UCR

Center for Plant Cell Biology



REU Students and their Summer 2018 Research Programs


Summer 2018 REU Students and their Research Projects

 The need for scientists in plant biology-related disciplines is growing. The converging global challenges of increasing demand for food, the need for renewable energy, environmental degradation, and limited natural resources require science-based solutions developed by appropriately trained plant scientists. Most life-science disciplines are experiencing a major shift towards the use of innovative high-throughput technologies. We think it is of critical importance that life-science undergraduates get exposed to this new style of biological research. The NSF-funded research experiences for undergraduates (REU) program hosted by the Center for Plant Cell Biology (CEPCEB) at UC-Riverside is focused on next-generation biology of plants and plant pathogens. Each summer 10 – 14 students from US colleges and universities are invited to pursue individual research projects in the areas of cellular and molecular biology of plants and their pathogens. The CEPCEB-REU targets a broad range of students from both two- and four-year colleges, including institutions that offer limited research opportunities or serve groups traditionally underrepresented in the sciences. Program participants get exposed to innovative experimental techniques and benefit from state-of-the-art genomics, bioinformatics, microscopy and proteomics facilities. In summer 2018, 12 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 2018 research projects in UCR laboratories.

  2018 CEPCEB REU students

 group

 2018 CEPCEB REU students

REU Student

College/University

Lab/PI

Jessica Adams 

University of Connecticut

Ian Wheeldon

Karen Ayetiwa

University of California, Riverside

Meng Chen

Veronica Batallones

California State University Fullerton

Kathy Borkovich

Hanah Chaudhry

New College of Florida

Thomas Eulgem

Audrey Habron

Rockhurst University

Carolyn Rasmussen

Luis Jaimes Santiago

California State University Northridge

Isgouhi Kaloshian

Elise Landsbergen

Denison University

David Nelson

Liberty Onia 

University of California, Berkeley

Linda Walling

Henry Richmond-Boudewyns

Rochester Institute of Technology

Jaimie Van Norman

William Sauers

University of Scranton

Katie Dehesh

Marakee Teshome Tilahun

San Francisco State University

Paul Nabity

Chris Roland R. Valdez

San Bernardino Valley College

Dan Koenig

Jessica Adams, Univ. of Connecticut (Wheeldon lab)

Jessica

Increasing the Versatility of Genome Editing in Yarrowia lipolytica through the Application of Type 5, Class 2 Nucleases in CRISPR-mediated Systems

The costly and detrimental nature of modern fuel sources necessitates the study of alternative methods to produce oil. Oleaginous microorganisms capable of accumulating a large amount of lipids are thus sought and studied with the objective of amplifying their lipid production. The yeast Yarrowia lipolytica demonstrates a capacity to generate a high degree of metabolic precursors for lipid biogenesis and a lipid mass amounting to 90% of its dry cell weight.  As a nonconventional yeast with a complete reference genome amenable to new gene integration, Y. lipolytica is a model candidate for studies involving the use of CRISPR-mediated genome editing technologies to enhance the productivity of lipid biogenesis pathways. A CRISPR-Cas9 system effective in driving new gene integration in Y. lipolytica was developed in 2016, however, the capability of Type V, Class 2 CRISPR nucleases such as Cms1 and Cpf1 with Y. lipolytica has yet to be demonstrated.  Cms1 and Cpf1 nucleases do not require a trans-activating crRNA, or tracrRNA, and are therefore shorter and easier to synthesize than Cas9 nuclease systems. Additionally, Cms1 and Cpf1 complexes provide versatility in cut-site sequence composition by cleaving target DNA located near T-rich protospacer-adjacent motif (PAM) sequences, while Cas9 requires a G-rich PAM sequence. These qualities, in conjunction with the capacity of Cpf1 nuclease to generate overhangs in cleaved DNA that aid new gene integration, illustrate the potential of Cms1 and Cpf1 nucleases to increase the versatility of genome-editing technology in model organisms. Here, four CRISPR-Cms1 and Cpf1 systems were proven functional in Y. lipolytica, allowing for their use in future investigations of lipid biogenesis in this key oleaginous organism.   

