Facilities and Services
Genomics Research Highlights
The following projects highlight the types of research supported by Genomics Core instrumentation and services.
Venugopala Reddy Gonehal, Department of Botany & Plant Sciences
Towards developing a genome-scale, cell type-specific gene expression map of Arabidopsis shoot apex. The regulation of stem-cell specification in the Arabidopsis shoot apical meristems (SAMs) is a dynamic process, mediated by cell-cell communication. The SAM stem-cell niche resides at the tip of each shoot and it has been divided into distinct functional domains (Fig. 1). The central zone (CZ) is at the tip and harbors a set of stem-cells. Progeny of stem-cells enter into differentiation pathways when they enter the flanking peripheral zone (PZ). The CZ also provides cells to the rib-meristem (RM), located beneath the CZ, where cells of the stem form. The stem-cell number and the relative proportions of functional domains remain constant, even though cells are being continually diverted to differentiation pathways. Thus, the SAM stem-cell niche represents a dynamic and interacting network of functionally distinct domains. The challenge is to understand how the interconnected network of cells interprets complex spatio-temporal signals to regulate gene expression patterns and cell behaviors. An approach that is capable of integrating information from multiple experimental platforms is crucial to generating integrated models of stem-cell homeostasis that includes the development of a genome-scale, cell type-specific expression patterns of genes, gene functions and their regulation.
Fig. 1: Functional sub-domains of stem-cell niche highlighted by fluorescent reporter constructs. (A), (B) and (D) are the 3-D re-constructed views of shoot apical meristems (SAMs), highlighting the stem-cell domain (CZ) marked by CLAVATA3 promoter (A), the adjacent peripheral zone (PZ) is marked by UNUSUAL FLORAL ORGANS (UFO) (B), A section of the Rib-meristem (RM) revealing WUSCHEL expression (C), and the sites of differentiation are marked by LEAFY (D). (E-G) are the re-constructed side views of SAMs represented in (A-C) respectively, revealing 3 clonal layers of cells (E,F) and RM (G). Cell outlines are highlighted by FM4-64 dye (red).
The SAM represents interacting networks of distinct functional domains. Understanding the genetic networks underlying cell identity transitions will require the description of genome scale expression patterns of genes at a single cell-type resolution. However, the stem-cell population accounts for a small fraction (there are about 35-40 stem-cells per several million cells of the SAM) of entire wild-type SAM consisting of differentiating leaves/flower buds and therefore represents a major challenge to isolate a pure population of stem-cells or any other specific cell types [Fig. 1A]. Our laboratory is exploring several different methods to isolate RNA from pure populations of individual cell types so that a cell type specific transcriptome map could be developed. Microarray expression data obtained for different cell types will be subjected to bioinformatic analysis utilizing the Bioinformatics Core with the aim of identifying the molecular code regulating cell type specification.
This project also makes significant use of the Microscopy Core facility as well where 4D data sets were collected on the Zeiss 510 confocal microscope, visualized with Amira software, and manipulated on custom matlab-based software, which is being developed for this purpose. Laser capture microdissection of cryosectins will be used for capturing deeper layers of meristem which are not so readily collected by protoplasts generation and sorting.
Julia Bailey-Serres, Department of Botany & Plant Sciences
Mechanisms of sugnal transduction in stress responses. The goal of research in the Bailey-Serres laboratory is to define mechanisms of signal transduction and gene regulation that are critical to the response of plants to adverse changes in the environment. Our focus is primarily on the sensing mechanisms and acclimation responses to cellular oxygen deprivation (hypoxia/anoxia) that is a major consequence of flooding, submergence or high metabolic activity (i.e., in meristems). We use molecular-genetic, biochemical, chemical genomics and systems-biological approaches to study these processes. The long-term goal is to increase crop tolerance of flooding/submergence and to contribute to the general understanding of low-oxygen sensing and translational regulation in eukaryotic cells. Current projects include the evaluation of mechanisms of submergence tolerance in rice (USDA, USAID), establishment of methods for analysis of cell-type specific populations of mRNAs and elucidation of post-transcriptional regulation of gene expression in Arabidopsis (NSF), systematic evaluation of stress-induced proteins of unknown biological function in Arabidopsis (NSF) and the study of low-oxygen sensing and response mechanisms in plants.
