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Current Research Projects

A. Elucidating the function of a U-box E3 ligase-mediated ubiquitination pathway in programmed cell death and disease resistance in rice supported by USDA-NRI Plant Biology Program
PI; Guo-Liang Wang

Diseases are major limitations in crop production. This project will identify important genes that control defense responses to pathogens. The ubiquitination pathway is a major selective protein degradation system in eukaryotes. In mammals, ubiquitination has been shown to play critical roles in regulating apoptosis and defense. However, little is known about the function of ubiquitination-mediated pathways in plant programmed cell death (PCD) and defense responses. The rice lesion mimic gene Spl11 encodes a novel U-box/ARM protein that confers E3 ligase activity. In the last two years, we have made significant progress in the Spl11 project. Eight SPL11-interacting proteins (SPINs) were identified in our yeast two hybrid screens. The interaction between SPIN1 and SPL11 was confirmed in rice protoplasts. SPIN1 is oligo-ubiquitinated but not targeted for degradation by SPL11 in rice cells. Nucleic acid binding assays showed that SPIN1 has DNA/RNA binding activity. Spin1 RNAi lines confer enhanced susceptibility to a bacterial pathogen. Seven spl11 suppressor mutants were identified. With these extensive preliminary results, we propose: 1) To confirm the disease reaction of the T4 Spin1 RNAi lines and identify SPIN1 interacting proteins and target mRNA and DNA sequences; 2) To isolate the genes that suppress the spl11 lesion mimic phenotypes; and 3) To unravel the relationship between Spl11 and key components of different defense pathways in rice. The successful completion of this project will lead to an in-depth understanding of the mechanism of the Spl11-mediated ubiquitination in rice cell death and disease resistance and the development of novel strategies to create durably resistant crop plants in the US.

B. Cloning and Molecular Analysis of the Broad-Spectrum Resistance Genes Pi9, Pi2, Piz and Piz-t.

To understand the molecular basis of broad-spectrum resistance to rice blast, we are cloning and characterizing the four broad spectrum resistance genes: Pi9, Pi2, Piz and Piz-t. To clone the Pi9 gene, three RAPD markers tightly linked to Pi9 were identified using the bulk segregant analysis technique. Twelve positive bacterial artificial chromosome (BAC) clones were identified and a BAC contig covering about 100 kb was constructed when the Pi9 BAC library was screened by one of the markers. A high-resolution map of Pi9 was constructed using BAC ends. After sequencing 76 kb fragment in the contig, six resistance candidate genes with a nucleotide binding site (NBS) and leucine rich repeats (LRRs) (named NBS1 to NBS6) were identified. Constructs made from each candidate gene were used in transformation of the susceptible cultivar TP309. Evaluation of transgenic plants showed that the candidate gene Nbs2-Pi9 is Pi9. Pi2 is linked to the Pi9 gene on chromosome 6. To construct a high-resolution map at the Pi2 locus, we used markers linked to the Pi9 gene to screen a Pi2 BAC linrary. The candidate genes from the Pi2 locus are being transformed in to a susceptible rice cultivar for disease resistance tests.