 

Karen Ayetiwa, University of California, Riverside (Meng Chen lab)

Karen

Characterization and Localization of PAP1

Anthocyanin is a pigment that protects plants by absorbing high-energy UV light, in response to environmental stress, improving the survival of plants. Production of Anthocyanin Pigment-1 (PAP1) is a transcription factor that regulates the production of the pigment anthocyanin. PAP1 has both a DNA binding domain and an activation domain that regulate gene expression. Although we have a broad understanding of the PAP1 protein, the activation domain has yet to be characterized. The transcription activation domain (TAD) is a short sequence of amino acids that helps trigger the activation of a particular set of genes. To identify the TAD of PAP1, we will construct clones incorporating various truncations of PAP1, then transform these constructs into yeast. By utilizing theYeast- two Hybrid (Y2H) system, we expect to identify if a particular truncation we insert into the plasmid is a TAD. Previous studies have revealed that PAP1 translocates from the cytoplasm to the nucleus, but requires a certain amino acid sequence to instruct the transcription factor where to move. This amino acid sequence is a nuclear localization signal (NLS), which tags a protein for import into the cell nucleus by nuclear transport. To investigate the NLS, we will clone potential NLS candidates and transiently transform tobacco via Agrobacterium. By attaching a fluorescent protein, mClover3, to our PAP1 truncations, we will use confocal microscopy to identify the localization of the protein. Through the characterization of PAP1, we can further understand gene regulation of plants under environmental stress.

 

Veronica Batallones, California State University Fullerton (Borkovich lab)

veronica

In Vivo Fluorescent Tagging to Determine Co-localization and Protein-Protein Interactions Between HeterotrimericGαSubunits and other Proteins in Neurospora crassa

Neurospora crassa is a filamentous, multicellular fungus with low nutrient requirements, a short-haploid life cycle, a sequenced genome and easy, tractable genetics, making it an ideal organism to study cell biology and signal transduction. In eukaryotes, G protein signaling regulates normal growth, development, and environmental sensing. The G protein is a heterotrimer consisting of an α, β, and γ subunit.  When a signal is detected, the Gα dissociates from the heterotrimer, leaving the Gβ and Gγ as a dimer. Both the Gα and the Gβ-Gγ dimer go on to perform downstream signaling. In N. crassa, there are three Gα, two Gβ, and one Gγ subunits. A previous study in N.crassa found that two of the three Gα proteins interacted with RIC8, a type of guanine-nucleotide exchange factor.  The aim of this study is to determine whether the three Gαs co-localize with other proteins, such as RIC8. The three Gα subunits will be tagged with mCherry (Red), while the other protein of interestwill be tagged with GFP (Green), all at the native chromosomal locus. The Gα-mCherry construct will insert mCherry into a conserved loop region of the Gα subunits. Assemblage of the Gα-mCherry construct will require the polymerase chain reaction, which will create specific DNA fragments. The fragments will be integrated into a plasmid via homologous recombination in yeast. The plasmid will be isolated and transformed into N. crassa. We will then utilize cellular fusion, a process in which different strains of N. crassa can grow as one, to bring together fluorescently tagged proteins together in the same cell. We will fuse a strain with the mCherry tag on the Gα protein and a strain with the GFP on the protein of interest and the resulting fused strain will produce both the mCherry tagged Gα protein and GFP-tagged target. These cells will then be analyzed using a fluorescent confocal microscope to determine whether the proteins co-localize.

