UCR

Center for Plant Cell Biology



REU Students and their Summer 2016 Research Programs


 Summer 2016 REU Students and their Research Projects

Undergraduate students were invited to apply to the Center for Plant Cell Biology (CEPCEB) at UC-Riverside to pursue individual research projects in the areas of cellular and molecular biology of plants and their pathogens. In 2016, 13 students were selected for this ongoing 10-week residential summer program. Please click on the following student links to see photos and read abstracts about their summer 2015 research projects in UCR laboratories.

 2016reu

 2016 CEPCEB REU students

REU Student

College/University

Lab/PI

Emily Bossard

Western Washington University

Kathy Borkovich

Brooke Gomez

San Bernardino Valley College

Meng Chen

Olenka Graham

Chaffey College

Isgouhi Kaloshian

Ashley Henry

Truman State University

Amy Litt

Cody Jacobs

Bennington College

Ian Wheeldon

Timothy Jang

University of Illinois

Jaimie Van Norman

McKenzie Pickle

UC-Riverside

Carolyn Rasmussen

Yonathan Mewail

San Diego Community College

Venu Reddy

Thomas Sokolich

California State Polytechnic University

Patty Manosalva

Emilie-Katherine Tavernier

Coastal Carolina University

Eugene Nothnagel

Guillermo David- Valero

University of Puerto Rico-Mayagüez

Thomas Eulgem

Joel A. Velasco

Boise State University

Bailey-Serres

Zoe Yeoh

Gettysburg College

Patty Springer

 

- Emily Bossard, Western Washington University

2016emily.bossard

 

The phenotypic effect of RIC8 point-mutations in Neurospora crassa (Borkovich lab)

G-protein signaling is essential to normal growth, sensing, and development in eukaryotes. G-Protein Coupled Receptors (GPCRs) are Guanine-Nucleotide Exchange Factors (GEFs) for Ga subunits that interact with Ga, Gb, and Gg proteins. Guanine Nucleotide Exchange Factors (GEFS) activate the signaling pathway of Ga subunits by instigating the release of GDP and subsequent binding of GTP. RIC8 is a non-GPCR GEF found in animals and filamentous fungi that is essential for the regulation of Ga subunits. Previous research has found that RIC8 interacts with Ga proteins GNA-1 and GNA-3 in Neurospora crassa. RIC8 knockout mutants (Dric8) possess significant defects in sexual and asexual development and growth. Dric8 mutants were also shown to have decreased levels of Ga (GNA-1, GNA-2, GNA-3) and Gb (GNB-1) subunit proteins and shown to regulate GEF activity of GNA-1 and GNA-3. Alignment of N. crassa RIC8 with proteins from animals identified numerous conserved amino acids. Mutation of these residues showed that several were essential for normal interaction with GNA-1 and/or GNA-3 and GEF activity. We are studying the phenotypic effects of 5 ric8 point-mutations in N. crassa. We will quantitate hyphal growth rate, aerial hyphae height and conidiation. We will also assess colony morphology and the development of sexual structures.  The effect of ric8 point-mutations on GNA-1 and GNB-1 production will be determined. RIC8 protein levels will be measured using Western blots and the quantity of mRNA will be assessed using Northern blots. This research will help us to further understand the role of RIC8 in Ga regulation in eukaryotic organisms.

 

Brooke Gomez, San Bernardino Valley College

2016brooke.gomez

Mechanism of Sorting Dual-Localized Proteins in the Plant Cell (M. Chen lab)

In the eukaryotic cell thousands of proteins need to be transported from the cytoplasm, in which they are synthesized, to their respective organelles. Each protein consists of one or more individual signals that target proteins to specific organelles. However, the sorting mechanisms of proteins localized to more than one organelle are still poorly understood. HEMERA (HMR) in Arabidopsis thaliana is a dual-localized protein necessary for light signaling in the nucleus and transcriptional regulation in the chloroplasts. HMR contains both a putative nuclear localization signal and a transit peptide for nuclear and plastidial import, respectively. To investigate the subcellular partitioning of dual-localized proteins to the nucleus and plastids, I will utilize HMR as the model to investigate the subcellular localization of proteins with dual localization signals in tobacco. This project will allow us to gain novel insight into the sorting mechanism of dual-localized proteins in plants.