C. Understanding the Rice Epigenome: From Genes to Genomes

PI: Blake Meyers; CoPIs: Guo-Liang Wang, Steven Jacobsen, Matteo Pelligrini

The goal of this project is to apply novel methods to understand the rice epigenome, with the fundamental objective of transferring the extensive knowledge about plant epigenetics to rice, perhaps the world's most important food crop. One recently proposed and updated definition of epigenetics states that it is "the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states." (Nature 447, 396-398). Epigenetic mechanisms have a demonstrated and important role in plant development, stress responses, and transcriptional regulation. The data generated by this project will include genome-wide measurements of DNA methylation, histone methylation, small RNA and mRNA profiles for a comparative set of rice tissues and genotypes. These data will enable functional and genomic studies of rice chromatin modifications, small RNAs that can direct these modifications, and their impact on gene expression. To enhance these studies and as a long-term resource, one aspect of the project is the development and characterization of mutants in rice genes critical to chromatin remodeling. The research will utilize novel laboratory and bioinformatics methods for whole-genome chromatin analysis and for the deep sequencing of small RNAs. The project will develop a comprehensive genomic resource for rice, suitable for comparative analyses with other plant genomic data.
More broadly, these data have an important impact by allowing the experimental characterization of chromatin modifications and epigenetic processes in rice, an agriculturally and scientifically important plant species that also serves as a model for other cereal crops. The data, mutants and particularly the sequencing-based methods that are developed will have tremendous utility to a broad set of plant biologists interested in development, stress responses, epigenetics, and plant genomics. The project will also include a novel education and outreach component focused on a proposal exchange system that can be used broadly by plant genetics and genomics courses at universities to build writing, communication, and critical thinking skills among graduate students. Finally, the project will build on existing websites to facilitate public use of these data and to assist in analyses of the rice genome and epigenome. The data will be accessible through our websites http://mpss.udel.edu/rice or http://epigenomics.mcdb.ucla.edu, and biological materials available through USDA-approved stock centers.

D. Deep Transcriptional Profiling of Rice Using Signature Sequencing  supported by NSF-Plant Genome Program

PI: Blake Meyers, Co-PI: Guo-Liang Wang

Project website: http://mpss.udel.edu/rice/rice_mpss.html.

The primary goal of this project is to demonstrate the utility of a novel technology called 'massively parallel signature sequencing' (MPSS) for the quantification of gene expression in plants. MPSS is a rapid method to produce 17 base pair sequence tags that are precisely representative of the population of messenger RNAs in a given tissue. Eight libraries from diverse plant tissues will be sequenced by MPSS, generating ~500,000 tags per library, for a total of four million tags. The 17-bp tag is derived from the 3' end of a messenger RNA or 'transcript' and provides a virtually unique, experimentally derived identifier for each expressed gene. The number of identical tags in a library for a given gene is precisely indicative of the level of expression of that gene. The MPSS sequence data provide quantitative or 'digital' expression information for the entire 'transcriptome', avoiding problems inherent in microarray analysis such as cross-hybridization, pre-selection of probe sequences and low signal. Statistical methods for the analysis of quantitative expression data have demonstrated that these data are robust.The MPSS sequence data is most informative when the tags are compared to either a completely sequenced genome or to large collections of ESTs. To take full advantage of the MPSS technology, the libraries will be generated from the crop plant rice (Nipponbare) and the MPSS tags compared to the complete genomic sequence. This comparison identifies the individual genes from which the tags are derived. These data can be used to quantify and confirm gene expression in different tissues, study alternative polyadenylation and assess global transcriptional changes or differences by comparing.

E. Use of Oligo Arrays to Dissect Rice Defense Response Pathways supported by USDA-Plant Functional Genomics Program

PI: Guo-Liang Wang, Co-PIs: Pam Ronald, Jan Leach, Hei Leung

Rice has emerged as the model for study of cereal genomes because of its importance as a food crop, its small genome size, the large amount of molecular genetic resources available, and its demonstrated synteny with the other cereal genomes. Compared with the advancement made in Arabidopsis during the last decade, research on dissection of defense pathways in cereals is just starting and new strategies are needed for efficient characterization of identified defense mutants. With the availability of a high quality draft rice genome sequence, large mutant collections, and whole-genome oligo arrays for rice, we are now well positioned to dissect rice defense pathways. Our specific objectives are to:
• Perform global expression analyses to identify genes that are differentially expressed in 15 mutant lines exhibiting altered defense responses
• Identify novel genes governing disease resistance and elucidate the rice defense response networks
• Capture, analyze and deposit the mutant expression data
• Develop an outreach program to enhance the knowledge base of high school teachers in Great Plains Region about modern genomic approaches to plant science
The consortium generated microarray information will be systematically cataloged for dissemination. All the processed hybridization data will be deposited at TIGR and the International Rice Information System (IRIS) for public data mining. The proposed activities will produce functional data for defense response mutants and generate of a novel forward genetics approach that are directly relevant to the goals in the International Rice Functional Genomics Consortium.
A High Throughput Protoplast System for Rice Functional Genomics and Proteomics: Protein-Protein Interactions at the Host-Pathogen Interface supported by the NSF Plant Genome Research Program