 

Hanah Chaudhry, New College of Florida (Eulgem lab)

Hanah

Using Arabidopsis to determine defense devices against the deadly and devastating fungal pathogen Macrophomina phaseolina

Plants have evolved sophisticated immune systems that allow them to recognize and respond to pathogens. A particularly devastating pathogen, Macrophomina phaseolina, is a necrotrophic fungus causing diseases like charcoal rot and stem blight on its broad range of over 500 hosts. As global temperatures rise M. phaseolina will have increased habitat availability. Although this pathogen causes significant economic losses globally, defense mechanisms against it are not well studied. We have established agar plate-based assays using Arabidopsis thaliana to study genes important in defense against M. phaseolina. Using mRNA sequencing, genes upregulated in infected plants were identified. I am using A. thaliana T-DNA mutant lines, which have a loss of function in one or more of these genes to analyze their role in host immunity. The mutants are grown and infected with M. phaseolina to determine if the gene contributes to disease resistance. By the end of this project, I expect to identify many key players in the defense system of A. thaliana to M. phaseolina, especially those controlled by plant stress hormones like ethylene, jasmonic acid and salicylic acid. With this information, I hope crop varieties that are more resistant to M. phaseolina will be developed.

  

Audrey Habron, Rockhurst University (Rasmussen lab)

Audrey

A Mutagenesis Approach to Identifying Division Plane Orientation Genes in Zea mays

Division plane orientation is an essential process in plant development and for relative cell alignment within the tissue. Symmetrical cell division is important for growth, while asymmetrical cell division allows for new cell type differentiation. Knowledge about genes involved is imperative to understanding the functionality of division plane orientation and its relationship with cell maintenance. However, a comprehensive identification of these genes and their respective roles is still incomplete. In a forward genetic screen approach, mutants were generated in maize (Zea mays), a model organism for plant biology research, with ethyl methanesulfonate (EMS). This produced mutants with defects in division plane orientation; next, a ranking and prioritization of defect mutants was conducted with light microscopy. Current research consists of a plant tissue screening for unknown mutant identification as well as the dismissal of known mutants. Screening techniques include leaf glue impressions of potential mutant specimens, imaging of dividing plant cells with a confocal microscope, and immunofluorescence techniques. Results will provide insight into cellular interactions related to division plane orientation. This may reveal additional proteins and genes involved in division plane orientation and promote deeper understanding of the division plane orientation.

 

Luis Jaimes Santiago, California State University Northridge (Kaloshian lab)

Luis

Enzymatic Activity of a Possible Cowpea Aphid Effector

The cowpea aphid, Aphis craccivora, is an insect pest that is found worldwide and is responsible for significant yield loss of legume plants, primarily the cowpea, Vigna unguiculata. Cowpea is an important crop that is cultivated in several developing countries due to its short growing period, drought tolerance, and high nutritional value. Cowpea aphids feed on plant phloem sap by inserting their stylets in plant tissues to reach the phloem where they feed on plant sap. In the stylet penetration path and during feeding they deposit saliva containing a cocktail of proteins and other molecules. Molecules found in the aphid saliva are speculated to interfere with plant defense biochemical pathways against aphids. Both plant monosaccharides and disaccharides have been found to play roles in regulation of defense gene expression and signaling in plants. One such disaccharide, trehalose, is involved in resistance to the aphid Myzus persicae in Arabidopsis thaliana.   trehalase, an enzyme that breaks down trehalose into glucose, identified in aphid saliva, could be countering the effect of trehalose. The aim of this project is to clone the A. craccivora trehalase and express the recombinant protein in E. coli for functional characterization. The purified trehalase will be tested for in vitro trehalase activity.

 

Elise Landsbergen, Denison University (Nelson lab)

 elise1

From Mutation to Mutant: identifying genes that may be involved in synthesizing a novel hormone