 

Olenka Graham, Chaffey College

2016olenka.graham  

Immunity against root-knot nematodes in Arabidopsis thliana and tomato (Kaloshian lab)

Plant parasitic nematodes are responsible for $US157 billion in crop losses annually. The Kaloshian lab studies immunity to the root-knot nematodes (RKN, Meloidogyne spp.) which infect a vast number of plant species. Plant immunity to pathogens relies on a two-tiered process: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). In the past, research on plant immunity against RKN has been centered mainly on ETI. To characterize PTI responses, the Kaloshian lab used mutants of the model plant Arabidopsis thaliana and identified a well-known co-receptor, BAK1, that is required for early RKN perception. To identify the RKN recognition receptor, they performed gene expression analysis with RNAseq in wild-type and mutant bak1 plants, RKN-infected and control, and identified 11 candidate genes. Screening mutants of a subset of these with RKN identified a negative regulator of immunity. This project aims to further investigate these PTI mutants and identify the tomato homolog of this negative regulator to evaluate conservation of its role in immunity. This negative regulator belongs to G-type lectin receptor kinase family with limited information. Therefore, as a first step, we identified the Arabidopsis members of this family. We are now currently investigating their tomato homologs and will use this information to develop constructs for virus-induced gene silencing (VIGS) in tomato to evaluate with RKN. With this research we aim to use negative regulators of immunity to develop broad-spectrum resistance in crops against RKN.

 

- Ashley Henry, Truman State University

2016ashley.henry

Role of FUL1 in desert tobacco and tomato fruit development (Litt Lab)

The nightshade family contains members that produce both dry fruits, such as the capsules of desert tobacco, and fleshy fruits, such as the edible tomato. The gene FRUITFULL has been discovered in Arabidopsis plants to be involved in the development of their dry fruits. Over time, the FRUITFULL gene underwent a gene duplication to produce 4 derivative genes, FUL1, FUL2, MBP10, and MBP20. FUL1 is the first to be studied in this lab, but eventually we will understand the importance of all four of these genes. The main goal is to knock out FUL1 through CRISPR technology, but first we need to know where FUL1 is expressed. We have sectioned and stained desert tobacco ovaries at multiple developing stages to discover the timing of anthesis, cell division triggered by fertilization, the lignification of cells, and when the cells dry to make a dry fruit. Next, we will extract RNA and sequence the transcriptome on the samples from each of the four developmental stages. Likewise, a fluorescent transcriptional reporter construct will be built to examine FUL1 transcription in tomato through confocal microscopy. Once the construct proves to be successful in the tomato plant, this method will be repeated in the lesser-known desert tobacco. Knowledge of the expression of the FUL1 gene in both tomato and desert tobacco could lead to an understanding of the evolution of dry and fleshy fruit development in the nightshade family.

 

 - Cody Jacobs, Bennington College

 2016cody.jacobs

Enhancing ER membrane production in the yeast Yarrowia lipolytica to increase P450 function and expression for use in biocatalysis (Wheeldon lab)

Y. lipolytica is an oleaginous yeast capable of producing vast amounts of intracellular lipids, which is useful as lipids serve as precursors to a number of valuable molecules, such as biofuels, steroids, and dicarboxylic acids. The cytochrome P450 family is a large, highly conserved family of membrane proteins that play a role in the catalysis of a wide variety of compounds, many of which require modification of lipids. The aim of this project is to genetically engineer a strain which is capable of enhanced production of ER-localized proteins; this first requires increasing the size of the endoplasmic reticulum, which can be done by exploiting Y. lipolytica’s natural lipid metabolism capabilities. This should in turn allow for the increased expression of the membrane bound P450s, which can then be used for biocatalysis of many useful molecules. To create a strain capable of increased P450 expression, we will knockout phosphatidic acid phosphatase, PAH1, which has previously been demonstrated to result in increased ER membrane production. We will also induce a constitutive unfolded protein response (UPR) to enhance the ER protein quality control, which is known to improve protein function. The UPR is regulated by the non-canonical splicing of HAC1 mRNA. We will introduce a modified HAC1, which has had this intron removed, under a TEF promoter, which induces constitutive expression, and thus will induce a constitutive UPR. Knockouts and insertions will be produced through use of the CRISPR-Cas9 genome editing tool. ALK2 is a member of the P450 family which is native to Y. lipolytica, and will be used to measure P450 expression in our genetically modified strain. We expect to see enhanced ALK2 and Alk2p expression, as well as Alk2p function as a result of this genome modification. This will be measured by analysis of ALK2 mRNA levels by qtPCR, western blot analysis of Alk2p levels, and assaying activity of Alk2p using a protocol developed by Guengerich et al.

