Source: OHIO STATE UNIVERSITY submitted to NRP
USE OF OLIGO ARRAYS TO DISSECT RICE DEFENSE RESPONSE PATHWAYS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0199122
Grant No.
2004-35604-14226
Cumulative Award Amt.
(N/A)
Proposal No.
2003-05373
Multistate No.
(N/A)
Project Start Date
Feb 15, 2004
Project End Date
Feb 14, 2008
Grant Year
2006
Program Code
[200.1]- (N/A)
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Except as one of the important food crops, rice is becoming the model monocot plant for genome studies. Rice blast and bacterial blight are major devastating diseases of rice, causing several billions of dollars of yield loss every year worldwide. The purpose of this project is to understand the molecular basis of disease resistance to these two pathogens. This study will lead to identification of novel genes governing disease resistance and creation of transgenic rice lines with enhanced disease resistance.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011530104090%
2124099116010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
4099 - Microorganisms, general/other; 1530 - Rice;

Field Of Science
1040 - Molecular biology; 1160 - Pathology;
Goals / Objectives
1. Perform global expression analyses to identify genes that are differentially expressed in 15 wild-type versus mutant lines exhibiting altered defense responses. 2. Identify novel genes governing disease resistance and elucidate the rice defense response networks 3. Capture, analyze and deposit the mutant expression data 4. Organize two summer workshops to enhance the knowledge base of high school teachers in Great Plains Region about modern genomic approaches to plant science.
Project Methods
To rapidly and efficiently classify the defense mutants, we will conduct gene expression profiling of a set of 15 mutants with the oligo arrays to be generated in the Ronald lab (separately funded by NSF). These carefully selected mutant lines exhibiting altered defense responses to two pathogens will be extensively profiled. We will use these lines with our microarray and bioinformatic tools to dissect the pathways leading to resistance in rice. This project will be directly linked to the International Rice Functional Genomics Consortium (IRFGC; http://www.iris.irri.org/IRFGC), which has a goal to functionally characterize 60% of all rice genes by 2010, a target similar to that of the Arabidopsis 2010 Project. This project will benefit from the collective resources from the IRFGC and at the same time contribute to its goal by producing data towards understanding rice gene functions. The microarray information generated from this project will be systematically cataloged for dissemination. Our data will be housed in a microarray expression database which is being developed at TIGR (through NSF funding) and will be available to the public for data mining. The mutant data generated by this project will be stored for public online access in the IRRI-hosted IRIS. Our innovative outreach program will enhance the knowledge base of a diverse student population about modern genomic approaches to science. Rural high school teachers in High Plains region will receive training in conceptual and practical genomics. A cadre of postdocs will receive excellent training in plant genomics, including molecular and classic plant genetics, pathology and bioinformatics at three participating institutions.