Wildfires are a common environmental stress that hinder plant growth. Fire following seeds have adapted to the stress of wildfires by sensing smoke. Wildfire smoke contains karrikins, a chemical that causes seeds to germinate and increases light sensitivity. Sensing karrikins requires the receptor KAI2, which is not fire follower specific; this suggests that KAI2 has an alternative purpose to karrikin sensing. Additionally, the KAI2 pathway shares many similarities with the D14 pathway. D14 mediates plant responses to strigolactones: endogenous plant hormones that share structural similarities with karrikins. Phylogeny suggests that D14 evolved from KAI2, yet KAI2 has not been observed to sense endogenous signals. Using the karrikin sensitive fire follower Whispering Bells, or Emmenanthe penduliflora, this study aims to gain a better understanding of the KAI2 pathway and KL, a hypothetical endogenous signal sensed by KAI2. We will extract the RNA of E. penduliflora seedsand use next generation sequencing to de novo assemble a transcriptome. This transcriptome will then be used to identify KAI2 genes within E. penduliflora, compare their protein sequences with other karrikin-recognizing plants, and locate related genes. Because of the similarities between KAI2 and D14, this study hypothesizes that KL is an endogenous signal recognized by KAI2, and transcription of KL genes will increase in tandem with KAI2 gene transcription.

  

Liberty Onia, University of California, Berkeley (Walling lab)

liberty1

Characterizing the Role of ERF72 in the Response of Arabidopsis to Whiteflies

Manihot esculenta, also known as cassava, is a tropical root crop that is the fourth highest source of calories in the world. In the past few decades, high densities of whiteflies have devastated cassava crops in Eastern and Central Africa. At high population densities, whiteflies are able to create physical damage to cassava through feeding alone as well as through their ability to vector cassava viruses. Previous RNA-sequencing studies from the Walling Lab identified candidate whitefly defense genes that are strongly induced in a whitefly-resistant cassava genotype, but not in whitefly susceptible cassava. One of these candidate genes is the ethylene response factor ERF72. We hypothesize that ERF72 plays an important role in whitefly resistance in cassava. We are also interested in understanding possible roles of this gene in regulating different defense hormone signaling pathways. As making transgenic cassava is time consuming, I will test the role of ERF72 in resistance to whiteflies using Arabidopsis ERF72 homozygous knock-out T-DNA lines (erf72-1 and erf72-2). I will infest erf72-1,erf72-2, and the wild-type with Bemisia tabaci B. As resistance to whiteflies is manifested as a slowing of whitefly development, the numbers and stages of nymphs (immature whiteflies) will be determined at day 21 after infestation. Additionally, I will use semi-quantitative RT-PCR to determine if transcript levels of defense sentinel genes in the erf72 lines and wild type change in response to whitefly infestation. These experiments will determine if ERF72 plays a role in the whitefly defense response of Arabidopsis. Future work can then confirm its role in resistance to whitefly in cassava. Possible findings will contribute to our understanding of the plant-whitefly interaction at the genetic level.

 

Henry Richmond-Boudewyns, Rochester Institute of Technology (van Norman lab)

 henry1

Characterizing the Localization and Function of PLK4 in Root Development

Transmembrane receptor-like kinases (RLKs) are a large family of transmembrane receptor kinases that are important for cell-to-cell signalling in plants. Due to the fact that plant cells are surrounded by rigid cell walls, signaling at the plasma membrane is predicted to be critical for plant development. Despite this, many RLKs remain poorly understood. The Van Norman lab has identified a specific RLK named POLARLY LOCALIZED KINASE 4 (PLK4) that is expressed in the endodermis and the central stele with stronger expression as cells begin to differentiate. Preliminary work reveals that PLK4-GFP fusions are polarly localized to the inner polar domain of undifferentiated endodermal cells, suggesting that PLK4 perceives a signal originating from the stele. Additionally, PLK4 appears to be expressed only in a subset of endodermal cells and is only present in cells that will form the Casparian Strip - a selectively permeable, belt-like structure that wraps around differentiated endodermal cells. Using laser scanning confocal microscopy, we will conduct a detailed analysis of PLK4 expression and localization in space and time. We predict that PLK4 is important for differentiation of endodermal cells that will form a Casparian Strip. Two T-DNA insertion alleles of PLK4 have been obtained; one of which is likely a null allele. To test our hypothesis for PLK4 function, we will examine plk4 mutant roots.. Specifically, we will examine the timing and function of the Casparian Strip and passage cells in the plk4 mutants as compared to wild type.