The Bailey-Serres lab has benefited from the array of services available through IIGB and the Center for Plant Cell Biology. Students and postdoctoral fellows have attended workshop sessions held by Drs. Girke (Bioinformatics), Pan (proteomics) and Carter (microscopy). The bioinformatics consulting service has been used and Thomas Girke is a Co-PI on our large multi-institutional Arabidopsis 2010 Collaborative Research project to explore proteins of unknown function in Arabidopsis. The proteomics services have been used in conjunction with that project, as well as another USDA-funded project that focuses on rice biology. The confocal and fluorescence microscopy facility and the expertise of the Academic Coordinators have been used by three members of the lab. The IIGB Core Instrumentation facility has been used regularly for DNA sequencing, estimation of low quantitites of nucleic acid, phosphoimaging as well as the Affymetrix Array Hybridization Service. To date, over 50 hybridizations have been performed. The quality of the data has been excellent. The lab is particularly pleased with the very professional and efficient technical service provided by Barbara Walters.
Peter Atkinson, Department of Entomology
Genentic contol of medically important insects. My laboratory has used the Genomics Core facility since its opening in 2001. My laboratory works on the genetic control of medically important insects such as mosquitoes. We aim to develop DNA recombination-based strategies that permit the effect genetic control of these insects both through the development of genetic technologies and more directly through translational research. We focus on the discovery, analysis and engineering of transposons and more recently have become involve in mosquito genome projects. We are deeply interested in the molecular basis of the the behavior of transposons and use biochemistry, yeast genetics and fly genetics to dissect their behavior.
We have utilized the core facility for DNA sequencing of numerous DNA clones. These have been used for clone verification and for determining the breakpoints of transposition in plasmids and genomic DNA. Sequencing has verified the sequences of the transposons we have cloned from genomic DNA and is used for identifying the genetic basis of the mutants we generate in their transposases. We have used the Typhoon phosphorimaging system in transposon display analysis and have used the Proteomics Core facility in preliminary experiments to identify host proteins bound specifically to transposon end fragments. We have also used the Microscopy Core to visualize fluorescent proteins localized to the testes of transgenic insects.
Sean Cutler, Department of Botany & Plant Sciences
Strain Selective Drugs: Exploiting Natural Variation in Chemical Genomics. Natural genetic variation within a species exists at levels ranging from simple “aphenotypic” molecular polymorphisms to large-scale differences in development such as flowering time. One facet of natural variation explored primarily in humans is pharmacogenetic variation, a clinically important form of inter-individual variation in drug sensitivity. We reasoned that if pharmacogenetic variation is biologically pervasive it could be used to identify genetic factors that modulate drug sensitivity in model systems. To survey this variation in Arabidopsis, several geographically diverse ecotypes were subjected to the same chemical genetic screen of ~13,500 small molecules to identify compounds that show differential effects on hypocotyl cell elongation. This screen uncovered 4 loci that act as simple Mendelian traits to modify drug sensitivity.
To better understand the molecular basis of the variation, we characterized a molecule called hypostatin which is a new small molecule inhibitor of Arabidopsis etiolated hypocotyl cell expansion that ~25% of Arabidopsis isolates possess natural resistance to. Hypostatin-resistant accessions carry recessive mutations in HYR1, a UDP-glycosyltransferase (UGT) that converts hypostatin from a pro-drug into an activated form by glycosylation. Notably, HYR1 is part of the large UGT-superfamily of enzymes that are important pharmacogenetic factors in humans. Thus, our work demonstrates that inter-specific variation in UGT function acts to modulate drug sensitivity across biological kingdoms, suggesting that Arabidopsis may be a good model for exploring the mechanisms of pharmacogenetic variation.
