and REN plant genes along with the different reporters. The yeast-2-hybrid approach relies on functional protein interaction, in this case between two known proteins, so that the promoter region, made active by the CArop2 and REN interaction, can facilitate the binding of RNA polymerase, which transcribes genes coding for histidine production- an essential amino acid for cell growth. Once chemicals are introduced to the cell suspension (CArop2_REN3) the growth of the cells is measured qualitatively by observing growth and quantitatively by measuring the light absorbance of each culture before and after incubation with the chemical. If the introduced chemical inhibits cell growth by inhibiting the histidine production, which is regulated by the CArop2-REN3 interaction, then the chemical moves on for further study. In the last portion of the research we will introduce the screened chemicals to live plant tissue and observe the phenotypic characteristics.

ZHEN QIN UNIV. OF CALIFORNIA, BERKELEY, CA

My project in Dr. Borkovich's lab this summer revolves around the G-protein alpha subunit 3 (GNA-3), which is found in the fungus Neurospora crassa.  G-proteins are a family of proteins that act to regulate cell processes.  Using a previously constructed mutant strain of N. crassa that had its copy of the gna-3 gene deleted, we were able to compare the phenotypical changes of the fungus.  Most notable was that the mutant strain would conidiate, or form spores, while submerged.  Under the guidance of my mentor, James Kim, we will further investigate the specific role that GNA-3 plays in cell development.  I will be screening the Spectrum Bioactive Compound Library for compounds that cause the wild-type strain to behave like the mutant strain (conidating under submerged conditions), and for compounds that cause the mutant strain to behave normally.  During this time, I will also use the yeast two-hybrid model, a method for testing physical interactions between different proteins, to identify proteins that interact with GNA-3.  At the end of my tenure here, I hope to find several compounds used to activate or deactivate GNA-3, and to utilize these compounds in probing the signaling pathway that involves GNA-3.

LAUREN QUEZADA LOYOLA MARYMOUNT, LOS ANGELES, CA

In the Ding lab, we are currently studying the interaction between RNA silencing and viral infection. RNAi is known as the viral innate immune pathway in insects that uses siRNAs to target viral RNAs for silencing.  This in turn inhibits viral replication and transcription, rendering the virus harmless.  However, to bypass RNAi, viruses have evolved various proteins that suppress RNAi, which collectively are called viral suppressors of RNAi, VSR for short.  One such VSR is the NS1 protein encoded by Influenza A virus; more commonly known as Avian flu.  NS1 has been shown to be active in suppressing RNAi and the interferon response in insect and mammalian cells respectively. 

This summer I will attempt to find inhibitors of the NS1 protein by screening a small molecule library. To do so I will be performing a cell-based screening assay in Drosophila S2 cell culture to find chemical inhibitors of NS1.  To do this the lab has constructed a chimeric plasmid that codes for an inducible viral amplicon (R1GFP) as well as a constitutively active promoter that drives NS1.  We will transfect this plasmid into S2 cells and then the small molecules will be added. Normally cells that carry this plasmid will fluoresce green from the virally driven production of GFP. However if NS1 function is inhibited, GFP fluorescence will be lost due to silencing via RNAi.  When this occurs, the dark wells will be marked as hits.  We then will re-screen the hits and confirm down-regulation of viral RNA with Northern blot analysis.  This technique will allow us to read the levels of RNA present in each of the wells not expressing GFP – thus affirming that the chemical “hits” found bound to and suppressed the expression of the VSR.

SONIA SO UNIVERSITY OF ARIZONA, TUCSON, AZ

The field of chemical genetics has rapidly grown in recent years due to the ability to discover drug-like molecules with high-throughput screening (Kolb et al, 2004).  The Cutler lab is focused on studying plant cell development and the pathways that control growth.  In using the principle of chemical genetics, I will aid in the effort to screen a library of small molecules, which will be fluorescently-labeled.  They will be screened and visualized using confocal microscopy to study the subcellular effects on cell expansion and growth in Arabidopsis.  We will harness click chemistry to attach the small molecules onto a biotin group via an azide-alkyne cycloaddition reaction.  As shown by Scheme 1 (Sharpless et al, 2003), catalytic amounts of Cu (I) are used to increase the rate of reaction, which can be accomplished in water.  This is significant because the small molecules in our library have a low enough ClogP such that they can be introduced into the cell and then tagged, thus ensuring that the cellular processes have not been disrupted by our efforts to visualize them.  The click library is composed of terminal acetylenes to ensure that the hit molecule can be tagged and introduced to the cell easily, relying on the fact that click chemistry reacts our acetylene and azide groups by a large thermodynamic force in a simple, specific, and high yielding manner.  The click reaction is done under water, which allows us to perform the reaction in vivo.  We are looking for a variety of phenotypes where, ultimately, the Arabidopsis seedlings have stunted growth in the hypocotyl, root, or cotyledon.  Once the chemical screen yields a hit, a dose curve will be made to measure the concentrations at which the chemical is active.  My project includes the screening and creation of dose curves of chemicals, the use of confocal microscopy, the organic synthesis of molecules as well as the synthesis of the fluorescent marker, Coumarin, and the use of a liquid handling robot programmed to make plates for biological and chemical screens.

DANIEL SWANK RIVERSIDE COMMUNITY COLLEGE, RIVERSIDE, CA

The Bailey-Serres Lab is focused on the study of low oxygen (hypoxia) stress in plants.  To this end, the lab is focusing on four ongoing projects: the evaluation of mechanisms of submergence tolerance in rice, the development of methods to analyze cell-type specific mRNAs to study cell type specific transcriptional regulation under hypoxia in Arabidopsis, the evaluation of hypoxia stress regulated proteins with no known biological function in Arabidopsis, and the project to which I have been assigned, the study of low-oxygen sensing and response mechanisms in plants.

Under the supervision of graduate student Charles Jang, I will be studying the role of the hypoxia-induced mitogen-activated protein (MAP) kinase, MAPK6, in Arabidopsis (AtMPK6). A purely classical genetic approach introduces complications as an AtMPK6 knockout produces no phenotype under hypoxia, most likely due to its partial functional redundancy with AtMPK3 and double knockouts of atmpk3/atmpk6 are embryo lethal. To avoid this complication and to more finely control the activity of AtMPK6, we will screen for a small molecule that specifically inhibits AtMPK6 kinase activity.

An in vitro assay was developed to monitor kinase activity.  Kinase activity consumes ATP in order to phosphorylate a substrate.  The assay will monitor the depletion of ATP in solution via luciferase, an enzyme that bioluminesces in the presence of ATP and luciferin.  A small molecule screen will be conducted using the Spectrum bioactive library to look for inhibitors of kinase activity.  A recombinant source of AtMPK6 will be used instead of native AtMPK6 due to the difficulty of obtaining sufficient quantities in sufficient concentration to be useful in a high throughput screen.  We expect to see lower luminescence as the kinase consumes ATP and in the presence of the inhibitor, the kinase consumes less ATP and the final luminescence will be higher.  Thus there is a direct correlation between inhibition of kinase activity and luminescence. 

Once an inhibitory compound is isolated it will then be used in planta to determine the role of AtMPK6 in low oxygen stress signaling by treating three genotypes of Arabidopsis  (Columbia, atmpk3 knockout, atmpk6 knockout) with the inhibitor and monitoring plant responses both in normoxic and hypoxic conditions.