McKell Dilg BRIGHAM YOUNG UNIV., PROVO, UT

In order to study more in depth FCA as an ABA receptor we will attempt to construct a fluorescent ABA sensor using a portion of the FCA protein. Fluorescence can be observed as an absorption of light at a given wavelength and then subsequent emission at a lower wavelength. We will be using two specific fluorescent indicators, CFP and YFP. CFP is a cyan fluorescent protein, and YFP is a yellow fluorescent protein. The special thing about these two fluorescence proteins is that together they create what is called FRET: fluorescence resonance energy transfer. Light is first absorbed by one fluorescent molecule (CFP), and then the energy is transferred to a different molecule (YFP) and finally emitted. The larger the FRET, the closer together CFP and YFP are.

In my experiments I will construct a fusion protein where CFP and YFP are attached to opposite ends of a portion of the FCA protein. We will look for changes in CFP-YFP FRET upon ABA binding in in-vitro ways. Preliminary experiments have shown a change in FRET upon ABA binding with this fusion protein. We will attempt to first, maximize the ABA binding dependent change in FRET by modifying the linkers between the CFP-FCA-YFP fusion protein and second, further define the ABA binding site by making additional truncated versions of FCA.

Protein expression and ABA binding assays with other potential ABA binding proteins are also underway.
References: Razem, A. Fawzi, et. al. The RNA-binding protein FCA is an abscisic acid receptor. Nature, 439, 290-294 (2006).

CALEB JONES EASTERN OREGON UNIV., LA GRANDE, OR

The Borkovich Lab is working on a five year project with Dartmouth Medical School and UCLA to learn the function of the Neurospora crassa genome. N. crassa is a filamentous fungus that is a model organism in which plays an important role in numerous accounts of research and discoveries. N. crassa has already been completely sequenced (there are 10,620 genes), but now the goal is to learn the function of each of the genes. In order to figure this out, knockout mutants have been created.

My project this summer will be analyzing at about twenty knockout mutants and phenotyping them. To analyze them I will be growing them on different types of medium, watching their growth, and keeping a photo record, using several forms of microscopy. The genes that will be “knocked out” in these mutants are protein phosphatases. Not much is known about the phophatases in N. crassa; they have been studied more in other organisms such as Saccharomyces cerevisae. Phosphatases are dephosphorylating negative regulators in several cell pathways. They regulate physiological responses to extracellular signals. My goal for the end of the summer will be to successfully phenotype the protein phosphatase genes to see what purpose they serve in N. crassa.

Fady Rofail CERRITOS COMMUNITY COLLEGE , NORWALK, CA

The major focus of my project is to examine how FHV is transported between plant cells to achieve systemic movement. The genome of FHV is bipartite: RNA 1 (F1) codes for a non structural protein that is required for viral replication and another non structural protein B2 expressed as a subgenomic RNA3 (sgRNA3). Protein B2 has been identified as a suppressor of RNA silencing. RNA2 (F2) encodes the structural capsid protein gene which results in co-packaging of RNA1 and 2 into a single virion. The sgRNA3 is not encapsidated into virions. Although FHV replicates efficiently in plant cells, it can not spread from the point of initial entry to neighboring cells and to distal part of the plant due to the lack of genes that are involved in this process (referred to as movement protein). This defect however can be circumvented, by complementing with movement proteins of either tobacco mosaic virus (TMV) or red clover necrotic mosaic virus (RCNMV). In the absence of complementing movement proteins the spread of FHV remain subliminal. In addition to movement protein, some viral systems also require additional proteins such as capsid protein.

My research will focus in addressing the following questions: (1) Does spread of FHV require both movement protein and capsid protein? (2) To what extent does FHV coat protein contribute to cell-to-cell spread? (3) Does FHV spread in virion or non-virion form? To find answers to these questions I will use a FHV RNA2 derived defective interfering RNA (DI-634) as an experimental vehicle in combination with green fluorescent protein (GFP) as a tracer molecule. DI-634 replicates in the presence of F1 and is also encapsidated by the FHV capsid protein. After subcloning GFP ORF into DI-634, the resulting DI-634/GFP will be inserted into pCass4 binary vector amenable for agrotransforamtion. Following agrotransformation, DI-634/GFP will be co-infiltrated with F1 and F2 to Nicotiana benthamiana leaves. Infiltrated leaves will be harvested at regular time intervals and are subjected to confocal laser scanning microscopy for monitoring temporal expression and localization of GFP. For studying encapsiadtion, virion RNA profile will be compared to that of total RNA by Northern hybridization using riboprobes specific for FHV RNAs. The inherent nature of the research project will expose me to various recombinant DNA techniques such as PCR, cloning, gel electrophoresis, agrotransformation, infiltration, virus purification, RNA isolation, confocal laser scanning microscopy, Northern and Western blot hybridizations.