Glycoactivation as a new approach for drug discovery
Our work demonstrated that hypostatin is a pro-drug that is activated in vivo by addition of a glucose moiety (see figure above). This “glycoactivation” is quite novel and provides a clear genetic and biochemical picture of glycoactivation at work to modulate the bioactivity of a small molecule. We are testing how pervasive glycoactivation is and whether it can be used to identify new bioactive molecules. We are screening a panel of 10 S. cerevisiae strains that each express a different Arabidopsis UGT against a large collection of potential substrate molecules. New glycoactivated molecules will be identified as those that induce toxicity in the presence of a plant UGT and can be followed up using in vitro synthesis of glucosides and bioassays to confirm the bioactivity. In commercial libraries, glucosides are virtually non-existent. We hope to discover classes of bioactive molecules with new mechanisms by searching this poorly explored area of chemical space. If our strategy is successful, we plan to use other assay systems (in addition to yeast) to explore the utility of this approach.
Our chemical genomic work relies almost daily on the core facilities provided by IIGB. In additional to standard services (DNA sequencing), our screens for new glycoactivated molecules rely on the robotics and small molecule libraries housed at IIGB. For example, in our screen for glycoactivated molecules, we currently perform about ~12,000 assays a day (1536 compounds tested on 8 strains in duplicate); this level of throughput would not be feasible without the advanced robotics provided by the IIGB.
Timothy J. Close, Department of Botany & Plant Sciences
Research in the Close laboratory is focused on several important aspects of crop plants for the purposes of education as well as translation of genome research into practical applications to meet the needs of agriculture. This includes integrative genome biology research on barley, cowpea (Vigna unguiculata), citrus, rice and wheat. Close's group produces fundamental genome resources such as cDNA libraries, ESTs, SNPs, high throughput genetic mapping systems and high density genetic linkage maps, sequence assemblies, content definition for microrarrays, software (HarvEST) for EST database browsing, and physical maps. In partnership with UCR Computer Scientist Stefano Lonardi and Statistician Xinping Cui, and through joint supervision of students and post-docs, Close's research also encompasses the development of new algorithms that support physical and genetic mapping. These genome resources are then used for biological studies including the development of nearby markers to facilitate marker assisted breeding and to provide an understanding of plant responses to biotic and abiotic environments including low temperature, drought and high salinity. Recent and current biological areas of interest are salinity tolerance mechanisms in rice and barley; citrus topics including easy-peeling, the effect of storage on flavor, citrus tristeza virus response and reactions to insect feeding; and deployment of high density SNP molecular marker maps into barley and cowpea breeding programs at UCR and worldwide.
Most publications from the Close lab in recent years have benefitted from technical support by the IIGB core facilities. This support has ranged from access to QPCR instrumentation and routine DNA sequencing to the production of microarray data from Affymetrix barley, rice, wheat, citrus and soybean GeneChips. The importance of the Genomics Core to research at UCR and perhaps beyond is highlighted by the fact that the Genomics Core produced about 20,000 citrus EST sequences during a phase of content development for the Affymetrix citrus GeneChip, which is now a commercial product as an outcome of a project of Close and fellow IIGB member Dr. Mikeal Roose. Development by the Close lab of HarvEST software, a gene browser downloadable for Windows from http://harvest.ucr.edu or operable online from www.harvest-web.ucr, has benefitted from access to the processing cluster in the Bioinformatics Core. Close's group helped benchmark and pay for the initial components of this processing cluster, developed under the leadership of Dr. Thomas Girke.
Richard Stouthamer, Department of Entomology
In our lab we have three main lines of research:
The interaction between bacterial symbionts and their hosts. In this work we study in particular the effects that the bacterial symbiont Wolbachia has on its insect host. This bacterium is found in many different insect species, estimates are as high as 70% of all insects are infected. Wolbachia causes a series of reproductive manipulations, such as parthenogenesis induction, male killing and crossing incompatibility. We study the interactions between the parthenogenesis inducing Wolbachia and their parasitoid wasp hosts.
