Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: This research project focuses on understanding the role of ethylene (ET) in rice disease resistance and how a stress-responsive MAP kinase (OsMPK5) may regulate ET biosynthesis and signal transduction. To elucidate the role of ET biosynthesis and signaling in rice disease resistance, a series of gene constructs were made and used to successfully generate transgenic rice lines with altered ET biosynthesis or signal perception. These include RNAi lines with suppression of OsACS2, OsACS1+2, OsACS5, OsEIN2a, OsEIN2b, OsEIN2a+b, or OsEIL1 and transgenic lines with inducible overexpression of OsACS2 and OsETO1. All of these transgenic lines have been characterized for altered expression of the target genes and downstream defense genes as well as changes in ET biosynthesis and/or sensitivity. These transgenic lines were also evaluated in 2nd, 3rd and/or 4th generations for altered resistance to rice blast and/or sheath blight pathogens. To gain insights into the role of OsMPK5 in regulating OsEIL1 and ET signaling, in vitro protein-binding, bimolecular fluorescence complementation and kinase assays were used to demonstrate that OsMPK5 interacts with and phosphorylates OsEIL1. A major phosphorylation site in OsEIL1 protein was determined based on the deletion analysis and the site-directed mutagenesis. Genetic, molecular and biochemical analyses were carried out to demonstrate that OsEIL1 protein and downstream OsERF1 and OsPR5 expression in rice plants are negatively regulated by OsMPK5. In addition, molecular and transgenic studies were conducted to demonstrate that OsMPK5 phosphorylates OsACS2 and negatively regulates ET biosynthesis. The findings from this project were presented in national and international meetings and submitted for publication. Besides the seven listed publications, four additional manuscripts derived from this project are under review or being prepared for submission. PARTICIPANTS: This research project was coordinated by Yinong Yang (PI) and conducted primarily by two PhD students (Mr. Jianping Chen and Ms. Emily E. Helliwell), a postdoctoral fellow (Dr. Kabin Xie) and a research technologist (Qin Wang). We have also collaborated with Drs. David Vleesschauwer and Monica Hofte at Ghent University in Belgium to elucidate the role of ethylene and abscisic acid in rice resistance to brow spot disease. TARGET AUDIENCES: The findings resulting from this project will be useful to plant pathologists and rice researchers who are interested in understanding plant defense mechanisms and developing novel approaches for managing diseases in rice and other cereal crops. Gene constructs and transgenic rice lines derived from this research project have been distributed and utilized by many labs in the US, Spain, Belgium, Germany, China and South Korea. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies reveal that both ET biosynthesis and signaling play a positive role in host resistance to rice blast and sheath blight pathogens. Suppression of OsACS2 or ACS1+2 in transgenic RNAi lines reduces the endogenous ET level and basal defense response. Similarly, suppression of ET signaling components such as OsEIN2 and OsEIL1 also negatively affects basal immunity. By contrast, transgenic rice with inducible overexpression of OsACS2 and elevated ET level exhibits enhanced resistance to multiple races of Magnaporthe oryzae and a field isolate of Rhizoctonia solani. Interestingly, OsMPK5 was shown to act as a negative regulator of ethylene signaling by impinging on signal transduction between OsEIN2 and OsEIL1 in rice. OsMPK5 was shown to phosphorylate both OsEIL1 and OsACS2 and negatively regulate their protein stability and downstream ethylene-responsive gene expression. OsMPK5 was also demonstrated to play a key role in mediating ET and abscisic acid crosstalk as well as biotic/abiotic interaction in rice. Rice is one of the most important food crops in the world. Understanding the defense signaling mechanism involved in biotic and abiotic stress tolerance will help develop novel strategies for managing rice diseases and minimizing crop loss. Since rice is a model plant for monocots, this study may also have broader impacts on other cereals, turf grasses and biofuel crops important to the U.S. agriculture.