Jason Schoneman CALIFORNIA STATE POLYTECHNIC STATE UNIV, POMONA, CA

Asexual spores are extremely important in the pathogenesis of P. infestans. They are actively involved in the infection of the plant and the dispersal of inoculum. Asexual spores consist of sporangia and the zoospores formed within the sporangia. In P. infestans, sporangia can detach from the hyphal body and act as a dispersal agent. At cooler temperatures six or more biflagellate zoospores can exit the sporangia and further travel to find their host. Before the zoospore can infect the host, it has to encyst and germinate. Essentially, all the steps from hyphae to germination of encysted zoospores may involve the expression of important sporogenesis genes.

The Judelson lab has assembled an EST dataset consisting of genes upregulated greater than ten times during asexual sporulation. They produced this dataset from microarray (Affymetry Genechip) analysis studies. I will use the EST dataset to construct correlating promoter regions in closely related Phytophthora species with bioinformatics tools. Using the BLAST (Basic Local Alignment Search Tool) of an annotated database, I will find gene and promoter homologs in these closely related Phytophthora species. Also, I will BLAST the EST clones on a raw sequence dataset for P. infestans (an annotated database for this species is not yet developed). This will allow me to move or “walk” in the sequence database to find promoter regions. After compiling the promoter homologs of the closely related Phytophthora species, I hope to find conserved blocks within these promoters using programs such as CLUSTAL, GIBBS MOTIF, SAMPLER, and others. Using Polymerase Chain Reaction, the promoters will be amplified and then cloned into the pOGUS reporter plasmid. I will then transform the plasmid into P. infestans.

CHERI TILBURG ROCHESTER INSTITUTE OF TECHNOLOGY, ROCHESTER, NY

The yeast two-hybrid system was developed using the GAL4 gene in E.coli to study protein interactions in all organisms. First we will attach the known gene of protein, X, to the binding domain (BD) of GAL4, creating the "bait". Next, we will attach each gene of the Arabidopsisthaliana cDNA library to an activating domain (AD), which creates the "hunters". When both of these plasmids are transformed into yeast the proteins in the cDNA library that interact with HEN1 will bring the BD and AD close together thus activating the GAL4 reporter gene. The GAL4 gene encodes for B-galactosidase activity, and when a lift filter assay is performed the colonies that are blue show the positive result of a protein's interaction with HEN1. The goal of this summer's REU project is to use the yeast two-hybrid system to identify the proteins that interact with HEN1 to methylate the siRNA in plants. For more information on the yeast two-hybrid system please visit http://www.biochem.arizona.edu/classes/bioc568/two-hybrid_system.htm

JOHN TRACEY ST. NORBERT COLLEGE, DEPERE, WI

We are screening over 11,000 Arabidopsis trap lines. The enhancer traps consist of a reporter gene fused to a minimal promoter, which were then inserted randomly into the genome. If the trap inserts near an enhancer, the reporter gene will be turned on. The reporter gene in the enhancer traps is known as the GUS gene and encodes for the enzyme ­β-glucoronidase. Since we are interested in the traps that are inserted near genes involved in the immune response, we “GUS-stain” the leaves and look for the staining of tissue near the Peronospera infection, along hyphae or next to a germinating spore. Individual leaves are examined using light microscopy and then tissue staining is verified with confocal microscopy. Once candidates are discovered, DNA will be isolated and amplified using TAIL-PCR (Thermal Asymmetric Interlaced), which amplifies DNA of unknown sequence upstream and downstream of the T-DNA insertion of the enhancer trap. We will then BLAST amplified sequences against the Arabidopsis genome and analyze them. Finally, a 5’deletion analysis will be initiated in order to examine the location and sequence of the enhancers used to regulate defense genes. A long term application of the research involves modifying these enhancers to help improve disease resistance in crops.

CARRIE WANG UNIV. OF COLORADO, BOULDER, CO

To understand the biochemical functions of PNY/PNF, I am developing a procedure to purify the PNY-transcriptional complex from isolated inflorescence SAMs. The goal is to identify the molecular mass of the complex and the components that constitute the complex. Cauliflower, a close relative of Arabidopsis, will be utilized as the source for inflorescence meristems due to its availability and ease of isolation. Techniques such as ion exchange, gel filtration, SDS-PAGE, and immuno-precipitation will be used in order to purify the PNY-transcriptional complex. Future research will focus on determining the function of these proteins during flowering, and the mechanisms of inflorescence development.