 

- Timothy Jang, University of Illinois

 2016timothy.jang

Exploring Unique Cellular Features of the Middle Cortex Using Fluorescent Reporters and Confocal Microscopy (Van Norman lab)

In the root of Arabidopsis thaliana, the outer cell layers are concentric around the central vasculature. From the outermost layer toward the vascular there are: epidermis, cortex, endodermis, and pericycle layers. The endodermis and the cortex make up the ground tissue and are derived from a single stem cell. The middle cortex is a ground tissue cell type that comes from a later periclinal division of the endodermis, and the middle cortex has been thought to simply be another, second cortex layer. However, the middle cortex seems to have properties that are distinct from the endodermis and cortex. We have identified a protein that shows unique localization in the middle cortex, suggesting that it is a distinctly different cell type and may have different functions than cortex cells. For my project, I am examining the spatial and temporal expression of PLK1 and CO2 in middle cortex cells. We predict this information could show how long it takes for the middle cortex to gain its own identity. To analyze changes in polar localized proteins during middle cortex formation, I am also exploring different methods to increase the number of middle cortex cells and decrease the time of initial formation. Among other treatments, I will be using paclobutrazol, a gibberellic acid biosynthesis inhibitor, to accelerate middle cortex formation. If the paclobutrazol successfully increases middle cortex formation by at least 50%, we can use fluorescence-activated cell sorting to isolate the middle cortex cells. Through my project, we hope to provide evidence that middle cortex is indeed a unique cell type and use its formation to further our understanding of the basic processes of development and how cell fate is specified during root growth.

 

- McKenzie Pickle, UC-Riverside

2016mckenzie.pickle

Role of Microtubule Dynamics in Cell Division using Maize tangled-1 Mutant (Rasmussen lab)

In maize, tangled1 (tan1) mutants exhibit shorter stature compared to wild-type, rougher leaves, and misplaced cell walls. Timelapse analysis shows that the tan1 mutant has slower division times in metaphase and telophase compared to wild-type. There is no correlation between longer metaphase division times and misoriented divisions, suggesting that mitotic progression does not affect placement of cell wall. Phragmoplast expansion and disassembly is slower in the tan1 mutant, suggesting a role for TANGLED in regulating microtubule disassembly. We are interested in testing if the TANGLED protein alters microtubule dynamics. Another undergraduate in the lab and I will be growing wild-type and mutant plants that express YFP-TUBULIN in taxol (a microtubule stabilizing drug) and propyzamide (a microtubule depolymerizing drug) and assessing growth by measuring root length. My hypothesis is that tan1 mutants have hyperstabilized microtubules so when grown in taxol media the mutant will have proportionally smaller roots compared to wild-type. tan1 mutants grown in low concentrations of propyzamide may have partially complemented root growth. Using live-cell imaging, we will assess phragmoplast dynamics in the tan1 mutant with and without taxol by measuring phragmoplast expansion and disassembly rates. We expect the mutant to be more sensitive to taxol so expansion and disassembly rates will be proportionally slower compared to wild-type at the same drug concentrations. These experiments will help us understand the role of the TANGLED protein in regulating microtubule dynamics in vivo.

  

- Yonathan Mewail, San Diego Community College

2016yonathan.mewail

The effect of drought stress on gene expression of the Arabidopsis shoot apical meristem (Reddy lab)

Water scarcity is a severe environmental problem that affects plant productivity. In 2015, California lost $1.8 billion and over 10,000 jobs due to years of severe drought impacting its agricultural productivity. With losses expected to increase due to climate change, it is important to understand how plants have adapted strategies to tolerate drought stress. While studies have characterized how drought impacts the development of the root, leaf, and plant, how shoot development is affected by drought is still not well understood. The development of the shoot system arises from a set of stem cells located at the center of the shoot apical meristem (SAM) of plants. As these stem cells undergo cell division and get pushed outwards, they begin to undergo differentiation to develop into the leaves, flowers, fruits, and buds. Genetic analysis has revealed a set of pathways involved in stem-cell maintenance, organ differentiation, and flowering, however there is limited information regarding how drought stress affects these pathways in the SAM. We will investigate how drought stress impacts growth patterns and gene expression in specific cell-types from the SAM using next-generation sequencing techniques. By monitoring how gene expression patterns change at a single-cell type resolution in Arabidopsis SAMs upon drought-induced stress, we can identify stress response genes for further characterization, focusing on how these genes are implicated in shoot development during stressful conditions.