  

William Sauers, University of Scranton (Dehesh lab)

will1

Delineating plastidial metabolite MEcPP regulated interorganellar signaling cascade

Plants have evolved elaborate endogenous signaling pathways which allow them to respond to environmental stresses. Plastids play a key role in sensing and responding to stress partially through altering nuclear gene expression. However, the underlying mechanisms of this communication are still unknown. MEcPP, the plastidial produced intermediate of MEP pathway, identified as retrograde signaling molecule, conveys information from the chloroplast to the nucleus in response to environmental stimuli. Upon stress, elevated MEcPP levels increase the expression of the nuclear encoded plastidial localized ICS1, thereby raising the levels of the stress related hormone Salicylic Acid (SA). To delineate the signaling cascade regulated by MEcPP, the second-round genetic screen was performed to identify mutants that can suppress SA accumulation in mutant plants (ceh1) that accumulates high endogenous levels of MEcPP and SA. One of the mutant designated recovery of ceh1 (rceh1) was screened out. To demonstrate the function of RCEH1 in MEcPP mediated retrograde signaling, genetic, cellular imaging, and biochemical analysis were preformed to identify interacting proteins of RCEH1 in the signaling cascade. Two proteins were identified that regulate cellular redox status and which may play a role in this pathway. We will use a split luciferase construct to identify and locate interactions. Further work will confirm the RCEH1 interacting proteins to elucidate the specific mechanism of MEcPP regulating the RCEH1 function in intraorganellar communication.

 

Marakee Teshome Tilahun, San Francisco State University, (Nabity lab)

 marakee2

Comparative phenomics at the plant-insect interface

Daktulosphaira vitifoliae (grape phylloxera) is a global pest of grapes, the world’s most culturally and economically significant fruit. When D. vitifoliae feeds on Vitis (species), proteins called effectors are released into the plant to manipulate the host plant systems. There are over two thousand predicted effectors determined by their secretory codes and expression in feeding life stages. To characterize the unknown function of candidate effectors we used bioinformatics to analyze gene expression in populations that differ in successful colonization of the same host plant. Several effector genes were differentially expressed and contained common protein structure motifs one of which being the EF Hand domain. The EF hand domains bind with calcium ions, suggesting a possible target in the Ca+ signaling pathways of the plant immune system. Our second step was taking the predicted effector genes containing EF hand domains and test them using yeast two hybrid (Y2H) experiments. Y2H experiments are used to identify the plant proteins that our effectors interact with in vitro. This process involved techniques in creating primers, conducting PCRs to amplify our effector genes, and cloning into yeast. I predict we will see a positive interaction in the yeast two hybrid that suggests our predicted effectors are true effectors that manipulate the calcium signaling pathways in grape.

 

Chris Roland R. Valdez, San Bernardino Valley College (Koenig lab)

 chris2

Elucidating the Molecular Basis of Genome Size Variation in Asian Rice (Oryza sativa)

The genome size of flowering plants varies dramatically between and within species. Most of this variation is not due to changes in the amount of genic DNA sequence but is instead due to changes in the amount of repetitive sequences such as tandem satellite repeats, microsatellites, and transposable elements. Repetitive sequences make up the majority of eukaryotic genomes and can contribute to phenotypic variation, yet the processes by which repeat content rapidly changes remain obscure. Using approaches in cutting-edge bioinformatics, we will analyze variation in genome content of over 3000 resequenced accessions of Asian Rice (Oryza sativa), an important staple crop that feeds over half of the world. We will use a novel approach based on counting K-mers – DNA sequences of length K – in various Asian Rice accessions to evaluate genome content variation. We will then determine the genetic regulation of variable sequences in the genome using association studies. This project will provide insight into the significance of the variation of these repetitive sequences, the regulation of this variation, and the role of this variation in genome size.

 


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