Publications
- Helliwell, E. H., Q. Wang, and Y. Yang. 2012. Transgenic rice with inducible ethylene production exhibits broad-spectrum disease resistance to the fungal pathogens Maganporthe oryzae and Rhizhoctonia solani. Plant Biotechnology J. http://dx.doi.org/10.1111/pbi.12004.
- Jia, Y., G. Liu, D.-S. Park, F. Correa-Vectoria, and Y. Yang. 2012. Inoculation and scoring methods for rice sheath blight disease. Ed. Y. Yang, Humana Press. Methods in Molecular Biology: Rice Protocols 956:257-268.
- Helliwell, E. H. and Y. Yang. 2012. Molecular strategies to improve rice disease resistance. Ed. Y. Yang, Humana Press. Methods in Molecular Biology: Rice Protocol 956: 285-309.
- Yang, D.-L., J. Yao, C.-S. Mei, X.-H. Xiao-Hong Tong, L.-J. Zeng, Q. Li, L.-T. Xiao, T.-P. Sun, F. Li, X.-W. Deng, C.-M. Lee, M. Thomashow, Y. Yang, Z.-H. He, and S. Y. He. 2012. Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade. Proc. Natl. Acad. Sci. 109:7152-7153.
- Helliwell, E. E. and Y. Yang. 2009. Systemic signaling in plant defense. In Encyclopedia of Life Sciences. John Wiley & Sons, Ltd. http://dx.doi.org/10.1002/9780470015902.a0001322.pub2.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The overall goal of this project is to determine the role of ethylene (ET) in rice disease resistance and how a stress responsive MAP kinase (OsMPK5) may regulate ET biosynthesis and signal transduction. Below are outputs regarding each of three specific objectives: 1) To elucidate the role of ET biosynthesis and signaling in rice disease resistance. All of the required gene constructs were made and successfully used to generated transgenic rice lines with altered ET biosynthesis or signal perception. These includes RNAi lines with suppression of OsACS2, OsACS1+2, OsACS5, OsEIN2a, OsEIN2b, OsEIN2a+b, or OsEIL1 and transgenic lines with inducible overexpression of OsACS2 and OsETO1. All of these transgenic lines have been characterized for transcript levels and ET biosynthesis and/or sensitivity. Most of the transgenic lines have also been evaluated in 2nd or 3rd generation for altered resistance to the rice blast fungus. Currently, transcriptome profiling is being conducted to further characterize these transgenic lines for altered defense gene expression. 2) To determine if OsMPK5 phosphorylates OsEIL1 and regulates ET signaling. Genetic, molecular and biochemical analyses have been conducted using transgenic rice lines with altered expression of OsMPK5, OsEIN2 and OsEIL1. In vitro protein binding and kinase assays were used to demonstrate that OsMPK5 interacts with and phosphorylates OsEIL1 in vitro. The site-directed mutagenesis was carried out to determine a major phosphorylation site in OsEIL1 protein. In addition, we are testing if OsEIL1 protein with the mutated phosphorylation site is more stable in rice protoplasts. 3) To examine if OsMPK5 phosphorylates an ACC synthase (OsACS2) and regulates ET biosynthesis. In vitro kinase assays were conducted to determine the phosphorylation of OsACS2 protein by OsMPK5. A specific phosphorylation site in OsACS2 was determined by the site-directed mutagenesis. In addition, transgenic analyses were conducted to demonstrate that suppression of OsMPK5 increased ET level whereas overexpression of OsMPK5 decreased ET level. We are also testing if OsACS2 protein with the mutated phosphorylation site is more stable in rice protoplasts. PARTICIPANTS: The project participants during the past year include Ms. Emily Helliwell (PhD student), Dr. Kabin Xie (postdoc) and Ms. Qin Wang (research technologist). Ms. Helliwell has been worked on Obj. 1 to determine the role of ET biosynthesis and signaling in rice disease resistance. Dr. Xie worked on Obj. 2 to characterize