FUNCTIONAL ANALYSIS OF NOVEL E3 LIGASE GENES INVOLVED IN DEFENSE RESPONSE TO MAGNAPORTHE ORYZAE IN RICE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0191511
• Project Start Date: Nov 1, 2012
• Project End Date: Oct 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Plant Pathology

Non Technical Summary
Rice diseases are the major threat for stable rice production and food security worldwide. Deep understanding of the disease resistance pathway in rice is essential for effective control of the diseases. We use rice as the experimental plant to understand the molecular basis of host resistance to pathogens because rice is the lifeline for about half of the world's population and the model plant for cereal crops. Although rice contains many E3 ubiquitin ligases, the function of their substrates in immune responses is still not fully understood. We previously characterized U-box E3 ligase SPL11 in rice that is involved in the regulation of programmed cell death (PCD), immune responses, and flowering. However, how SPL11 interacts with its substrates to control cell death and immunity is not clear. Recently, we found that the SPL11 interacts with SPIN6, ubiquitinates, and degrades the protein via the 26S proteasome pathway. Both the Spin6 RNAi and mutant plants show enhanced resistance to rice pathogens and activate defense gene expression and ROS generation. Importantly, we found that SPIN6 is the RhoGAP of the small GTPase OsRac1, which is a key component in rice immunity. Our study provides further insights into the relationship between SPIN6 and its interacting proteins SPL11 and OsRac1, and its function in the control of cell death and immunity in rice.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1530	1040	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1530 - Rice;

Field Of Science
1040 - Molecular biology;

Keywords

plant diseases

rice

host resistance

rice blast

Goals / Objectives
Plant diseases are important limiting factors in crop production worldwide, causing billions of dollars in yield loss and tremendous historical human suffering. Infected crops have reduced nutritional and aesthetic value and may contain potent toxins. Many plant diseases can be controlled by chemical pesticides but these can pollute soil, water, and air, and thereby endanger human and animal health and ultimately the sustainability of crop production. Use of resistant varieties is the most effective, economical, and environmentally sound way to control plant diseases. Because of the instability in pathogen populations, however, the most resistant cultivars often become susceptible within a few years of release. A thorough understanding of plant-pathogen interactions and host defense responses to diverse microorganisms is essential for developing new approaches to control plant diseases. Rice, the stable food for nearly 50% of the worlds population, continues to be the nutritional, financial, and social lifeline for many countries. Therefore, rice diseases that decrease yields or destroy entire harvests are major global problems. Rice blast is one of the most devastating diseases of this crop. The causal agent, the fungus Magnaprothe oryzae, occurs in virtually all rice-growing areas with periodic epidemics affecting entire rice-growing regions. Although much progress has been made in the last decade, our understanding of how rice plants activate defense response to defend rice blast infection remains unclear. The long-term goal of the proposed project is to understand the function of the host ubiquitination proteasome system (UPS) to interact with rice pathogens and how to trigger defense response in rice. The specific objectives in the project are to: 1) Determine the function of the E3 ligase genes in the regulation of Piz-t or Pi9-mediated immunity and 2) Determine the function of the SPL11-mediated cell death and defense pathway in rice and 3) Identify new genes for the development of broad-spectrum resistance to pathogens. The expected outputs of the research are: 1) We will obtain new results about the function of a host target gene involved in plant-fungal interactions and how this gene work with its partners in rice immunity. These results will be communicated in high impact journals; 2) We will train postdoctoral scientists and PhD students with comprehensive training in molecular plant pathology, who might continue to work on this important disease in the future; Last, while this research is focused on rice, the insights gained these projects will have broad impacts in plant disease control. We fully expect this research to advance the understanding of host plant resistance and spur the discovery of similar genes in other important cereals such as wheat and corn in the US.

Project Methods
Objective 1: Determine the function of the E3 ligase gene APIP10 in the regulation of Piz-t mediated HR and PTI. 1. Characterize the resistance phenotypes of APIP10 RNAi and OX lines after infection by strains with or without AvrPiz-t. We will select two homozygous T3 OX or RNAi lines for blast inoculation with an AvrPiz-t-containing strain and an AvrPiz-t-lacking strain. Three-week old seedlings will be used for spray inoculation and two month-old plants will be used for punch inoculation. Disease reactions will be carefully recorded and analyzed. 2. Test if the PTI response is altered in the APIP10-NPB RNAi plants. We will measure the ROS response of the APIP10 RNAi and OX plants to flg22 and chitin. Plants will be grown in growth chambers and leaf tissue will be collected at both seedling (3 week-old) and adult (2 month-old) stages. 3. Detect whether APIP10 is degraded in the Piz-t and non-Piz-t backgrounds after blast inoculation. Because the reduction of APIP10 transcripts in rice plants caused cell death and defense gene activation, we speculate that AvrPiz-t may interact and target APIP10 for degradation. To test this hypothesis, we will use western blot analysis to detect the changes of APIP10 in Toride and NPB plants when inoculated with a blast strain with or without AvrPiz-t. 4. Isolate APIP10 interacting proteins using Y2H and affinity purification methods. Because no direct interaction between APIP10 and Piz-t was detected in yeast, we speculate that APIP10 might interact with a substrate(s) that may directly regulate Piz-t activity or that the three proteins form a protein complex to respond to AvrPiz-t attack and to mediate defense responses. As we did for APIP identification, we will use the cDNA library to identify APIP10 interacting proteins using Y2H. In vivo interaction between APIP10 and the identified interacting proteins will be assessed using BiFC in rice protoplasts. Objective 2: Elucidate the genetic relationships between APIP10 and Piz-t by genetic and biochemical assays. 1. Test the stability of Piz-t during blast infection when APIP10 is expressed at normal or reduced levels. To detect whether Piz-t is stable during infection with AvrPiz-t-containing and -lacking strains, total protein will be isolated from the infected Piz-t-HA plants at 0, 12, 24, 48, and 72 h after inoculation. To test the stability of Piz-t as affected by a partial reduction of APIP10, we will cross APIP10 RNAi-NPB plants with the Piz-t-HA-tagged plants. IP-ed Piz-t-HA proteins from different time points will be subjected to western blot analysis using anti-HA antibody. 2. Perform Co-IP analysis to determine whether Piz-t and APIP10 are located in the same protein complex and to identify new partners in the APIP10-mediated defense pathway. We plan to cross Piz-t-HA transgenic plants with APIP10-Flag OX NPB plants to generate double transgenic mutants. Homozygous double transgenic lines will be selected and used for protein isolation. The protein will be first IP-ed with the anti-APIP10 antibody or anti-Flag antibody. The western blot will be probed with both antibodies to detect whether APIP10 and Piz-t are present in the same protein complex.