Progress 02/15/04 to 02/14/08

Outputs
OUTPUTS: 1). Identification of novel genes for the resistance to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae. Using a log2 ratio value > 1.0 (induced) or <1.0 (suppressed), about 700 genes were induced in the resistant plants of Xa21, GR978, spl11, and spl17, and 320 genes were repressed in the same plants after Xoo inoculation. A total of 88 genes were induced by M. oryzae in Pi9, MPK5, and GR978 plants. Interestingly, most of these genes were suppressed in the resistant reaction of Xa21, GR978, spl17, NH1-OX to Xoo. Many genes (138) that were induced in both resistant reactions to Xoo and M. oryzae were identified. 2). Identification of activation tagging mutants involved in resistance to Xoo and M. oryzae. A total of 47 genes showing at least 1.74 fold induction in Xa21, GR978, spl11, and spl17 lines relative to the wild type 24 hr after Xoo inoculation were chosen for functional characterization. Thirty two activation tagging lines of 22 genes were identified from rice mutant collections. Among the tested lines, seven lines conferred enhanced resistance to Xoo and/or blast strains. RT-PCR analysis confirmed that seven genes were activated in the mutants. Similarly, candidate genes with at least 2.0 fold induction after M. oryzae inoculation in the Pi9 and Pi2 plants were selected for functional analysis. In total, 25 activation tagging lines were found and are being tested for their resistance to M. oryzae. 3). Validation of candidate genes by RT-PCR and protoplast-based array. The function of RING finger or U-box E3 ligase proteins in plant defense is largely unknown. Many E3 genes showed differential expression after inoculation. Over 10 E3 ligase genes were selected for RT-PCR analysis using RNA from Xoo and M. oryzae infected tissues. For the positive clones, their biological function was examined with RNAi method in rice protoplasts. Silencing of two candidate genes (Ring 4 & 6) caused cell death in more than 50% of rice protoplast compared to the control construct. Stable inducible-RNAi plants of the two genes will be generated. 4). Development of a high throughput transient protoplast system for rapidly screening and characterizing defense genes. A transient GUS, GFP and luciferase-based reporter system for defense-related genes expression analysis was established. Candidate genes can be silenced by transfecting siRNA constructs into the protoplasts. The established method can be used for large-scale screening of candidate defense genes from our microarray analysis. 5). Development of protocols for rapid detection of genomic deletions in the rice genome. Hybridization of labeled genomic DNA directly onto oligonucleotide arrays allows rapid localization of deleted regions in rice mutants derived by physical and chemical means. 6). Development of an outreach program to enhance the knowledge base of high school teachers in Great Plains Region. Teachers and students learn and use the laboratory techniques such as DNA extraction, gel electrophoresis, PCR, microarray technology etc. Dr. Stephens, who manages the project outreach program, has made many presentations to over 1000 K-12 teachers and students. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Rice is the staple food for over half of the world population and the model plant for cereal functional genomics. Diseases caused by pathogens are a major threat to food security world-wide. A thorough understanding of the molecular basis of the defense response to pathogens is essential for designing effective strategies to control the rice diseases. In this study, we conducted gene expression profiling of a set of 10 defense-related mutants using the rice 20K oligo arrays. Comprehensive microarray analyses of the mutants led to classification of related mutants in several subgroups and identification of a set of novel genes that play important roles in disease resistance. The microarray information generated from this project has been systematically cataloged and deposited on the NCBI-GEO database for public dissemination. The optimized array-based deletion detection method opens the door for development of rice mutants rapidly and cheaply. It can be also used as a means to characterize mutants that underlay a QTL. The outreach program has received wide attention on the Colorado State University campus which be linked to other such programs to make them more effective and productive.