OsMPK substrates such as OsEIL1. Ms. Qin Wang worked part-time on the project and was responsible for tissue culture and generation of transgenic rice lines. We have also collaborated with Drs. David Vleesschauwer and Monica Hofte at Ghent University in Belgium to understand the role of ethylene and abscisic acid in rice resistance to brow spot disease. TARGET AUDIENCES: The findings resulting from this project should be useful to plant pathologists and rice researchers who are interested in novel approaches for managing diseases in rice and other cereal crops. Gene constructs and transgenic rice lines derived from this research projects have been distributed and utilized by many labs in the US, Spain, Belgium, China and South Korea. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Transgenic analyses reveal that both ET biosynthesis and signaling play a positive role in rice resistance to Magnaporthe oryzae infection. Transgenic rice with inducible overexpression of OsACS2 and elevated ET level shows enhanced resistance to multiple races of rice blast fungus. By contrast, suppression of OsACS2 or ACS1+2 in transgenic RNAi lines reduces endogenous ET levels and increase blast susceptibility. Similarly, suppression of ET signaling components such as OsEIN2 and OsEIL1 results in increased host susceptibility to rice blast fungus. Interestingly, the ABA-inducible OsMPK5 appears to act as a negative regulator of ethylene signaling by impinging on signal transduction between OsEIN2 and OsEIL1 in rice. OsMPK5 was shown to phosphorylate both OsEIL1 and OsACS2 and negatively regulate their protein stability and downstream ethylene-responsive gene expression. OsMPK5 is also a key gene mediating ABA and ET crosstalk as well as biotic/abiotic interaction in rice. Rice is one of the most important food crops in the world. Understanding the defense signaling mechanism involved in biotic and abiotic stress tolerance will facilitate the development of novel strategies for managing rice diseases and minimizing crop loss.
Publications
- Helliwell, E. E., Q. Wang, and Y. Yang. 2011. Transgenic rice with inducible overproduction of ethylene exhibits broad-spectrum disease resistance. Phytopathology 101:S71.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: This research project focuses on the role of ethylene (ET) in rice disease resistance and how a stress responsive MAP kinase (OsMPK5) may regulate ET biosynthesis and signal transduction. Below are outputs for each of three specific objectives: 1) To elucidate the role of ET biosynthesis and signaling in rice disease resistance. We have made all of the required gene constructs and successfully generated transgenic rice lines with altered ET biosynthesis or signal perception. These includes RNAi lines with suppression of OsACS2, OsACS1+2, OsACS5, OsEIN2a, OsEIN2b, OsEIN2a+b, or OsEIL1 and transgenic lines with inducible overexpression of OsACS2 and OsETO1. Up to date, most of the transgenic lines have been characterized for transcript levels, hormone biosynthesis and/or sensitivity. About a half of transgenic lines have also been characterized in 2nd or 3rd generation for altered resistance to the rice blast fungus. We are further characterizing these transgenic lines for downstream defense gene expression and elucidating the role of ET pathway in rice disease resistance. 2) To determine if OsMPK5 phosphorylates OsEIL1 and regulates ET signaling. We conducted in vitro protein binding and kinase assays and showed that OsMPK5 interacts with and phosphorylates OsEIL1 in vitro. A major phosphorylation site was determined based on the site-directed mutagenesis. In addition, we carried out molecular, biochemical and genetic analyses using transgenic lines with altered expression of OsMPK5, OsEIN2 and OsEIL1. 