Progress 11/01/12 to 10/30/17

Outputs
Target Audience:Plant pathologists, plant breeders, farmers and extension specialists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have focused on providing comprehensive training for the next-generation scientists in plant pathology and molecular biology in the project. In the last five years, two postdoctoral fellows, four PhD students, two visiting scholars and one undergraduate students participated in the project.They have learned how to perform advanced molecular and biochemical analyses ofrice genes in the lab as well as grow rice plants in growth chambers and greenhouses for disease resistance evaluations. In addition, these trainees presented oral talks and posters at the professional meetings. They were also involved in paper and grant writing. How have the results been disseminated to communities of interest?Our results have been disseminatd to the community through presentations at growers meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Results obtained for Objective 1 "Determine the function of the E3 ligase gene APIP10 in the regulation of Piz-t mediated HR and PTI" 1) AvrPiz-t Interferes with the E3 Ligase Activity of APIP10 and is Ubiquitinated by APIP10 in vitro To assess whether AvrPiz-t has any biochemical functions in the ubiquitination process, we included the purified GST:AvrPiz-t:HA protein in the in vitro E3 ligase assay of APIP10. Surprisingly, a band above the GST:AvrPiz-t protein was detected by western blot analysis with the anti-HA antibody in the presence of the GST:AvrPiz-t:HA recombinant protein. In contrast, no such signal was detected in the presence of the GST:AvrPi-ta:HA protein, an unrelated effector protein from M. oryza, in the E3 ligase reaction. Furthermore, the western blot analysis that used the anti-Myc antibody to detect ubiquitins showed that the E3 ligase activity of APIP10 was significantly reduced when the GST:AvrPiz-t:HA fusion protein was included in the reaction, suggesting that AvrPiz-t interferes with APIP10 E3 ligase activity. These results showed that AvrPiz-t interferes with APIP10 E3 ligase activity and that APIP10 ubiquitinates AvrPiz-t in vitro. 2) APIP10 Promotes the Degradation of AvrPiz-t in N. benthamiana Since AvrPiz-t is ubiquitinated by APIP10 in vitro, we hypothesized that AvrPiz-t may be a substrate of APIP10 in planta. To test this, we co-expressed GFP:AvrPiz-t:HA with Myc:APIP10 in N. benthamiana leaves. The western blot analysis revealed that the GFP:AvrPiz-t:HA protein level was significantly lower in the tissue in which Myc:APIP10 was co-expressed than in the control tissue in which Myc:APIP10 dRING was co-expressed. This result suggested that APIP10 promotes the degradation of AvrPiz-t in plant cells. Pre-treatment of the leaves with MG132 inhibited the degradation of GFP:AvrPiz-t:HA, confirming the earlier in vitro finding that APIP10 degrades AvrPiz-t via the 26S proteasome system. 3) AvrPiz-t Promotes the Degradation of APIP10 in vivo Because we previously reported that AvrPiz-t promotes the degradation of APIP6 when they are co-expressed in N. benthamiana (Park et al., 2012), we reasoned that AvrPiz-t might also affect the accumulation of APIP10 in plant cells. The western blot analysis showed that APIP10 was reduced by about 30-40% (relative to the controls) when GFP:AvrPiz-t:HA and Myc:APIP10 were co-expressed. Furthermore, the degradation was inhibited by the MG132 treatment, suggesting that AvrPiz-t promotes the degradation of APIP10 in planta, likely through the 26S proteasome system. Results obtained for Objective 2 "Elucidate the genetic relationships between APIP10 and Piz-t by genetic and biochemical assays". 1) Knockdown of APIP10 in the Piz-t Background Causes Severe Spontaneous Cell Death Phenotypes To understand the relationship between APIP10 and Piz-t, we transformed APIP10 RNAi construct in the Piz-t:HA background (NPB Piz-t:HA). Surprisingly, most of the APIP10 RNAi lines in the NPB Piz-t:HA background showed severe cell death and dwarf phenotypes compared with the APIP10 RNAi lines in the NPB background. To assess whether the severe cell death is related to the Piz-t:HA protein level, we conducted immunoblot analysis for Piz-t:HA with the anti-HA antibody and semi-quantitative (sq)RT-PCR for the APIP10 transcripts with three independent lines that had different levels of cell death: one line had relatively mild cell death, and two had severe cell death and dwarf phenotypes. The transcript levels of APIP10 in the three lines were inversely correlated with the cell death and dwarfism phenotypes, indicating that the cell death observed in the transgenic plants was caused by the silencing of APIP10. Strikingly, the lines with fewer APIP10 transcripts in the NPB Piz-t:HA background showed a higher expression level of the Piz-t protein, suggesting that APIP10 might be a negative regulator of the accumulation of Piz-t and Piz-t-mediated cell death. 2) APIP10 Promotes Degradation of the Piz-t Protein through the 26S Proteasome System Because we found that silencing of APIP10 in transgenic rice leads to accumulation of the Piz-t protein, we speculated that APIP10 may promote the degradation of Piz-t in rice. To test this hypothesis, we co-expressed either Myc:APIP10 or Myc:APIP10 dRING (as a control) with Piz-t:HA using agro-infiltration in N. benthamiana leaves. The assay showed that less Piz-t:HA protein accumulated when it was co-expressed with APIP10 than when it was co-expressed with APIP10 dRING, and the accumulation was recovered almost to the control level by treatment with MG132. To further confirm these results, an semi in vivo degradation assay was performed in which total protein extracted from the Piz-t:HA rice plants was mixed with total protein extracted from N. benthamiana leaves agro-infiltrated with either APIP10 or APIP10 dRING. The samples were collected at different times before 1X SDS loading buffer was added to stop the reaction. The assay showed that the level of Piz-t was decreased by APIP10 over time, and after 2 h, less Piz-t protein was detected in the presence of APIP10 than in the presence of APIP10 dRING. The decrease in the level of the Piz-t protein at 2 h was partially inhibited by treatment with MG132. Together, these data suggested that APIP10 promotes Piz-t degradation through the 26S proteasome system.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, X, Jiang, N. Liu, J., Liu, W, and Wang, GL. 2014. The role of effectors and host immunity in plantnecrotrophic fungal interactions. Virulence 5:7, 111.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, W. Liu, J. Triplett, L Leach, J. and Wang GL. 2014. Novel insights into the rice innate immunity against bacterial and fungal pathogens. Annu. Rev. Phytopathol. 52:213-41.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Venu RC, Ma J, Jia Y, Liu G, Jia MH, Nobuta K, Sreerekha MV, Moldenhauer K, McClung AM, Meyers BC, Wang GL. 2014. Identification of candidate genes associated with positive and negative heterosis in rice. PLoS One. 17;9(4):e95178.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Cai Y, Vega-Sanchez ME, Park CH, Bellizzi M, Guo Z, Wang GL, 2014. RBS1, an RNA binding protein, interacts with SPIN1 and is involved in flowering time control in rice. PLoS ONE 9, e87258.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Shirsekar G, Vega-Sanchez ME, Bordeos A, et al., 2014. Identification and characterization of suppressor mutants of spl11-mediated cell death in rice. Molecular Plant and Microbe Interactions, 27:528-36.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Liu, W., J Liu, Y Ning, B Ding, X Wang, Z Wang, GL Wang (2013) Recent progress in understanding PAMP- and effector-triggered immunity against the rice blast fungus Magnaporthe oryzae. Mol. Plant, 6(3):605-620.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yang, H. Melissa H Jia, Yulin Jia, Junjie Xing, Venu, R-C, Maria Bellizzi, Longping Yuan, Zhilong Wang, Chuanqing Sun and Guo-Liang Wang. (2013) Molecular mapping of four blast resistance genes using recombinant inbred lines of 93-11 and Nipponbare. J. of Plant Biology, 56:91-97
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Chen, S., Songkumarn, P., Venu, R., Gowda, M., Bellizzi, M., Hu, J., Liu, W., Ebbole, D., Mitchell, T. & Wang, G.L. (2013). Identification and characterization of in-planta expressed secreted effectors from Magnaporthe oryzae that induce cell death in rice. Mol Plant Microbe Interact, 26(2):191-202.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li Y, Ye Z, Nie Y, Zhang J, Wang GL, Wang Z. 2015. Comparative phosphoproteome analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars. J Proteomics. 6;115:66-80.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ning Y, Shi X, Wang R, Fan J, Park CH, Zhang C, Zhang T, Ouyang X, Li S, Wang GL. 2015. OsELF3-2, an Ortholog of Arabidopsis ELF3, Interacts with the E3 Ligase APIP6 and Negatively Regulates Immunity against Magnaporthe oryzae in Rice. Mol Plant. S1674-2052(15)00327-5.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang H, Liu J, He F, Wang Z, Ning Y, Wang GL. 2015. OsHUB1 and OsHUB2 interact with SPIN6 and form homo- and hetero-dimers in rice. Plant Signal Behav. 3;10(8):e1039212
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Xie X, Kang H, Liu W, Wang GL. 2015. Comprehensive profiling of the rice ubiquitome reveals the significance of lysine ubiquitination in young leaves. J Proteome Res. 14(5):2017-25.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu J, Park CH, He F, Nagano M, Wang M, Bellizzi M, Zhang K, Zeng X, Liu W, Ning Y, Kawano Y, Wang GL. 2015. The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice. PLoS Pathog. 11(2):e1004629.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ding B, Wang GL. 2015. Chromatin versus pathogens: the function of epigenetics in plant immunity. Front Plant Sci. 6:675.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mahesh, H.B., Shirke, M.D., Singh, S., Rajamani, A., Hittalmani, S., Wang, G.L., and Gowda, M. (2016). Indica rice genome assembly, annotation and mining of blast disease resistance genes. BMC Genomics 17, 242
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mgonja, E.M., Balimponya, E.G., Kang, H., Bellizzi, M., Park, C.H., Li, Y., Mabagala, R., Sneller, C., Correll, J., Opiyo, S., Talbot, N.J., Mitchell, T., and Wang, G.L. (2016). Genome-wide Association Mapping of Rice Resistance Genes Against Magnaporthe oryzae Isolates from Four African Countries. Phytopathology, 106(11):1359-1365
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, W., and Wang, G.-L. (2016). Plant innate immunity in rice: a defense against pathogen infection. National Science Review, doi: 10.1093/nsr/nww015
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ning, Y., Wang, R., Shi, X., Zhou, X., and Wang, G.L. (2016). A Layered Defense Strategy Mediated by Rice E3 Ubiquitin Ligases against Diverse Pathogens. Mol. Plant 9, 1096-1098.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kang, H., Wang, Y., Peng, S., Zhang, Y., Xiao, Y., Wang, D., Qu, S., Li, Z., Yan, S., Wang, Z., Liu, W., Ning, Y., Korniliev, P., Leung, H., Mezey, J., McCouch, S.R., and Wang, G.L. (2016). Dissection of the genetic architecture of rice resistance to the blast fungus Magnaporthe oryzae. Mol. Plant Pathol. 17, 959-972.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, J., Peng, H., Cui, J., Huang, W., Kong, L., Clarke, J.L., Jian, H., Wang, G.L., and Peng, D. (2016). Molecular Characterization of A Novel Effector Expansin-like Protein from Heterodera avenae that Induces Cell Death in Nicotiana benthamiana. Sci Rep 6, 35677
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, D., Liu, J., Li, C., Kang, H., Wang, Y., Tan, X., Liu, M., Deng, Y., Wang, Z., Liu, Y., Zhang, D., Xiao, Y., and Wang, G.L. (2016a). Genome-wide Association Mapping of Cold Tolerance Genes at the Seedling Stage in Rice. Rice 9, 61
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, R., Ning, Y., Shi, X., He, F., Zhang, C., Fan, J., Jiang, N., Zhang, Y., Zhang, T., Hu, Y., Bellizzi, M., and Wang, G.L. (2016b). Immunity to Rice Blast Disease by Suppression of Effector-Triggered Necrosis. Curr. Biol. 26, 2399-2411
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Xiong, Y., Peng, X., Cheng, Z., Liu, W., and Wang, G.L. (2016a). A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses. J Proteomics 138, 20-29
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu, D., Kang, H., Li, Z., Liu, M., Zhu, X., Wang, Y., Wang, D., Wang, Z., Liu, W., and Wang, G.L. (2016). A Genome-Wide Association Study of Field Resistance to Magnaporthe Oryzae in Rice. Rice, 9, 44
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ning Y, Liu W, Wang GL. 2017. Balancing Immunity and Yield in Crop Plants. Trends Plant Sci. 22(12):1069-1079. doi: 10.1016/j.tplants.2017.09.010.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen Z, Cheng Q, Hu C, Guo X, Chen Z, Lin Y, Hu T, Bellizzi M, Lu G, Wang GL, Wang Z, Chen S, Wang F. 2017, A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice. Front Plant Sci. 8:1447. doi: 10.3389/fpls.2017.01447.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mgonja EM, Park CH, Kang H, Balimponya EG, Opiyo S, Bellizzi M, Mutiga SK, Rotich F, Ganeshan VD, Mabagala R, Sneller C, Correll J, Zhou B, Talbot NJ, Mitchell TK, Wang GL. 2017. Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa. Phytopathology. 107(9):1039-1046. doi: 10.1094/PHYTO-12-16-0421-R.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mutiga SK, Rotich F, Ganeshan VD, Mwongera DT, Mgonja EM, Were VM, Harvey JW, Zhou B, Wasilwa L, Feng C, Ou�draogo I, Wang GL, Mitchell TK, Talbot NJ, Correll JC. 2017. Assessment of the Virulence Spectrum and Its Association with Genetic Diversity in Magnaporthe oryzae Populations from Sub-Saharan Africa. Phytopathology. 2017 Jul;107(7):852-863. doi: 10.1094/PHYTO-08-16-0319-R.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang GL, Valent B. 2017. Durable resistance to rice blast. Science. 3;355(6328):906-907. doi: 10.1126/science.aam9517.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhong X, Yang J, Shi Y, Wang X, Wang GL. 2017. The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae. Mol Plant Pathol. doi: 10.1111/mpp.12546
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Selisana SM, Yanoria MJ, Quime B, Chaipanya C, Lu G, Opulencia R, Wang GL, Mitchell T, Correll J, Talbot NJ, Leung H, Zhou B. 2017. Avirulence (AVR) Gene-Based Diagnosis Complements Existing Pathogen Surveillance Tools for Effective Deployment of Resistance (R) Genes Against Rice Blast Disease. Phytopathology. 107(6):711-720. doi: 10.1094/PHYTO-12-16-0451-R.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu Q, Ning Y, Zhang Y, Yu N, Zhao C, Zhan X, Wu W, Chen D, Wei X, Wang GL, Cheng S, Cao L. 2017. OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice. Plant Cell. 29(2):345-359. doi: 10.1105/tpc.16.00650.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Li Z, Ding B, Zhou X, Wang GL. 2017. The Rice Dynamin-Related Protein OsDRP1E Negatively Regulates Programmed Cell Death by Controlling the Release of Cytochrome c from Mitochondria. PLoS Pathog. 12;13(1):e1006157. doi: 10.1371/journal.ppat.1006157.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Plant pathologist, geneticists, breeders and farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We aim at training of next-generation scientists in plant pathology and molecular biology in the project. In the past year, two postdoctoral fellows, four PhD students, two visiting scholars participated in the project.They have learned how perform advanced molecular and biochemical analyses ofrice genes in the lab as well as grow rice plants in growth chambers and greenhouses for disease resistance evaluations. How have the results been disseminated to communities of interest?We presented the following seminars at the national and international conferences: 36th Rice Technical Working Group Meeting, March 1-4, 2016, Galveston, TX, USA. Title: Molecular mechanism and marker-assisted breeding of rice resistance to Magnaporthe oryzae Department of Botany, University of British Columbia, Vancouver, Canada, March 24-25, 2016, Title: Unraveling the molecular mechanism of rice immunity against Magnaporthe oryzae Plant Biology Mini-Symposium at the University of Maryland, May 26, 2016, College Park, MD, USA, Title: Unraveling the molecular mechanism of rice immunity against Magnaporthe oryzae XVII International Congress on Molecular Plant-Microbe Interactions, July 18-23, 2016, Title: Unraveling the molecular mechanism of rice immunity against Magnaporthe oryzae 7th International Rice Blast Conference, Oct. 9-14, the Philippines, Title: Positive and negative regulation of the NLR immune receptor Piz-t in rice Workshop of Genetic Basis and Breeding Application on High-yield Superior Quality Rice. China National Rice Research Institute, Hangzhou, Oct. 14-15, 2016, Title: Molecular Mechanisms of Rice Resistance and Breeding StrategiesagainstMagnaportheoryzae Symposium on rice disease and insect control. October 22-23, 2016, Fuzhou, China Title: Molecular Mechanisms of Rice Resistance and Breeding StrategiesagainstMagnaportheoryzae. Department of Plant Pathology, UC Davis, USA, Nov. 7, 2016, Title: Negative and positive regulation of the NLR immune receptor Piz-t in rice What do you plan to do during the next reporting period to accomplish the goals?1. Continue to analyze the function of genes associated with Piz-t, APIP5, APIP6 and APIP10 and determine their relationship with other defense signaling genes. 2. Identify the SPL11 and SDS2-interacting proteins and characterize their functions.