F. A High Throughput Protoplast System for Rice Functional Genomics and Proteomics: Protein-Protein Interactions at the Host-Pathogen Interface supported by the NSF Plant Genome Research Program

PI: Ralph Dean, CoPIs: Daniel Ebbole, Yinong Yang, Thomas Mitchell and Guo-Liang Wang
ABSTRACT
Rice is the food staple for over half the world's population. Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is highly devastating and persists as a major threat to global food security. The ability of host and pathogen to detect and respond to each other's presence is governed by protein interactions, however knowledge of these interactions remains fragmentary at best. Access to near complete genomes of both rice and M. grisea, along with the extensive biological resources and a rich history of investigation, provides the opportunity to investigate interactions between host and pathogen. This research project will develop and test a novel system based on the reassembly of dissected fragments of light/fluorescence generating proteins fused to associating fungal and/or rice peptides to directly visualize protein interactions in living plant cells. Rice protoplasts (cells devoid of their cell wall) will be using since they are easy to manipulate, are amenable to large-scale analyses and typically mimic whole plants. As a proof-of-concept, the system will be first tested using rice proteins that are known to interact. The applicability of the system to interrogate the interaction of proteins from rice with those of the rice blast fungus will be subsequently tested using a known defense elicitor (avirulence) protein and its cognate rice resistance receptor. The system will be finally scaled up to screen for unknown fungal and plant protein associations that are predicted to control the deployment of host defenses. The high throughput protoplast system will be also applied in a feasibility study to screen for unknown fungal elicitors of plant host defense responses, of which a select few will advance into the novel screen developed to identify interacting plant proteins. An extensive education plan for high school students and activities promoting recruitment and participation by members of underrepresented groups are planned. In addition to furthering our basic understanding of plant-pathogen interactions, the technologies to be developed will have far-reaching utility for the discovery and evaluation of other protein-protein or protein-ligand interactions in plants systems outside rice and rice blast.

Data from this project will be made available in refereed publications. Data, protocols, and reagents will also be made avaiable via www.fsgc.net (Fungal Genetics Stock Center) and www.mgosdb.org (Magnaportha grisea Oriza sativa database) as they are generated and qualities checked.

G. Proteome analysis of chromatin associated proteins during endosperm development in rice
PI: Zhaohua Peng, Co-PI: Guo-Liang Wang, J. Li, J.; C. Boyle

Cereal endosperm is the economically most important organ in crops. The progress in rice (Oryza sativa) genome sequencing and functional genomics projects has made it possible to examine endosperm development with proteome approach. Recent evidence has shown that chromatin associated proteins and chromatin related events, such as genome imprinting, play a central role in endosperm development. This project uses a genome-wide approach to analyze the chromatin-associated proteins and examine endosperm specific post-translational modifications. Our long-term goal is to identify the regulatory genes and manipulate these genes for grain yield and quality improvement. The specific objectives are: 1) Generation of a proteome database of chromatin-associated proteins in rice root and endosperm, with emphasis on the identification of novel chromatin-associated proteins and endosperm specific chromatin proteins. 2) Quantitative analysis of chromatin associated proteins using difference gel electrophoresis (DIGE) method during endosperm development. 3) Identification of possible histone modifications that are concomitant with endosperm development. 4) Identification of at least 45 T-DNA/transposon insertion mutants of endosperm specific chromatin genes, or generation of the corresponding RNAi mutants or overexpression transgenic lines if insertion mutants are not available for some of the genes.
This project focuses on elucidation of the chromatin regulation in endosperm development. The specific objectives are: 1) Generation of a proteome database of chromatin associated proteins in rice (Oryza sativa) root and endosperm using chromatin purification, 2-D gel separation, and followed by mass spectrometry analyses. 2) Identification and quantification of chromatin proteins whose expression is up or down regulated during endosperm development using difference gel electrophoresis (DIGE) method. 3) Identification of possible histone modifications that are concomitant with endosperm development. 4) Identification of T-DNA/transposon insertion mutants of endosperm specific chromatin genes, or generation of RNAi mutants and gene overexpression lines if insertion mutants are not available in public mutant libraries.