Publications

  • Lirong Zeng, Chan Ho Park, R.C. Venu, Julian Gough and Guo-Liang Wang. 2008. Classification, Expression pattern, and E3 ligase activity assay of Rice U-box-containing proteins. Molecular Plant, in press.
  • Leach, J.E., Rebecca Davidson, Bin Liu, Patricia Manosalva, Ramil Mauleon, Gay Carrillo, Myron Bruce, Janice Stephens, Maria Genaleen Diaz, Rebecca Nelson, Casiana Vera Cruz, and Hei Leung. 2007. Understanding Broad-Spectrum, Durable Resistance in Rice. Pages 191-209 in Rice Genetics V, D S Brar, D Mackill & B Hardy, eds. World Scientific Publ. Co.
  • Stephens, J. and J. E. Leach. 2007. Classroom activities in plant biotechnology. Stephens, J.L. 2008 Classroom activities in plant biotechnology. APSnet http://www.apsnet.org/education/K-12PlantPathways/TeachersGuide/Activ ities/PlantBiotechnology/default.htm
  • C. H. Park, K.H. Jung, J. Phetsom, M. R. Babu, M. Bruce , R. Mauleon, A. Bordeos, M. Bellizzi, J. Leach, H.Leung, P. Ronald, G.L. Wang. 2008. Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. Poster presentation at the Plant and Animal Genome Meetings, SanDiego, CA. January 12-16 http://www.intl-pag.org/16/abstracts/PAG16_P07a_694.html
  • Songbiao Chen, Malali Gowda, RC Venu, Pattavipha Songkumarn, Chan Ho Park, Maria Bellizzi, Daniel Ebbole, Guo-Liang Wang. 2008.Isolation and Functional Analysis of Putative Effectors from Magnaporthe oryzae Using Integrated Genomic Approaches. In Advances in genetics, genomics and control of rice blast disease, Eds. G.L. Wang and B. Valent, Springer Publishing Company. In press.
  • Bo Zhou and Guo-Liang Wang. 2008. Functional and evolutional analysis of the Pi2/9 locus in rice. In Advances in genetics, genomics and control of rice blast disease, Eds. G.L. Wang and B. Valent, Springer Publishing Company. In press.
  • Ki-Hong Jung, Gynheung An and Pamela Ronald. 2008. Towards a better bowl of rice: Assigning function to tens of thousands of rice genes. Nature Reviews Genetics. 9, 91-101.
  • Miguel E. Vega- Sanchez, Malali Gowda and Guo-Liang Wang. 2007. Tag-based approaches for deep transcriptome analysis in plants. Plant Sciences, 173:371-380.
  • Songbiao Chen, Lizhen Tao, Lirong Zeng, Miguel Vega-Sanchez and Guo-liang Wang. 2006. Protoplast-based transient assay system for gene expression and protein-protein interaction study in rice. Mol Plant Pathol 7(5). 417-427, Front cover of the issue.
  • Leung, H., S. Madamba, A. Bordeos, M. Baraoidan, Y. Liu, X. Zhu, S. Zhang2, Bin Liu, M. Bruce, J. E. Leach. 2008. Using Rice Mutants And Near-Isogenic Lines For Dissecting Rice-Blast Interactions. Presentation at the Plant and Animal Genome Meetings, SanDiego, CA. January 12-16. http://www.intl-pag.org/16/abstracts/PAG16_W62_434.html.
  • Bruce, M.A., P. M. Manosalva, S. Lee, R. Davidson, J. Snelling, H. Leung, J. E Leach. 2008. Silencing 14-3-3 Protein Gene GF14e In Rice Causes Lesion Mimic Phenotype And Enhanced Resistance To Bacterial Blight. Poster presentation at the Plant and Animal Genome Meetings, SanDiego, CA. January 12-16. http://www.intl-pag.org/16/abstracts/PAG16_P07b_753.html
  • Chan Ho Park, Jung K, Phetsom J, Babu MR, Bruce M, Mauleon R, Bordeos A, Bellizzi M, Leach J, Leung H, Ronald P and Wang GL. Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. 2007 PMBB Symposium. March 31, 2007
  • Young-Su Seo , Laura Bartley , Todd Richter , Mawsheng Chern , Chris Dardick , Kihong Jung , Johann Chen , Jirapa Phetsom , Geun Cheol Lee , Randy Ruan , Ying Peng , Xuewei Chen, Chang Jin Park, Rajeshwari Ramanan, Chan Ho Park, Guo-Liang Wang, Pamela Ronald. Mapping the Rice Innate Immunity Interaction And Transcription Network. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.
  • Young Su Seo, Laura Bartley, Todd Richter, Mawsheng Chern, Chris Dardick, Kihong Jung, Johann Chen, Jirapa Phetsom, Geun Cheol Lee, Randy Ruan, Ying Peng, Xuewei Chen, Chang Jin Park, Rajeshwari Ramanan, Chan Ho Park, Guo-Liang Wang, Pamela Ronald. The Rice Innate Immunity Interaction Map. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.
  • Chan Ho Park, Ki-Hong Jung, Jirapa Phetsom, Mohan R Babu, Myron Bruce, Ramil Mauleon, Alice Bordeos, Maria Bellizzi, Jan Leach, Hei Leung, Pam Ronald, Guo-Liang Wang. Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.
  • Myron A Bruce, Tong Zhu, Sean Coughlan, Alice Bordeos, Guo-Liang Wang, Hei Leung, Jan E Leach. Microarrays As A Tool To Identify Untagged Deletions In Rice. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA
  • Kihong Jung, Jirapa Phetsom, David Rocke, H. H. Chou, C. Robin Buell, Patrick Schnable, Gynheung An, Pamela Ronald.. A publicly available whole genome oligonucleotide array for functional analysis of rice. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA
  • Leach, J. , P. Manosalva, R. Davidson, S. Lee, M. Bruce, M. Genaleen Diaz, B. Liu, C. Vera Cruz, H. Leung. Approaches to Durable Resistance in Rice. Invited Symposim Speaker, Cold Spring Harbor Symposium: Plant Genomics. Cold Spring Harbor, NY March 13-15, 2007
  • C. H. Park, K.H. Jung, J. Phetsom, M. R. Babu, M. Bruce , R. Mauleon, A. Bordeos, M. Bellizzi, J. Leach, H.Leung, P. Ronald, G.L. Wang. 2006. Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. Poster presentation at the Plant and Animal Genome Meetings, SanDiego, CA. January 14-18. http://www.intl-pag.org/pag/14/abstracts/PAG14_P757.html
  • Chan Ho Park, Ki-Hong Jung, Jirapa Phetsom, Mohan R. Babu, Myron Bruce, Ramil Mauleon, Alice Bordeos, Maria Bellizzi, Jan Leach, Hei Leung, Pam Ronald and Guo-Liang Wang. Characterization of rice defense mutants using whole genome expression profiling, Presented at the 2006 Plant Molecular Biology and Biotechnology Research Symposium, 3/31-4/1, 2006, Wooster, OH
  • Leach, J.E., B. Liu, R. Mauleon, P. Manosalva, R. Davidson, G. Carrillo, M. G. Diaz, M. Bruce, R Nelson, and C. Vera Cruz, H. Leung. 2005. Understanding broad-spectrum, durable resistance in rice. 5th International Rice Genetics Symposium and the 3rd International Rice Functional Genomics Symposium, Manila, Philippines. Nov. 2005.
  • Bruce, M. S. Coughlan, T. Zhu, A. Bordeos, C.J. Wu, H. Leung, and J. E. Leach. 2005. Expression profiling and gene localization of rice lesion mimic mutant spl1. 5th International Rice Genetics Symposium and the 3rd International Rice Functional Genomics Symposium, Manila, Philippines. Nov. 2005.