Impacts
What was accomplished under these goals? 1. A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses: We report high quality proteome-scale data for lysine-acetylation (Kac) sites and Kac proteins in rice (Oryza sativa). A total of 1337 Kac sites in 716 Kac proteins with diverse biological functions and subcellular localizations were identified in rice seedlings. About 42% of the sites were predicted to be localized in the chloroplast. Seven putative acetylation motifs were detected. Phenylalanine, located in both the upstream and downstream of the Kac sites, is the most conserved amino acid surrounding the regions. In addition, protein interaction network analysis revealed that a variety of signaling pathways are modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways are significantly enriched. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Kac functions in growth, development, and abiotic and biotic stress responses in rice and other plants. 2. Immunity to Rice Blast Disease by Suppression of Effector-Triggered Necrosis: We show that the M. oryzae effector AvrPiz-t interacts with the bZIP-type transcription factor APIP5 in the cytoplasm and suppresses its transcriptional activity and protein accumulation at the necrotrophic stage. Silencing of APIP5 in transgenic rice leads to cell death, and the phenotype is enhanced by the expression of AvrPiz-t .Conversely, Piz-t interacts with and stabilizes APIP5 to prevent necrosis at the necrotrophic stage. At the same time, APIP5 is essential for Piz-t stability. These results demonstrate a novel mechanism for the suppression of effector-triggered necrosis at the necrotrophic stage by an NLR receptor in plants. 3. Quantification of hydrogen peroxide in plant tissues using Amplex Red: We optimized an Amplex Red-based quantitation method for H2O2 estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H2O2 in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg-1 FW range. Commonly used additives for H2O2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H2O2 directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue. 4. A Genome-Wide Association Study of Field Resistance to Magnaporthe Oryzae in Rice: To identify loci associated with field blast resistance (LAFBRs), we conducted a genome-wide association study (GWAS) using the rice diversity panel 1 (RDP1) cultivars. These cultivars were evaluated in the field in three major rice production areas of China. GWAS identified 16 LAFBRs. Among them, 13 are novel and the other three are co-localized with known blast resistance regions. Seventy-four candidate genes are identified in the 16 LAFBR regions, which encode receptor-like protein kinases, transcription factors, and other defense-related proteins. Using the rice transcriptome data, compared with the rice-rice blast compatible interaction, we identified seven candidate genes that are significantly up-regulated and five genes that are significantly down-regulated in the incompatible interaction among the candidate genes. In conclusions, we identified 16 LAFBRs involved in field resistance to M. oryzae and 20 cultivars that exhibit high levels of resistance in both the field and growth chamber. The resistant cultivars and the SNP markers identified in this study should be useful for marker-assisted selection of new rice cultivars that confer high levels of resistance against M. oryzae field populations. 5. Performed a Genome-Wide Association Study of Resistance Genes to African M. Oryzae populations: We conducted a genome-wide association study (GWAS) to rapidly map rice genes conferring resistance against eight M. oryzae isolates from four African countries. We inoculated 162 rice cultivars, which were part of the Rice Diversity Panel 1 (RDP1) and were previously genotyped with the 44,000 SNP chip, with the eight isolates. The GWAS identified 31 genomic regions associated with blast resistance (RABRs) in the rice genome. In addition, we used PCR analysis to confirm the association between the Pish gene and a major RABR on chromosome 1 that was associated with resistance to four M. oryzae isolates. Our study has demonstrated the power of GWAS for the rapid identification of rice blast R/QTL genes that are effective against African populations of M. oryzae. The identified SNP markers associated with RABRs can be used in breeding for resistance against rice blast in Africa