The geographical origin of invading insect pests and the recognition of species and biotypes of both pest insects and their natural enemies. At least 6 new invasive insect pest species establish in California. We use various molecular techniques to determine their origin, their species status and how we can distinguish them from closely related species or biotypes.
The evolution of virulence in the plant pathogen Xylella fastidiosa. The insect vectored plant pathogen Xylella fastidiosa, became a large problem in California after the introduction of the leafhopper, the Glassy Winged Sharp Shooter, from the eastern USA. Before the introduction of the GWSS the plant pathogen was present but was only vectored by one native sharp shooter with a low population size. Because the GWSS has reached very high population densities, the bacterium is now vectored at a high rate, which allows for the evolution of more virulent bacterial forms. Our lab is working on typing different Xylella strains using the DNA sequence of several different house keeping genes to determine recombination rates between different Xyllela “subspecies”, and to help in the detection of new recombinant strains that infect different host plants.
We use the genomics Core facility for all our sequencing needs, which are considerable. At the moment we are working on 1) A molecular key to the slugs of North America based on their Internally Treanscribes Spacer sequences, 2) A key to the approximately 150 Thrips species of California also based on several gene sequences (28Sd2, ITS2, ITS2 and COI), 3) Determine the origin of the invasive Avocado Lace Bug using microsatellites, 4) Characterization of Wolbachia symbionts using 6 Wolbachia MLST sequences, 5) Characterization of Xylella strains using 7 Xylella MLST sequences, and 6) Molecualr characterization of scale insect species found on imported Avocado fruit. We are very content with the service we are receiving at the core facility.
Jian-Kang Zhu, Department of Botany & Plant Sciences
Sensing and response of plants to environmental perturbations. One of the most important distinguishing features of plants is that they are sessile and have to endure environmental challenges. Our lab is interested in the molecular mechanisms underlying plant responses to harsh environments such as soil salinity, drought and cold temperatures. In addition, we are interested in the mechanisms of transcriptional gene silencing and the role of epigenetic gene regulation in stress adaptation. We use a combination of genetic, biochemical, genomic and proteomic approaches to analyze various levels of gene regulation (chromatin level/epigenetic, transcriptional, posttranscriptional, and protein activity) and to understand stress signaling and stress tolerance. Our long-term goals are to elucidate the signaling pathways used by plants in responding to environmental stresses and to identify key genes for modifying the responses of crops to environmental stresses. This knowledge ultimately will lead to major contributions to agriculture and the environment.
Among the projects in my lab are the following:
Salt stress. Soil salinity, by inhibiting growth and crop yield, is a severe and increasing constraint on agricultural productivity. A critical aspect of salt tolerance is the maintenance of low concentrations of sodium ion in the cytosol. Recently, through the identification of Arabidopsis mutants that are salt overly sensitive (sos) and the cloning and characterization of the SOS genes, we discovered a novel signaling pathway that mediates ion homeostasis and is in part responsible for salt tolerance in Arabidopsis. We have extended our work to the family of 9 SOS3-like calcium-binding proteins (designated as SCaBPs) and 24 SOS2-like protein kinases (PKS). Members of the two protein families interact specifically to form distinct protein kinase complexes, and we have implicated several in decoding calcium signals elicited by environmental and hormonal stimuli. The function of the remaining SCaBP and PKS proteins are being investigated using biochemical and reverse genetics approaches. Our very recent work has provided new evidence for small RNA species that can modulate gene expression under stress; their role in stress tolerance will be an important facet of our future research.