3) To examine if OsMPK5 phosphorylates an ACC synthase (OsACS2) and regulates ET biosynthesis. We conducted transgenic analysis and showed that suppression of OsMPK5 increased ET level whereas overexpression of OsMPK5 decreased ET level, suggesting that OsMPK5 involves regulation of ET biosynthesis. Preliminary studies also indicate that OsMPK5 can phosphorylate OsACS2 in vitro. Additional biochemical and transgenic analyses will be conducted to further determine if OsMPK5 phoshorylates and destabilize OsACS2 in vivo. PARTICIPANTS: Two PhD students (Ms. Emily Helliwell and Mr. Jianping Chen), one postdoctoral research associate (Dr. Kabin Xie) and one research technologist (Ms. Qin Wang) have been involved in this research project. Ms. Helliwell works on Obj. 1 to elucidate the role of ET biosynthesis and signaling in rice disease resistance. Mr. Jianping Chen works on Obj. 2 to determine the phosphorylation of OsEIL1 and regulation of ET signaling by OsMPK5. Dr. Kabin Xie works on characterizing OsMPK substrates. Ms. Qin Wang was responsible for tissue culture and generation of transgenic rice lines. In addition, we are collaborating with Drs. David Vleesschauwer and Monica Hofte at Ghent University in Belgium for the role of ethylene and abscisic acid in rice resistance to brow spot disease. TARGET AUDIENCES: The findings from this research should be useful to plant pathologists and rice researchers who are interested in developing new strategies for plant disease control. Research materials derived from this project (e.g., DNA constructs and rice RNAi lines) have already been distributed to many labs in the US, China, Belgium and South Korea. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies show that ET signaling and its antagonistic interaction with ABA may play an important role in rice blast resistance. The ABA-inducible OsMPK5 appears to act as a negative regulator of ethylene signaling by impinging on signal transduction between OsEIN2 and OsEIL1 in rice. Suppression of OsEIN2 was shown to increase OsMPK5 mRNA, protein, and kinase activity. In addition, OsMPK5 protein or kinase activity was negatively correlated with the OsEIL1 protein level and downstream OsERF1 and OsPR5 expression in transgenic rice, suggesting that OsMPK5 mediates biotic and abiotic stress responses by negatively regulating OsEIL1 stability and ethylene signaling. Transgenic analysis showed that OsEIL1 positively regulated OsERF1 and OsPR5 expression and increased rice disease resistance against the rice blast infection. Furthermore, suppression of OsACS2 or ACS1+2 was shown to reduce endogenous ET levels and increased blast susceptibility, whereas inducible overexpression of OsACS2 led to enhanced resistance to rice blast. Therefore, both ET biosynthesis and signal transduction contribute to the activation of defense genes and rice blast resistance. Since rice is one of the most important food crop in the world, understanding the host defense mechanism against rice blast will help develop novel strategies for controlling the rice disease and reducing crop loss.
Publications
- Vleesschauwer, D. D., Y. Yang, C. V. Cruz, and M. Hofte. 2010. Abscisic acid-induced resistance against the brown spot pathogen Cochliobolus miyabeanus in rice involves MAPK-mediated repression of ethylene signaling. Plant Physiology 152:2036-2052.
- Helliwell, E. E., Q. Wang, and Y. Yang. 2010. Ethylene biosynthesis and its effect on rice resistance to fungal infection. Phytopathology 100:S49.
- Zhang, J., D. S. Park, and Y. Yang. 2010. Antagonistic role of ethylene and abscisic acid in mediating rice sheath blight resistance. Phytopathology 100:S146.