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu, D., Kang, H., Li, Z., Liu, M., Zhu, X., Wang, Y., Wang, D., Wang, Z., Liu, W., and Wang, G.L. (2016). A Genome-Wide Association Study of Field Resistance to Magnaporthe Oryzae in Rice. Rice (N Y) 9, 44.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chakraborty, S., Hill, A.L., Shirsekar, G., Afzal, A.J., Wang, G.L., Mackey, D., and Bonello, P. (2016). Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods 109, 105-113.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: He, F., Chen, S., Ning, Y., and Wang, G.-L. (2016). Rice (Oryza sativa) Protoplast Isolation and Its Application for Transient Expression Analysis. In Current Protocols in Plant Biology (John Wiley & Sons, Inc.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kang, H., Wang, Y., Peng, S., Zhang, Y., Xiao, Y., Wang, D., Qu, S., Li, Z., Yan, S., Wang, Z., Liu, W., Ning, Y., Korniliev, P., Leung, H., Mezey, J., McCouch, S.R., and Wang, G.L. (2016). Dissection of the genetic architecture of rice resistance to the blast fungus Magnaporthe oryzae. Mol. Plant Pathol. 17, 959-972.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, J., Peng, H., Cui, J., Huang, W., Kong, L., Clarke, J.L., Jian, H., Wang, G.L., and Peng, D. (2016). Molecular Characterization of A Novel Effector Expansin-like Protein from Heterodera avenae that Induces Cell Death in Nicotiana benthamiana. Sci Rep 6, 35677.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, W., and Wang, G.-L. (2016). Plant innate immunity in rice: a defense against pathogen infection. National Science Review. doi: 10.1093/nsr/nww015
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mahesh, H.B., Shirke, M.D., Singh, S., Rajamani, A., Hittalmani, S., Wang, G.L., and Gowda, M. (2016). Indica rice genome assembly, annotation and mining of blast disease resistance genes. BMC Genomics 17, 242.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mgonja, E.M., Balimponya, E.G., Kang, H., Bellizzi, M., Park, C.H., Li, Y., Mabagala, R., Sneller, C., Correll, J., Opiyo, S., Talbot, N.J., Mitchell, T., and Wang, G.L. (2016). Genome-wide Association Mapping of Rice Resistance Genes Against Magnaporthe oryzae Isolates from Four African Countries. Phytopathology, 106(11):1359-1365
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ning, Y., Wang, R., Shi, X., Zhou, X., and Wang, G.L. (2016). A Layered Defense Strategy Mediated by Rice E3 Ubiquitin Ligases against Diverse Pathogens. Mol. Plant 9, 1096-1098.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, D., Liu, J., Li, C., Kang, H., Wang, Y., Tan, X., Liu, M., Deng, Y., Wang, Z., Liu, Y., Zhang, D., Xiao, Y., and Wang, G.L. (2016a). Genome-wide Association Mapping of Cold Tolerance Genes at the Seedling Stage in Rice. Rice (N Y) 9, 61.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, R., Ning, Y., Shi, X., He, F., Zhang, C., Fan, J., Jiang, N., Zhang, Y., Zhang, T., Hu, Y., Bellizzi, M., and Wang, G.L. (2016b). Immunity to Rice Blast Disease by Suppression of Effector-Triggered Necrosis. Curr. Biol. 26, 2399-2411.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Xiong, Y., Peng, X., Cheng, Z., Liu, W., and Wang, G.L. (2016a). A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses. J Proteomics 138, 20-29.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Plant pathologist, geneticists, breeders and farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We aim at training of next-generation scientists in plant pathology and molecular biology in the project. In the past year, two postdoctoral fellows, two PhD students, one visiting scholars participated in the project.They have learned how perform advanced molecular and biochemical analyses ofrice genes in the lab as well as grow rice plants in growth chambers and greenhouses for disease resistance evaluations. How have the results been disseminated to communities of interest?We presented the following seminars at the national and international conferences: Invited talks January 19, 2015. Roles of the Ubiquitination Proteasome System in Rice Immunity, Institute of Genetics and Developmental Biology, CAS, Beijing. January 21, 2015, Dissection of the rice immunity pathway against Magnaporthe oryzae. Xiangshan Scientific Meeting on Rice Functional Genomics. April 8, 2015, Functions of the host proteins targeted by the fungal effector AvrPiz-t in rice, Seminar in the department of Plant Pathology and Microbiology, Texas A&M University April 20, 2015, Genome-wide association mapping of new rice resistance genes to Magnaporthe oryzae isolates from four African countries. ARC Workshop organized by the B&M Gates Foundation, London, UK May 12, 2015, Recent Progress in Dissecting the PTI and ETI Pathways in Rice, Institute of Plant Protection, CAAS, Beijing, China May 14, 2015, Molecular Basis of Rice Immunity Against Magnaporthe oryzae, College of Life Sciences, Wuhan University, Wuhan, Hubei, China May 16, 2015, Function of two rice E3 ligases targeted by the fungal effector AvrPiz-t, Sichuan Agricultural University, Chengdu, Sichuan, China June 5, 2015, Genome-wide association mapping of new rice resistance genes to Magnaporthe oryzae isolates from four African countries, Annual meeting of the African Rice Blast Project, Kenya, Africa June 10, 2015, Molecular Dissection of Rice Innate Immunity to Magnaporthe oryzae, Department of Soil and Crop Sciences, Sokoine University of Agriculture, Tanzania, Africa. July 29, 2015, Dissecting the molecular mechanism of AvrPiz-t-Piz-t-triggered resistance against Magnaporthe oryzae, 3rd Beijing International Symposium on Molecular Plant Pathology (3rd BISMPP), Beijing China, July 23, 2015, Molecular Mechanism of Rice Resistance and Breeding Strategies against Magnaporthe oryzae, HuaZhi Biotech Company, Changsha, Hunan, China. August 5, 2015, Dissection of the U-box E3 Ligase SPL11-mediated Cell Death and Innate Immunity Pathway in Rice, APS CSPP-APS Joint Symposium, Pasadena, CA, USA October 31, 2015, Principles and Applications of Plant Genomics, Hainan University, Haikou, Hainan, China. November 13, 2015, Molecular Mechanism of Rice Resistance and Breeding Strategies against Magnaporthe oryzae China Agricultural University. Beijing, China. What do you plan to do during the next reporting period to accomplish the goals?1. Continue to analyze the function of APIP6, APIP10 and SPIN6 associated genes and determine their relationship with other defense signaling genes. 2. Identify the APIP10 and SDS2-interacting proteins and characterize their functions.