Progress 01/01/07 to 12/31/07

Outputs
1). Identification of novel genes for the resistance to Xoo and M. oryzae. Using a log2 ratio value > 1.0 (induced) or <1.0 (suppressed), about 700 genes were induced in the resistant plants of Xa21, GR978, spl11, and spl17, and 320 genes were repressed after Xoo inoculation. A total of 88 genes were induced by M. oryzae in Pi9, MPK5, and GR978 plants. Interestingly, most of these genes were suppressed in the resistant reaction of Xa21, GR978, spl17, NH1-OX to Xoo. Many genes (138) that were induced in both resistant reactions to Xoo and M. oryzae were identified. 2). Identification of activation tagging mutants involved in resistance to Xoo and M. oryzae. A total of 47 genes showing at least 1.74 fold induction in Xa21, GR978, spl11, and spl17 lines relative to the wild type 24 hr after Xoo inoculation were chosen for functional characterization. Thirty two activation tagging lines of 22 genes were identified from rice mutant collections. Among the tested lines, seven lines conferred enhanced resistance to Xoo strains. RT-PCR analysis confirmed that seven genes were activated in the mutants. Similarly, candidate genes with at least 2.0 fold induction after M. oryzae inoculation in the Pi9 and Pi2 plants were selected for functional analysis. From POSTECH's mutant collection, 25 activation tagging lines were found and will be tested for their resistance to M. oryzae. 3). Validation of candidate genes by RT-PCR and protoplast-based array. The function of RING finger or U-box E3 ligase proteins in plant defense is largely unknown. Many E3 genes showed differential expression after inoculation. Over 10 E3 ligase genes were selected for RT-PCR analysis using RNA from Xoo and M. oryzae infected tissues. For the positive clones, their biological function was examined with RNAi method in rice protoplasts. Silencing of two candidate genes (Ring 4 & 6) caused cell death in more than 50% of rice protoplast compared to the control construct. Stable inducible-RNAi plants of the two genes will be generated. 4). Development of a high throughput transient protoplast system for rapidly screening and characterizing defense genes. A transient GUS, GFP and luciferase-based reporter system for defense-related genes expression analysis was established. Candidate genes can be silenced by transfecting siRNA constructs into the protoplasts. The established method can be used for large-scale screening of candidate defense genes from our microarray analysis. 5). Development of protocols for rapid detection of genomic deletions in the rice genome. Hybridization of labeled genomic DNA directly onto oligonucleotide arrays allows rapid localization of deleted regions in rice mutants derived by physical and chemical means. 6). Development of an outreach program to enhance the knowledge base of high school teachers in Great Plains Region. Teachers and students learn and use the laboratory techniques such as DNA extraction, gel electrophoresis, PCR, microarray technology etc. Dr. Stephens, who manages the project outreach program, has made many presentations to over 1000 K-12 teachers and students.