 

- Thomas Sokolich, California State Polytechnic University

2016thomas.sokolich

Physical interaction between MORC1 proteins and putative targets of MORC1-regulated miRNA (Manosalva lab)

Robust immune systems protect plants from various pathogens, but immune activity also competes for finite resources required for growth and development. Thus, immune responses are often highly controlled to balance defense with other physiological needs. Microchidia (MORC) proteins, members of the GHKL ATPase superfamily, have recently been described as players in RNA-directed DNA methylation (RdDM) and heterochromatin silencing. MORC1 and its closest homologs regulate multiple layers of plant immunity in Arabidopsis thaliana (Arabidopsis) and Solanaceous plants (tomato, potato and tobacco). Interestingly, the effect of MORC1 is species specific, positively regulating immunity in Arabidopsis and potato and negatively regulating immunity in tomato and tobacco. While MORC1 proteins have highly conserved N-termini amongst the four species, their C-terminal regions, comprised of a linker (disordered region) and a coil coil (CC) domain, harbor key amino acid differences. These CC domains are known to mediate protein-protein interactions, and recombinant protein analysis has attributed the species specificity of MORC1 to this region. In addition, MORC proteins have been shown to physically interact with some RdDM factors and Resistance (R) proteins. Small RNA deep sequencing conducted at Dr. Manosalva’s laboratory identified several microRNAs (miRNAs) targeting additional regulators of the RdDM pathway and plant immunity. By tracing these miRNAs to their predicted target genes, four MORC1 candidate binding partners were selected to test for MORC1 interactions. Three of them correspond to proteins involved in the RdDM pathway, and the fourth target is a tomato R protein. Yeast-two-hybrid (Y2H) screens will be implemented to test for physical interaction between tomato/potato MORC1 proteins and the aforementioned candidate proteins. Various Y2H pairings of full-length and truncated proteins also aim to identify particular domains responsible for binding activity. Characterization of MORC1's molecular interactions will help elucidate its role in epigenetic regulation and plant immunity.

 

- Emilie-Katherine Tavernier, Coastal Carolina University

2016emilie.katherine.tavernier

Analysis of singly methylated sugars and their biosynthesis in wild type and transgenic tobacco cell walls (Nothnagel lab)

This project is focused on the occurrence and biosynthesis of methylated sugars in plant cell walls.  Methylated sugars have one or more of the ring hydroxyl groups modified by attachment of an O-methyl ether substituent.  Some methylated sugars appear in pectic polysaccharides nearly throughout the plant kingdom, while other methylated sugars are much more common in relictual than in derived plants. This mixed occurrence of methylated sugars presents questions regarding their function and evolutionary significance. Research into methylated sugars is also motivated by their potential importance towards efficiency of biofuel production from cell wall biomass. While several methylated sugars occur in plant cell walls, the 4-O-methyl-glucuronosyl residue is the only such sugar for which the gene encoding the methyltransferase that adds the O-methyl ether is known. Physcomitrella patens, a moss which produces abundant 3-O-methyl-rhamnosyl residues in its arabinogalactan proteins, and Nicotiana tabacum, which is a facile system for transgenic expression of candidate moss methyl transferase genes, are the two model systems in this project. The specific goal is to identify all possible singly methylated sugars within cell walls of the wild type and transgenic tobaccos. Cell wall fractions prepared from leaves have been cleaved by acid catalysis and the resulting monosaccharides derivatized for analysis by gas chromatography-mass spectrometry. Thus far, three methylated sugars have been found in wild type tobacco, with the transgenic tobacco additionally expressing 3-O-methyl-galactosyl residues. The project is being continued with alternative cell wall preparations and alternative derivatization chemistries to assure comprehensive analysis.

  

- Guillermo David- Valero, University of Puerto Rico-Mayagüez

2016guillermo.david.valero

A super synthetic elicitorand a better understanding of the plant immune system  (Eulgem lab)