Impacts
What was accomplished under these goals? APIP10 promotes degradation of the Piz-t protein through the 26S proteasome system We co-expressed either Myc:APIP10 or Myc:APIP10 dRING (mutant as a negative control) with Piz-t:HA using agroinfiltration in N. benthamiana leaves. Less Piz-t:HA protein accumulated when it was co-expressed with APIP10 than when it was co-expressed with APIP10 dRING, and the accumulation was recovered almost to the control level by treatment with MG132. The result was confirmed in a semi-in vivo degradation assay AvrPiz-t promotes the accumulation of the Piz-t protein in planta Because we found that AvrPiz-t promotes degradation of APIP10 and that silencing of APIP10 leads to accumulation of Piz-t, we reasoned that expression of AvrPiz-t in planta may lead to the accumulation of Piz-t. To determine the relationship between AvrPiz-t and Piz-t, we co-expressed either empty vector, GFP, or GFP:AvrPiz-t with Piz-t:HA in N. benthamiana and observed the accumulation of both proteins by immunoblot analysis. Intriguingly, the Piz-t protein level was ~1.6-fold greater when Piz-t:HA was co-expressed with GFP: AvrPiz-t:HA than with the empty vector or with GFP, suggesting that AvrPiz-t stabilizes the Piz-t protein. This result was confirmed in the inducible AvrPiz-t transgenic lines in which the Piz-t level was increased after the β-estradiol treatment Map-based cloning of the Sds2 gene To dissect the Spl11-mediated cell death and defense pathway, we identified and characterized three spl11 cell death suppressor (sds) mutants. Crosses were made between spl11 and sds1-3 mutants. F1 plants were self-pollinated to produce F2 seeds. About 200 F2 plants from each cross were selected for gene mapping using DNA markers (SSR, SNP and InDel markers). Because we first found the linked markers on chromosome 1 for the Sds2 gene in the spl11 and sds2 (line #1902) cross, we began to clone the gene using a map-based cloning strategy. In a large F2 population (about 5000 plants), we identified about 1200 no-lesion mimic homozygous plants. The phenotype of these plants was confirmed in the F3 generation. Fine-mapping with seven InDel, SNP and SSR markers revealed that the Sds2 gene is located between markers indel3658 and SNP36628, where five genes are present. A G-A point mutation was found to be in the 3' region of the gene that encodes a S-domain receptor-like kinase. RT-PCR analysis of the Sds2 candidate gene showed that the sds2 mutant contains 3 splicing variants as opposed to the single band in the wilt-type plants. We sequenced the mutated region in about 100 F3 lesion-suppression plants, and all of the lines contained the same point mutation. To confirm the fine-mapping and gene sequencing results, we made an RNAi construct that targets both Spl11 and Sds2 and another construct that only targets Spl11. The transgenic plants from these two constructs were transformed into the Nipponbare calli. Lesion evaluation in the two types of transgenic lines showed that the Spl11 and Sds2 double RNAi had many fewer cell death lesions than that of the Spl11 RNAi lines when grown in tissue culture media. Sds2 encodes for a receptor like kinase BLAST searches using the Sds2 gene sequence as a query showed that it encodes a receptor-like kinase with a B-lectin domain and an S-domain in the extracellular region. We fused the MBP tag with wild-type Sds2 fragments and with Sds2-mutant (a kinase-dead mutation at the ATP binding site) fragments and expressed the fusion proteins in E. coli for in vitro kinase activity. Immunoblot analysis with anti-phospho-Threnine (anti-pThr) and Pro-Q staining showed that SDS2 is an active kinase but that the SDS2 mutant is not. We then fused the GFP tag to the C terminus of SDS2 and transformed the derived construct into rice protoplasts. The transient expression analysis showed that SDS2 localizes on the plasma membrane. These results show that SDS2 is a membrane-associated receptor-like kinase. SDS2 interacts with SPL11 in vitro and in vivo To test whether SDS2 interacts with SPL11 in yeast, we fused the wild-type, mutated (M12 and M34 deletions in the U-box), and the N terminus (NT) and ARM repeat domain (Arm) of the Sds2 gene with the activation domain (AD) in the bait vector. The SDS2 kinase domain (SDS2-KD) was fused with the binding domain in the prey vector. Y2H assays showed that SDS2 KD interacts strongly with the M34 mutant protein (a three amino-acid deletion in the U-box) and weakly with the M12 mutant protein (one amino-acid deletion in the U-box). Perhaps the wild-type SPL11 is degraded in yeast cells due to auto-ubiquitination or trans-ubiquitination by another E3 ligase. GST pulldown assays confirmed that SDS2 interacts with SPL11 in vitro. Finally, we fused the SDS2 fragment with the YFP C terminal and the SPL11 mutant fragment with the YFP N terminal in the BiFC vectors, respectively. The BiFC analysis clearly showed that SDS2 interacts with SPL11 in rice protoplast cells. These results demonstrate that the cell death suppressor protein SDS2 interacts with SPL11.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li Y, Ye Z, Nie Y, Zhang J, Wang GL, Wang Z. 2015. Data set from the phosphoproteomic analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars. Data Brief. 27;3:7-11. doi: 10.1016/j.dib.2014.12.009.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li Y, Ye Z, Nie Y, Zhang J, Wang GL, Wang Z. 2015. Comparative phosphoproteome analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars. J Proteomics. 6;115:66-80. doi: 10.1016/j.jprot.2014.12.007.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ning Y, Shi X, Wang R, Fan J, Park CH, Zhang C, Zhang T, Ouyang X, Li S, Wang GL. 2015. OsELF3-2, an Ortholog of Arabidopsis ELF3, Interacts with the E3 Ligase APIP6 and Negatively Regulates Immunity against Magnaporthe oryzae in Rice. Mol Plant. S1674-2052(15)00327-5. doi: 10.1016/j.molp.2015.08.004.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang H, Liu J, He F, Wang Z, Ning Y, Wang GL. 2015. OsHUB1 and OsHUB2 interact with SPIN6 and form homo- and hetero-dimers in rice. Plant Signal Behav. 3;10(8):e1039212. doi: 10.1080/15592324.2015.1039212
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Xie X, Kang H, Liu W, Wang GL. 2015. Comprehensive profiling of the rice ubiquitome reveals the significance of lysine ubiquitination in young leaves. J Proteome Res. 14(5):2017-25. doi: 10.1021/pr5009724.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu J, Park CH, He F, Nagano M, Wang M, Bellizzi M, Zhang K, Zeng X, Liu W, Ning Y, Kawano Y, Wang GL. 2015. The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice. PLoS Pathog. 11(2):e1004629. doi: 10.1371/journal.ppat.1004629
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ding B, Wang GL. 2015. Chromatin versus pathogens: the function of epigenetics in plant immunity. Front Plant Sci. 6:675. doi: 10.3389/fpls.2015.00675.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Plant pathologist, geneticists, breeders and farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training of next-generation scientists in plant pathology and molecular biology is one of the main goals in the project. In the past year, two postdoctoral fellows, four PhD students, four visiting scholars and two undergraduate students participated in the project.They have learned how perform advanced molecular and biochemical analyses ofrice genes in the lab as well as grow rice plants in growth chambers and greenhouses. How have the results been disseminated to communities of interest? Invited talk at the annual meeting of China Society of Plant Biologists, 8/5-8, 2014, Guiyang, China. Title: New insights into the innate immunity against M oryzae in rice Invited talk at the Nanjin Agricultural University,7/28, 2014, Nanjin, China. Title: Current advances in dissecting rice resistance to M. oryzae Invited talk at OSU CAPS, 5/9/2014, Columbus. Title: Molecular dissection of rice immunity to the blast fungus M. oryzae Invited talk at Xilinghaote, Joint State Key Laboratory meeting, 7/24-26, Xilinghaote, China, Title: Current advances in dissecting rice resistance to M. oryzae Invited talk at the International Congress-MPMI in Rhodes, 7/5-10/2014, Title: Recognition of the M. oryzae Effector AvrPiz-t by Multiple Host Targets in Rice Invited talk at the Rice Functional Genomics workshop, 1/12/2014, San Diego, CA, title: Genome-Wide Association Study (GWAS) of Rice Innate Immunity to M. oryzae Invited talk at MSU-CAAS Workshop (10/14). Title: Molecular dissection of the rice-M. oryzae interaction Invited talk at the Sichuan Agricultural University, 10/17/2014. Genetic dissection of the rice immunity to pathogens Invited talk at the South China Agricultural University (10/21). Title” Molecular dissection of the rice-M. oryzae interaction Invited talk at China Agricultural University, 10/31/2014, Beijing China. Title: Mechanisms of Ubiquitination-mediated Programmed Cell Death and Innate Immunity in Rice Invited talk at 12th International Symposium on Rice Functional Genomics. Nov. 16-19, 2014, Tucson, USA. Talk title: Dissection of the resistance pathway to M. oryzae in rice Invited talk at the Hunan Agricultural University, 10/22/2014. Title: Molecular dissection of the rice-M. oryzae interaction What do you plan to do during the next reporting period to accomplish the goals? 1. Continue to analyze the function of SPIN6-interacting genes and determine their relationship with the OsRac1 and other related gene. 2. Identify the SPIN6 interacting proteins and characterize their functions. 3. Investigate the relationship between SPL11, SPIN6 and other PAMP-related receptors in rice immunity.