Impacts
Rice is the staple food for over half of the world population and the model plant for cereal functional genomics. Diseases caused by pathogens are a major threat to food security world-wide. A thorough understanding of the molecular basis of the defense response to pathogens is essential for designing effective strategies to control the rice diseases. In this study, we conducted gene expression profiling of a set of 10 defense-related mutants using the rice 20K oligo arrays. Comprehensive microarray analyses of the mutants led to classification of related mutants in several subgroups and identification of a set of novel genes that play important roles in disease resistance. The microarray information generated from this project is systematically cataloged and deposited on the NCBI-GEO database for public dissemination. The optimized array-based deletion detection method opens the door for development of rice mutants rapidly and cheaply. It can be also used as a means to characterize mutants that underlay a QTL. The outreach program has received wide attention on the CSU campus which be linked to other such programs to make them more effective and productive.

Publications

  • Miguel E. Vega- Sanchez, Malali Gowda and Guo-Liang Wang. 2007. Tag-basedapproaches for deep transcriptome analysis in plants. Plant Sciences, 173:371-380.
  • Leach, J.E., Rebecca Davidson, Bin Liu, Patricia Manosalva, Ramil Mauleon, Gay Carrillo, Myron Bruce, Janice Stephens, Maria Genaleen Diaz, Rebecca Nelson, Casiana Vera Cruz, and Hei Leung. 2007. Understanding Broad-Spectrum, Durable Resistance in Rice. Pages 191-209 in Rice Genetics V, D S Brar, D Mackill & B Hardy, eds. World Scientific Publ. Co.
  • Stephens, J. and J. E. Leach. 2007. Classroom activities in plant biotechnology. The Plant Health Instructor, Dec. Issue.
  • Young Su Seo, Laura Bartley, Todd Richter, Mawsheng Chern, Chris Dardick, Kihong Jung, Johann Chen, Jirapa Phetsom, Geun Cheol Lee, Randy Ruan, Ying Peng, Xuewei Chen, Chang Jin Park, Rajeshwari Ramanan, Chan Ho Park, Guo-Liang Wang, Pamela Ronald. The Rice Innate Immunity Interaction Map. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.
  • Chan Ho Park, Ki-Hong Jung, Jirapa Phetsom, Mohan R Babu, Myron Bruce, Ramil Mauleon, Alice Bordeos, Maria Bellizzi, Jan Leach, Hei Leung, Pam Ronald, Guo-Liang Wang. Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.
  • Chan Ho Park, Jung K, Phetsom J, Babu MR, Bruce M, Mauleon R, Bordeos A, Bellizzi M, Leach J, Leung H, Ronald P and Wang GL. Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. 2007 PMBB Symposium. March 31, 2007
  • Myron A Bruce, Tong Zhu, Sean Coughlan, Alice Bordeos, Guo-Liang Wang, Hei Leung, Jan E Leach. Microarrays As A Tool To Identify Untagged Deletions In Rice. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA
  • Kihong Jung, Jirapa Phetsom, David Rocke, H. H. Chou, C. Robin Buell, Patrick Schnable, Gynheung An, Pamela Ronald.. A publicly available whole genome oligonucleotide array for functional analysis of rice. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA
  • Young-Su Seo , Laura Bartley , Todd Richter , Mawsheng Chern , Chris Dardick , Kihong Jung , Johann Chen , Jirapa Phetsom , Geun Cheol Lee , Randy Ruan , Ying Peng , Xuewei Chen, Chang Jin Park, Rajeshwari Ramanan, Chan Ho Park, Guo-Liang Wang, Pamela Ronald. Mapping the Rice Innate Immunity Interaction And Transcription Network. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.