- Helliwell, E. E., J. Chen, X. Zhou, and Y. Yang. 2009. Involvement of ethylene biosynthesis and signaling in host resistance to rice blast disease. The 6th International Rice Genetics Symposium. Manila, Philippines. Nov. 16-19. p. 105.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: To determine the role of ethylene (ET) biosynthesis and signaling in rice disease resistance, we have generated a number of rice RNAi lines impaired in ET biosynthesis or signal perception. These includes RNAi lines with suppression of OsACS2, OsACS1+2, OsACS5, OsETO1, OsEIN2a, OsEIN2b, OsEIN2a+b, or OsEIL1 expression and transgenic lines with overexpression of OsACS2 and OsETO1. Up to date, about a half of these transgenic lines have been characterized for transcript levels, hormone biosynthesis and/or sensitivity, downstream gene expression, and resistance to the rice blast fungus. To gain insights into the role of the stress-responsive MAP kinase (OsMPK5) in ethylene signaling and rice disease resistance, we have conducted molecular, biochemical and genetic analyses using transgenic lines with altered expression of OsMPK5, OsEIN2 and OsEIL1. Suppression of OsEIN2 was shown to increase OsMPK5 mRNA, protein, and kinase activity. In vitro protein binding and kinase assays reveal that OsMPK5 interacts with and phosphorylates OsEIL1 in vitro. In addition, OsMPK5 protein or kinase activity is negatively correlated with the OsEIL1 protein level and downstream OsERF1 and OsPR5 expression in transgenic rice, suggesting that OsMPK5 mediates biotic and abiotic stress responses by negatively regulating OsEIL1 stability and ethylene signaling. Transgenic analysis showed that OsEIL1 positively regulated OsERF1 and OsPR5 expression and increased rice disease resistance against the rice blast infection. The findings from this project were presented in national and international meetings and shared with plant biologists and rice researchers. A number of rice RNAi lines developed from this research were distributed to many labs in the US, China, Belgium and South Korea. Besides the listed publications, two manuscripts are being prepared for submission. In addition, one postdoctoral associate and one PhD student have been trained under this research project. PARTICIPANTS: Yinong Yang, principal investigator, responsible for experimental design, technical guidance, report and manuscript preparation. Emily Helliwell, PhD student, responsible for generating OsACS, OsETO RNAi constructs and transgenic analysis. Jianping Chen, PhD student, responsible for studying the OsMPK5/OsEIL1 interaction and analyzing the OsMPK5 and OsEIL1 RNAi lines. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Emerging evidence suggests that ethylene (ET) and its antagonistic interaction with abscisic acid (ABA) may play a crucial role in rice disease resistance. We demonstrate that OsMPK5 likely acts as a negative regulator of ethylene signaling by impinging on signal transduction between OsEIN2 and OsEIL1 in rice. Our data indicate that OsMPK5 plays a significant role in rice blast resistance by negatively regulating ET biosynthesis and signal transduction. Transgenic analysis also suggests that both ET biosynthesis and signal transduction contribute to the activation of defense genes and rice disease resistance. It was previously proposed that flood- or hypoxia-induced ET biosynthesis in rice is critical for field resistance and management of rice blast disease in the southern U.S. A better understanding of OsMPK5 and ET's role in rice disease resistance will help improve rice breeding strategies and cultural practices for effective disease control. Since rice is an important model for monocots, the data and discovery from this study will have broader impacts on other cereals, turf grasses and biofuel crops important to the U.S. agriculture.
Publications
- Chen, J., X. Zhou, and Y. Yang. 2009. OsMPK5 phosphorylates OsEIL1 and regulates ethylene signal transduction in rice. The Annual Meeting of American Society of Plant Biologist, Honolulu, Hawaii. July 17-23.
- Yang, Y. 2008. Interplay among ethylene, abscisic acid and MAP kinase pathways modulates rice disease resistance and drought tolerance. 6th International Symposium on Rice Functional Genomics, Jeju, Korea. Nov. 10-12, 2008.
- Bailey, T.A., X. Zhou, J. Chen, and Y. Yang. 2009. Role of ethylene, abscisic acid and MAP kinase pathways in rice blast disease resistance. In "Advances in Genetics, Genomics and Control of Rice Blast Disease". pp. 185-190. Eds. G. Wang and B. Valent, Springer.
- Yang, Y., T.A. Bailey, J. Chen, E.E. Helliwell, and X. Zhou. 2008. Ethylene pathway and disease resistance in rice are negatively regulated by a stress-responsive MAP kinase. Phytopathology 98: S176.
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