Impacts
What was accomplished under these goals? 1. SPIN6 interacts with SPL11 in vitro and in vivo: The interaction between SPIN6 and SPL11 in yeast was confirmed with a Y2H experiment with full-length SPIN6, full-length SPL11, the SPL11 ARM domain (ARM), and a truncated SPL11 with a three-amino-acid deletion at C314P315T316 in the U-box domain causing loss of E3 ligase activity (SPL11m). To verify the interaction between SPIN6 and SPL11, we performed an in vitro GST pull-down assay. The results showed that both GST fusion SPL11 and ARM proteins bind to SPIN6. Subsequently, a bimolecular fluorescence complementation (BiFC) assay in N. benthamiana revealed that SPIN6 interacts with SPL11 at the plasma membrane in plant cells. 2. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 through the 26S proteasome pathway in planta: To determine whether SPIN6 is a substrate of SPL11, we performed an in vitro ubiquitination assay. The immuneblotting analysis showed high molecular weight bands were only observed in the reaction with MBP:SPL11 but not with MBP:SPL11m with the anti-GST antibody, indicating that SPIN6 is ubiquitinated by SPL11 in vitro. To investigate whether the ubiquitination of SPIN6 by SPL11 can lead to instability of SPIN6 in vivo, we co-infiltrated different combinations of agrobacteria carrying the Spl11:Myc and GFP:Spin6 plasmids into N. benthamiana leaves with or without the 26S proteasome inhibitor MG132. The immunoblot analysis revealed that the GFP:SPIN6 was degraded by SPL11:Myc but that the SPIN6 degradation was inhibited by MG132, suggesting that SPIN6 degradation is mediated by the 26S proteasome pathway. 3. Knock-down of Spin6 transcripts causes PCD and enhances the resistance to rice pathogens and rapid generation of ROS after flg22 and chitin treatments. To understand the biological function of Spin6, we made an RNAi construct that targets the 302-bp 3’ UTR region in Spin6. About 4 weeks after planting, obvious cell death-like lesions were evident in the RNAi lines but not in the NPB plants. When rice leaves were inoculated with the compatible M. oryzae isolate RO1-1, the lesion size, spore number, and relative fungal biomass in the lesions were significantly lower in the two Spin6 RNAi lines. To further investigate which pathways are involved in the Spin6-mediated defense signaling pathway, we used chemical luminescence to monitor the dynamics of ROS generation in the Spin6 RNAi plants treated with the PAMPs chitin and flg22. The results revealed that ROS accumulation was significantly higher in the two Spin6 RNAi lines than in NPB in response to both chitin and flg22 treatments, indicating that silencing of Spin6 in rice enhances chitin- and flg22-triggered ROS accumulation. 4. SPIN6 interacts with the small GTPase OsRac1, catalyzes the GTP-bound OsRac1 to the GDP-bound state in vitro and inactivates OsRac1 in rice protoplasts. We performed a Y2H experiment to determine whether SPIN6 can interact with OsRac1 or not. The assay showed that SPIN6 interacts with OsRac1 in yeast. This interaction was further confirmed by both Co-IP and BiFC assays in N. benthamiana. To determine whether SPIN6 possesses the RhoGAP activity that facilitate the hydrolysis of the GTP-bound OsRac1, we performed an in vitro RhoGAP activity assay. The result showed that the fluorescence signal in the reaction containing the OsRac1 and SPIN6 proteins was as low as that in the two negative controls because the fluorescent GTP-bound OsRac1 was hydrolyzed to GDP-bound forms in the presence of the SPIN6 protein. To monitor in vivo GAP activity of SPIN6 toward OsRac1, we used the FRET sensor called Raichu-OsRac1. Using Raichu-OsRac1, we monitored the activation level of OsRac1 in the presence of SPIN6 in vivo. The ratio of YFP/CFP fluorescence was significantly higher in the cells expressing OsRacGEF1 PRONE than in the cells expressing the control GUS vector, which showed that OsRacGEF1 PRONE activates OsRac1 in rice protoplasts. Taken together these results provided evidence that SPIN6 has in vitro and in vivo GAP activity towards OsRac1.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: 1. Liu, W. Liu, J. Triplett, L Leach, J. and Wang GL. 2014. Novel insights into the rice innate immunity against bacterial and fungal pathogens. Annu. Rev. Phytopathol. 52:213-41. 2. Venu RC, Ma J, Jia Y, Liu G, Jia MH, Nobuta K, Sreerekha MV, Moldenhauer K, McClung AM, Meyers BC, Wang GL. Identification of candidate genes associated with positive and negative heterosis in rice. PLoS One. 2014 Apr 17;9(4):e95178. doi: 10.1371/journal.pone.0095178. 3. Cai Y, Vega-Sanchez ME, Park CH, Bellizzi M, Guo Z, Wang GL, 2014. RBS1, an RNA binding protein, interacts with SPIN1 and is involved in flowering time control in rice. PLoS ONE 9, e87258. 4. Shirsekar G, Vega-Sanchez ME, Bordeos A, et al., 2014. Identification and characterization of suppressor mutants of spl11-mediated cell death in rice. Molecular Plant and Microbe Interactions, 27:528-36.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Plant pathologist, geneticists, breeders and farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? An important goal of this project is to train next-generation scientists in plant pathology and molecular biology. During this year of the project, three postdoctoral fellows, four PhD students, three visiting scholars and three undergraduate students participated in the project.They have learned how perform advanced molecular and biochemical analyses ofrice genes in the lab as well as grow rice plants in growth chambers and greenhouses. How have the results been disseminated to communities of interest? We presented the following seminars at the national and international conferences: Invited talk in the Department of Plant Pathology, University of Arkansas, Fayetteville, AR, on 4/11-12, 2013, entitled, “Molecular Dissection of Innate Immunity to Magnaporthe oryzae in Rice.” Invited talk at the 3rd International meeting on plant and biotic interactions, on 8/18-21, 2013, Yangling, China, entitled, “The novel function of E3 ubiquitin ligases in rice innate immunity against Magnaporthe oryza.” Invited talk at the 6th International Rice Blast Conference on 8/20-23, 2013, Juju, Korea, entitled, “Molecular dissection of rice innate immunity to Magnaporthe oryzae.” Invited talk at The Second International Symposium on Molecular Plant Pathology on 8/24-25, 2013, entitled, “New insights into the rice immunity to Magnaporthe oryzae.” Invited talk at The 10th International Congress of Plant Pathology on 8/25-30, 2013, Beijing, China, entitled, “The fungal effector AvrPiz-t targets the ubiquitination proteasome system for its virulence and avirulence activities in rice.” Invited talk at the 7th International Symposium on Rice Genetics on 11/4-8, 2013, Manila, Philippines, entitled, “Molecular dissection of rice immunity to the blast fungus Magnaporthe oryzae.” We published three papers in referred journals: Liu, W., J Liu, Y Ning, B Ding, X Wang, Z Wang, GL Wang (2013) Recent progress in understanding PAMP- and effector-triggered immunity against the rice blast fungus Magnaporthe oryzae. Mol. Plant, 6(3):605-620. Yang, H. Melissa H Jia, Yulin Jia, Junjie Xing, Venu, R-C, Maria Bellizzi, Longping Yuan, Zhilong Wang, Chuanqing Sun and Guo-Liang Wang. (2013) Molecular mapping of four blast resistance genes using recombinant inbred lines of 93-11 and Nipponbare. J. of Plant Biology, 56:91-97 Chen, S., Songkumarn, P., Venu, R., Gowda, M., Bellizzi, M., Hu, J., Liu, W., Ebbole, D., Mitchell, T. & Wang, G.L. (2013). Identification and characterization of in-planta expressed secreted effectors from Magnaporthe oryzae that induce cell death in rice. Mol Plant Microbe Interact, 26(2):191-202. What do you plan to do during the next reporting period to accomplish the goals? 1. Continue to analyze the function of APIP10-interacting genes and determine their relationship with the Piz-t gene. 2. Identify the Piz-t-associating proteins using Co-IP/MS methods and determine their function in the Piz-t mediated resistance. 3. To investigate the Piz-t cyto-nuclear dynamics during Magnaporthe oryzae infection and identify interacting proteins that are associated with the cytoplasm and nuclear transfer.