Progress 01/01/06 to 12/31/06

Outputs
The goal of this project is to use rice oligo arrays to rapidly and efficiently classify defense mutants in rice. Ten carefully selected mutant lines that exhibit altered defense responses to fungal (Magnaporthe grisea) and bacterial blight (Xanthamonas oryzae pv. oryzae, Xoo) pathogens are selected to profile the gene expression before and after infections. So far, 156 hybridizations have been performed and 144 had good hybridization quality. After the data were analyzed using the ANOVA method, significant genes were identified and used for hierarchical clustering, K-means clustering (KMC) and Self-Organizing map (SOM). Comparative analysis of the expression profiles performed revealed significant differences among the tested mutants. Mutants in the Jasmonic acid and salicylic acid pathways exhibited no overlap for up or down-regulated genes with the other mutants tested. Lesion mimic mutants spl11 and spl17 showed highly similar expression patterns before and after pathogen inoculations. Twenty six genes showed at least a two-fold induction in the enhanced resistant transgenic line NH1ox and at least two-fold reduction in the enhanced susceptible transgenic line NRR1ox. For the validation of the microarray results, we selected 47 genes which are commonly induced in resistance lines after Xoo infection. The activation tagging lines of these genes were searched in the rice activation database generated by Dr Gyn An's lab in Postech, Korea. A total of 32 activation tagging candidate lines were found for 22 genes. The resistance of these lines was evaluated in our growth chambers. Among them, 13 lines showed enhanced resistance phenotypes to Xoo race 6. RT-PCR analysis confirmed that seven genes were activated in the mutants. These seven genes encode defense related proteins that are involved in basal resistance to pathogens. We developed a significantly improved transient expression method that can be used for rapidly screening and characterizing candidate genes that are involved in defense signaling pathways. High-level co-expression of multiple genes and efficient suppression of exogenous and endogenous genes have been observed in the transformed rice protoplasts. A transient GFP and luciferase-based reporter system for defense-related genes expression analysis has been established. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells has been optimized. We hybridized genomic DNA from rice deletion mutants generated by gamma ray and fast neutron. Hybridization results from both Syngenta and Affymetrix rice arrays detected deletions in the previously mapped region of chromosome 12 in spl1 lesion mimic mutant. We have confirmed using PCR that the predicted genes are deleted. An outreach program called Classroom Activities in Plant Biotechnology (http://lamar.colostate.edu/jsteph) has been developed at CSU. Dr. Stephens, who manages the project outreach program, has made presentations that included demonstrations as well as student experimental activities, to over 170 children in classroom situations in 2006.