Drought and ABA signaling. Upon drought stress, plants accumulate the phytohormone abscisic acid (ABA), which controls many adaptive responses. Our research is focused on how plants perceive drought stress as well as the signal transduction cascade leading to induction of ABA biosynthetic genes. We have constructed transgenic plants with drought stress- and/or ABA-inducible bioluminescence via chimeric genes consisting of drought/ABA-responsive promoters fused with the firefly luciferase reporter gene. A large collection of mutants that respond abnormally to water stress or ABA were recovered, and characterization of these mutants has provided new insights into osmosensing and osmotolerance. We cloned LOS5/ABA3, a major genetic locus controlling ABA biosynthesis and showed that this and several other ABA biosynthetic genes are positively regulated by the end product, ABA. Our work on sad1 (sensitive to ABA and drought 1) and several related mutants contributed to the discovery of a surprising role of RNA metabolism in regulating ABA sensitivity and biosynthesis. Our work on the fiery1 mutant provided the first mutational evidence indicating that inositol-1, 4, 5-triphosphate is a second messenger for ABA as well as for osmotic and cold-stress signaling.
Cold stress signaling and tolerance. Plants can increase their freezing tolerance by a pre-exposure to low, non-freezing temperatures, a process known as cold acclimation. During cold acclimation, the expression of hundreds of genes is either up- or down-regulated. Many of the cold up-regulated genes are also up-regulated by drought, high salt or ABA. Facilitated by the firefly luciferase reporter gene driven by cold-responsive promoters (e.g. RD29A, ZAT10 or CBFs), we have isolated Arabidopsis mutants that are defective in cold signal transduction and cold tolerance. Several of these mutants have led to the discovery of novel regulators of cold-responsive gene transcription, and of chilling and freezing tolerance. We cloned an important negative regulator of cold-responsive gene expression, HOS1, and found that it is a RING finger protein with an ubiquitin E3 ligase activity, thus implicating protein degradation in cold signaling. HOS1 also provides the first example of a cellular protein that exhibits cold-regulated nucleocytoplasmic partitioning. More recently, we identified the ICE1 protein, a key upstream transcription factor that binds to the CBF3 promoter and controls the expression of CBF genes in the cold. Other work in our laboratory has shown a complex regulation of cold signaling and tolerance by an RNA helicase, a bifunctional enolase, and by the functional state of mitochondria.
Gene silencing and stress adaptation. Epigenetic control of gene expression plays vital roles in development and in cellular responses to viruses, transposons and transgenes in eukaryotes. The silencing of transgenes and endogenous genes can occur at either the transcriptional (transcriptional gene silencing, TGS) or posttranscriptional (posttranscriptional gene silencing, PTGS) levels. The mechanism of TGS is poorly understood in particular. Little is known about the initial trigger for DNA methylation that is important for stable TGS. In particular, the cellular mechanisms for the active suppression of TGS are not known. We have developed a unique TGS system in the model organism Arabidopsis thaliana. In this system, an active transgene and a homologous endogenous gene become silenced when cellular ROS (repressor of silencing) factors are mutated. We have shown that ROS1 encodes a DNA glycosylase/lyase that reverses hypermethylation and TGS of homologous genes by active DNA demethylation via a base excision repair mechanism. We hypothesize that double-stranded RNA (or its small RNA products) from the transgene repeat triggers the silencing of the homologous genes and the ROS factors counter the production or action of the silencing RNA to prevent RNA-dependent DNA methylation or participate in the active demethylation of the DNA. To test this hypothesis, we plan to characterize the putative DNA demethylation activity of ROS1, to clone other ROS loci, to identify ROS1-interacting proteins, and to isolate and clone ros1 suppressor mutations. In related projects, we are investigating the potential role of miRNAs and other small RNAs in the regulation of stress-responsive genes and in stress adaptation.
An important aspect of all of our research has been the facilities and expertise at the IIGB. These include the sequencing of Arabidopsis and rice small RNA libraries, sequencing of rice kinase clones, bisulfite sequencing to determine methylation sites for several projects. In addition, we have taken advantage of the Affymetrix Genchip services at the Genomcs Core for several gene expression studies. Other gene expression studies have been supported by the use of imaging & typhoon and qPCR instrumtention. Overall, the Genomics Core and other IIGB facilities such as the Microscopy and Bioinformatics Core facilites have supported my laboratory’s research helping us to remain an extremely productive and dynamic laboratory.