Impacts
What was accomplished under these goals? AvrPiz-t Interferes with the E3 Ligase Activity of APIP10 and is Ubiquitinated by APIP10 in vitro To assess whether AvrPiz-t has any biochemical functions in the ubiquitination process, we included the purified GST:AvrPiz-t:HA protein in the in vitro E3 ligase assay of APIP10. Surprisingly, a band above the GST:AvrPiz-t protein was detected by western blot analysis with the anti-HA antibody in the presence of the GST:AvrPiz-t:HA recombinant protein. In contrast, no such signal was detected in the presence of the GST:AvrPi-ta:HA protein, an unrelated effector protein from M. oryza, in the E3 ligase reaction. Furthermore, the western blot analysis that used the anti-Myc antibody to detect ubiquitins showed that the E3 ligase activity of APIP10 was significantly reduced when the GST:AvrPiz-t:HA fusion protein was included in the reaction, suggesting that AvrPiz-t interferes with APIP10 E3 ligase activity. These results showed that AvrPiz-t interferes with APIP10 E3 ligase activity and that APIP10 ubiquitinates AvrPiz-t in vitro. APIP10 Promotes the Degradation of AvrPiz-t in N. benthamiana Since AvrPiz-t is ubiquitinated by APIP10 in vitro, we hypothesized that AvrPiz-t may be a substrate of APIP10 in planta. To test this, we co-expressed GFP:AvrPiz-t:HA with Myc:APIP10 in N. benthamiana leaves. The western blot analysis revealed that the GFP:AvrPiz-t:HA protein level was significantly lower in the tissue in which Myc:APIP10 was co-expressed than in the control tissue in which Myc:APIP10 dRING was co-expressed. This result suggested that APIP10 promotes the degradation of AvrPiz-t in plant cells. Pre-treatment of the leaves with MG132 inhibited the degradation of GFP:AvrPiz-t:HA, confirming the earlier in vitro finding that APIP10 degrades AvrPiz-t via the 26S proteasome system. AvrPiz-t Promotes the Degradation of APIP10 in vivo Because we previously reported that AvrPiz-t promotes the degradation of APIP6 when they are co-expressed in N. benthamiana (Park et al., 2012), we reasoned that AvrPiz-t might also affect the accumulation of APIP10 in plant cells. The western blot analysis showed that APIP10 was reduced by about 30–40% (relative to the controls) when GFP:AvrPiz-t:HA and Myc:APIP10 were co-expressed. Furthermore, the degradation was inhibited by the MG132 treatment, suggesting that AvrPiz-t promotes the degradation of APIP10 in planta, likely through the 26S proteasome system. Knockdown of APIP10 in the Piz-t Background Causes Severe Spontaneous Cell Death Phenotypes To understand the relationship between APIP10 and Piz-t, we transformed APIP10 RNAi construct in the Piz-t:HA background (NPB Piz-t:HA). Surprisingly, most of the APIP10 RNAi lines in the NPB Piz-t:HA background showed severe cell death and dwarf phenotypes compared with the APIP10 RNAi lines in the NPB background. To assess whether the severe cell death is related to the Piz-t:HA protein level, we conducted immunoblot analysis for Piz-t:HA with the anti-HA antibody and semi-quantitative (sq)RT-PCR for the APIP10 transcripts with three independent lines that had different levels of cell death: one line had relatively mild cell death, and two had severe cell death and dwarf phenotypes. The transcript levels of APIP10 in the three lines were inversely correlated with the cell death and dwarfism phenotypes, indicating that the cell death observed in the transgenic plants was caused by the silencing of APIP10. Strikingly, the lines with fewer APIP10 transcripts in the NPB Piz-t:HA background showed a higher expression level of the Piz-t protein, suggesting that APIP10 might be a negative regulator of the accumulation of Piz-t and Piz-t-mediated cell death. APIP10 promotes Degradation of the Piz-t Protein through the 26S proteasome system Because we found that silencing of APIP10 in transgenic rice leads to accumulation of the Piz-t protein, we speculated that APIP10 may promote the degradation of Piz-t in rice. To test this hypothesis, we co-expressed either Myc:APIP10 or Myc:APIP10 dRING (as a control) with Piz-t:HA using agro-infiltration in N. benthamiana leaves. The assay showed that less Piz-t:HA protein accumulated when it was co-expressed with APIP10 than when it was co-expressed with APIP10 dRING, and the accumulation was recovered almost to the control level by treatment with MG132. To further confirm these results, an semi in vivo degradation assay was performed in which total protein extracted from the Piz-t:HA rice plants was mixed with total protein extracted from N. benthamiana leaves agro-infiltrated with either APIP10 or APIP10 dRING. The samples were collected at different times before 1X SDS loading buffer was added to stop the reaction. The assay showed that the level of Piz-t was decreased by APIP10 over time, and after 2 h, less Piz-t protein was detected in the presence of APIP10 than in the presence of APIP10 dRING. The decrease in the level of the Piz-t protein at 2 h was partially inhibited by treatment with MG132. Together, these data suggested that APIP10 promotes Piz-t degradation through the 26S proteasome system.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: 1. Liu, W., J Liu, Y Ning, B Ding, X Wang, Z Wang, GL Wang (2013) Recent progress in understanding PAMP- and effector-triggered immunity against the rice blast fungus Magnaporthe oryzae. Mol. Plant, 6(3):605-620. 2. Yang, H. Melissa H Jia, Yulin Jia, Junjie Xing, Venu, R-C, Maria Bellizzi, Longping Yuan, Zhilong Wang, Chuanqing Sun and Guo-Liang Wang. (2013) Molecular mapping of four blast resistance genes using recombinant inbred lines of 93-11 and Nipponbare. J. of Plant Biology, 56:91-97 3. Chen, S., Songkumarn, P., Venu, R., Gowda, M., Bellizzi, M., Hu, J., Liu, W., Ebbole, D., Mitchell, T. & Wang, G.L. (2013). Identification and characterization of in-planta expressed secreted effectors from Magnaporthe oryzae that induce cell death in rice. Mol Plant Microbe Interact, 26(2):191-202.