Impacts
Plant diseases cause tremendous yield loss every year in the US. Our understanding on the activation and networking of the defense signaling pathways in plants is limited, especially in cereals. In this study, we are using microarray technique to rapidly profile defense mutant lines that exhibit altered defense responses to rice pathogens. From our hybridizations, we identified many commonly or uniquely regulated genes in the mutants. Further functional analysis of these genes will provide insights into the networking of defense pathways in rice. Our newly developed protoplast-based method can be possibly used as an alternative to the yeast two-hybrid system for large-scale detection of protein-protein interactions in living plant cells. The application of the above newly-developed method will accelerate large-scale functional analysis of the completely sequenced genome of rice and other important crops including corn, wheat, barley, and sorghum to increase knowledge for crop improvement. The paper was the front cover of the September issue of Molecular Plant Pathology. The story was also featured on USDA-NRI website as one of 2006 USDA-NRI cover stories. We developed a unique outreach program, called Classroom Activities in Plant Biotechnology, and reached out to over 700 students in 2005 and 2006. Many of the students are from Hispanic families. In addition, two teacher training sessions were conducted to instruct teachers in how to perform the series of biotechnology experiments.

Publications

  • S Chen, L Tao, L-R Zeng, M Vega-Sanchez and GL Wang. (2006). Protoplast-based transient assay system for gene expression and protein-protein interaction study in rice. Molecular Plant Pathology, 7:417-427


Progress 01/01/05 to 12/31/05

Outputs
A. Microarray analysis of the 10 defense mutants. Seeds of the 10 defense mutants were multiplied and their resistance to M. grisea and Xoo and unique phenotypes were confirmed under identical growth chamber conditions. Before performing large-scale hybridizations for all the mutants, we chose two lesion mimic mutants, spl11and spl17, for a pilot experiment. RNA was isolated from leaves with lesion mimics and without lesion mimics of spl11 and spl17, respectively. Microarray data analyses indicate that there are about 150-200 genes highly induced or repressed (>3.0 fold) when the lesions appear on the mutant leaves. Less than 10% of the identified genes are common in both spl11 and spl17 mutants, suggesting that the mutation in these two mutants may be in different pathways. Recently, all the RNA samples (208) were prepared from treated and untreated leaf tissues of the 10 mutants. Over 120 hybridizations have been performed. Detailed analysis of the large microarray data set is being conducted. Through pairwise crosses among selected mutants, our collaborator Dr. Leung at the International Rice Research Institute has produced 70 double mutants and evaluated 50 double mutants for bacterial blight resistance. At least 20 double mutants showed enhanced resistance higher than that shown by single parental mutants. Some of these selected double mutants will be used for gene expression analysis in early 2006. B. Establishment of an efficient protoplast transient system for defense gene analysis. Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. To rapidly characterize the candidate genes identified from our microarray analysis of rice defense mutants, we optimized the rice protoplast isolation protocol and developed a procedure to rapidly screen defense genes in the signal transduction pathway. To obtain a large amount of viable protoplasts, we used two-week old etiolated seedlings, and applied vacuum for longer time for enzyme solution infiltration. About a million protoplast cells can be easily obtained from 100 seedlings which are sufficient for RT-PCR, plasmid DNA transfection and reporter gene assay. We have also established an efficient PEG-mediated transfection method which has 50%-70% transformation efficiency. Using the established rice protoplast assay system, the defense response gene PBZ1 was selected as a marker gene for screening for candidate genes. Two PBZ1 promoter constructs were made in which the 1.1 kb PBZ1 promoter is fused with the luciferase gene and the GFP gene, respectively. After 10 hr treatment with Magnaprothe grisea cell wall elicitor, the relative LUC activity of the elicitor treated sample was 6.0-fold of that of the non-elicitor treated sample. A similar result was observed in the elicitor-treated protoplasts transfected with the PBZ1::GFP construct. These results suggest that the established method can be very useful for large-scale screening of candidate defense genes from our microarray analysis.