Plants are exposed to different environmental factors that they must cope with to be able to survive in their respective agro/ecosystems. To be able to defend themselves from one of the major threats, phytopathogens, plants have evolved a complex inducible immune system. Phytopathogens typically produce elicitors that can be recognized by plant immune receptors. Elicitors are small molecules or fragments of macromolecules that are specific to certain pathogens or microorganisms. Some of the genes and pathways required for plant immune responses have been studied, but are still poorly understood. Synthetic elicitors are drug-like compounds that can induce plant immune response, but are distinct from natural defense elicitors. Previously, our lab had performed a high-throughput screening of 60,000 diverse organic compounds for defense inducers and identified 114 new synthetic elicitors. This project is focused on the analysis of possible synergistic effects triggered by co-application of some of these compounds. Combinations of different synthetic elicitors applied at varying concentrations will be tested for their efficiency in protecting susceptible wild type Arabidopsis thaliana (Col-0) plants against virulent strains of Hyaloperonospora arabidopsidis (Hpa). Synergism can result from interactions between two or more agents that results in quantitatively greater effects than the sum of the individual effects of each agent. Such synergistic effects may allow for the design of new super synthetic elicitor cocktails containing ultra low doses of bioactive chemicals. In order to gain a better understanding of the genes involved in synthetic elicitor triggered immune response, I am testing different Arabidopsis T-DNA mutants bearing mutations in genes previously found by our lab to be highly expressed after synthetic elicitor treatment. I will test if any of these mutants exhibit enhanced susceptibility against Hpa and possibly other phytopathogens. Taken together, this study can lead us to a super synthetic elicitor that may serve as prototype for new pesticide alternatives avoiding excessive use of toxic pesticides. In addition, we may identify new components of the plant immune system that are important for the bio-activity of synthetic elicitors.

 

- Joel A. Velasco, Boise State University

2016joel.a.velasco

Profiling the nuclear transcriptome and translatome of discrete cell populations in Oryza sativa exposed to waterlogging and water deficit conditions (Bailey-Serres lab)

Plants are multicellular organisms composed of distinct cell types, each cell with a unique composition of RNA populations. Technologies such as INTACT (Isolation of Nuclei TAgged in specific Cell Types) and TRAP (Translating Ribosome Affinity Purification) facilitate access to nuclear mRNA (nuclear transcriptome) or translating mRNA (translatome) populations from specific cell types. Here we examine next-generation sequencing data obtained through the use of these technologies in transgenic Oryza sativa lines. These lines were developed and selected for the examination of cell types from the meristematic and differentiated zones in roots and meristematic region in shoots. We also examine data generated from lines for root barrier cells such as exodermis and endodermis, to evaluate their plasticity in production of suberin in roots subjected to waterlogging and water deficit conditions. The purpose of this project is to use bioinformatic tools to analyze the RNA populations of specific cell types of O. sativa in order to answer the following three questions: How do RNA populations differ between cell types in the context of their biological functions? How do nuclear RNA populations differ from those involved in translation? How do RNA populations in exodermal and endodermal cells differ between control, waterlogged, and water deficit treatments? We aim to identify genes with enriched expression in these cell types and under these conditions. This will aid understanding of how genes and gene networks specify cell functions, as these networks are dynamically modified in response to water stress in this important crop species.

 

Zoe Yeoh, Gettysburg College

2016zoe.yeoh

LOF1 and Interacting Transcription Factors in Plant Development (Springer lab)

Transcription factors (TFs) help ensure proper gene expression in developing tissues, and thus play a role in plant development and plant architecture.  LATERAL ORGAN FUSION1, or LOF1, is a TF expressed in the organ boundaries of Arabidopsis thaliana. lof1 mutants have fused axillary branches and cauline leaves, which indicates importance in boundary development.  Because transcription factors are known to act in complexes, we wanted to discover what other proteins interact with LOF1.  We executed a yeast-2-hybrid (Y2H) screen that identified several TFs as potential interactors: WHIRLY 3 (WHY3), MYB DOMAIN PROTEIN32 (MYB32), HOMEOBOX-LEUCINE ZIPPER PROTEIN4 (HB4), and LIGHT RESPONSE BTB2 (LRB2).  WHIRLY1 (WHY1) and HOMEOBOX ARABIDOPSIS THALIANA3 (HAT3) are thought to be redundant with WHY3 and HB4, respectively, and are included in our study. To gain evidence that the interactions between the potential protein interactors and LOF1 is biologically relevant in planta, we will characterize T-DNA insertion lines in which the genes that encode these interactors are disrupted.  Our goal is three-fold: genotype the T-DNA lines to identify homozygous mutants; characterize the phenotypes of these mutants and compare to known phenotypes; and create double- and triple-mutants between lof1 and the other TFs. Because the boundary region is involved in determining leaf angle and leaf angle affects planting density, changes in leaf angle have the potential to impact crop yield. In the future, we may be able to apply the knowledge we obtain in the model plant Arabidopsis thaliana to crop species in order to improve crop yield. 


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