Impacts
Although many rice defense mutants have been identified by forward genetic screens or generated by transgenic approaches, few of them are well categorized. It would be prohibitively time consuming to identify all the important components in the defense pathway of rice using traditional approaches because rice has a relatively long growth period and map-based cloning is still slow. In this project, we are using the microarray method to rapidly group the selected 10 mutants. This project will deliver a catalog of annotated novel genes governing disease resistance, a reference set of well-characterized rice mutant lines with altered defense responses, in-depth gene expression profiles of rice following challenge with the two most significant rice pathogens, JA and SAR inducer BTH and a novel method for identifying genes corresponding to deletion mutations.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
1. Seed multiplication and re-evaluation of mutant phenotype. Since we only obtained limited seeds of all 15 mutants from our collaborators in the US and Philippines, mutants were grown in the greenhouse for seed increase in the summer. The resistance of these mutants to rice blast fungus Magnaporthe grisea and bacterial blight pathogen Xanthomonas oryzae pv oryzae were evaluated in growth chamber conditions. The inoculation results were consistent with what have reported previously by our collaborators. 2. Gene expression analysis using rice oligo arrays Before the NSF-funded rice oligo array was available in November, 2004, we have conducted preliminary expression analysis of several well-characterized mutants such as lesion mimic mutants spl11, spl17 and spl25 and enhanced resistant mutant GR978 using the rice microarrays from Agilent, Beijing Genomics Institute and Incyte. The purpose of these hybridizations was to evaluate different array platforms using a common genetic stock and get experience in RNA isolation, labeling and microarray hybridizations. Preliminary analysis of these microarray hybridizations showed that about 100-200 genes were induced or repressed in these mutants when compared with the expression profiles in the corresponding wild types. When these mutants were challenged with rice blast fungus, about 300-400 genes were differentially up- or down-regulated in the mutant plants. Further analysis revealed that a small set of genes (about 30-40) were commonly induced or suppressed in all mutants. Many of these genes are involved in defense and cell death pathways. RT-PCR and northern blot confirmation analysis is being conducted. Recently, we started to use the NSF-funded rice oligo array developed by the Ronald lab at UC Davis for profiling of all 15 defense mutants. RNA isolation, labeling and microarray hybridization procedures have been optimized. We used lesion mimic mutants spl11, spl17 and their wild type IR64 as testing materials. To investigate whether gene expression patterns were related to the lesion formation, three types of leaves (young leaves without lesions, fully-expanded leaves with many lesions and old leaves with many lesions) were harvested from both mutants and IR64. Microarray data analysis showed that more genes were induced or repressed when the number of lesions were increased on the mutant leaves. Less than 10% of genes are commonly induced or repressed in both spl11 and spl17 mutants, suggesting that the mutation in these two mutants may be on different pathways. More hybridizations for all the mutants are being conducted. 3. Developing genetic resources Through pairwise crosses among selected mutants, our collaborator Dr. Leung has produced 70 double mutants and evaluated 50 double mutants for bacterial blight resistance. At least 20 double mutants showed enhanced resistance higher than that shown by single parental mutants. Some of these selected mutants will be available for expression analysis for our project in mid 2005.

Impacts
Rice is the food staple for over half the world's population and the model plant for cereal functional genomics. Although considerable progress has been made on the development of new resistant cultivars and new control methods, diseases caused by pathogens are still a major threat to food security world-wide. A thorough understanding of the molecular basis of the defense response to pathogens is essential for designing effective strategies to control the rice diseases. With funding from the CSREES NRI Plant Genome Program in 2004, we assembled a team of researchers at Ohio State University, University of California at Davis, Colorado State University and the International Rice Research Institute to conduct gene expression profiling of a set of 15 defense-related mutants using the rice oligo arrays. Preliminary microarray analysis show that many defense genes had different expression patterns in these lines before and/or after inoculation. Comprehensive microarray analysis of all the mutants will lead to identification of a set of novel genes that play important roles in disease resistance. The microarray information generated from this project will be systematically cataloged for dissemination on TIGR's rice expression database (www.ricearray.org), Gramene database (www.gramene.org) and the International Rice Information System (IRIS) (www.iris.irri.org/home.html).

Publications

  • Wang GL, Wu C, Zeng L, He C, Baraoidan M, William CE, Ronald P, Leung H (2004) Isolation and characterization of rice mutants susceptible to Xoo and rice blast. Theoretical and Applied Genetics, 108:379-84.