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

Outputs
OUTPUTS: Plant fungal diseases are a major limitation in crop production worldwide and cause significant yield loss every year. Although chemicals are effective on disease control, repeated application in crop plants has damaged the environment and caused contamination of food. Use of host resistance has been proved to be the most economical and environmentally-sound way to control plant diseases. However, the molecular basis of host resistance to fungal diseases has been fully unraveled. We focus on understanding the molecular basis of host defense to the fungal pathogen Magnaporthe oryzae, which causes the devastating rice blast disease in rice production. We made good progress torwards understanding the molecular mechanism of rice immunity to the pathogen in 2012. The results are summarized below: 1. We characterized the function of OsHDT701, a member of the plant-specific HD2 subfamily of HDACs, in rice innate immunity. Transcription of OsHDT701 is increased in the compatible reaction and decreased in the incompatible reaction after infection by the fungal pathogen Magnaporthe oryzae. Over-expression of OsHDT701 in transgenic rice leads to decreased levels of histone H4 acetylation and enhanced susceptibility to the rice pathogens M. oryzae and Xanthomonas oryzae pv. oryzae (Xoo). In contrast, silencing of OsHDT701 in transgenic rice causes elevated levels of histone H4 acetylation and elevated transcription of pattern recognition receptor (PRR) and defense-related genes, increased generation of reactive oxygen species (ROS) after pathogen-associated molecular pattern (PAMP) elicitor treatment as well as to enhanced resistance to both M. oryzae and Xoo. We also found that OsHDT701 can bind to defense-related genes to regulate their expression. Taken together, these results demonstrate that OsHDT701 negatively regulates innate immunity by modulating the levels of histone H4 acetylation of PRR and defense-related genes in rice. 2. We found that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into Oryza sativa (rice) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and APIP6 are both degraded when co-expressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The information from this project is useful for rice breeders and farmers to select resistant cultivars to control rice blast disease in the US. We have published our research results in international journals and present them in various rice meetings to rice breeders and farmers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Rice, the stable food for nearly 50% of the worlds population, continues to be the nutritional, financial, and social lifeline for many countries. Therefore, rice diseases that decrease yields or destroy entire harvests are major global problems. Rice blast is one of the most devastating diseases of this crop. The causal agent, the fungus M. oryzae, occurs in virtually all rice-growing areas with periodic epidemics affecting entire rice-growing regions. Moreover, the high variability of M. oryzae pathogenecity determinants has caused most resistant cultivars to be short-lived. The functional analysis of the AvrPiz-t gene in M. oryzae and the APIP6 gene in rice has provided new insights into the intricate relationship between a fungal effectors and a host E3 ligase. The new results from the OsHDT701 project have demonstrated the essential role of epigenetic regulation in rice innate immunity. Since rice blast is a model fungal pathosystem, we fully expect this research to advance the understanding of host plant resistance and spur the discovery of similar genes in other important cereals such as wheat and corn. Moreover, this project has provided extensive training opportunities for a postdoctoral fellow, a PhD student and two undergraduate students.

Publications

• Wang, Y., Wang, D., Deng, X., Liu, J., Sun, P., Liu, Y., Huang, H., Jiang, N., Kang, H., Ning, Y., Wang, Z., Xiao, Y., Liu, X., Liu, E., Dai, L. & Wang, G.L. (2012). Molecular mapping of the blast resistance genes Pi2-1 and Pi51(t) in the durably resistant rice 'Tianjingyeshengdao'. Phytopathology 102, 779-86
• Wang, W.M., Ma, X.F., Zhang, Y., Luo, M.C., Wang, G.L., Bellizzi, M., Xiong, X.Y. & Xiao, S.Y. (2012). PAPP2C interacts with the atypical disease resistance protein RPW8.2 and negatively regulates salicylic acid-dependent defense responses in Arabidopsis. Mol Plant 5, 1125-37 Wang JL, W.L., Liu JF,Dai LY,Liu XL,Xiao YH, Xie HJ,Liu QE,Li T, Jia XY, Wang GL, Yuan LP. (2012). Mapping of the Resistant Gene to Rice Blast in the Dual Purpose Genic Male Sterile Rice, LongS. Acta Agron Sin 38, 408-415
• Park, C.H., Chen, S., Shirsekar, G., Zhou, B., Khang, C.H., Songkumarn, P., Afzal, A.J., Ning, Y., Wang, R., Bellizzi, M., Valent, B. & Wang, G.L. (2012). The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern-Triggered Immunity in Rice. Plant Cell, 24: 4748-4762
• Liu, J., Li, W., Ning, Y., Shirsekar, G., Cai, Y., Wang, X., Dai, L., Wang, Z., Liu, W. & Wang, G.L. (2012). The U-Box E3 ligase SPL11/PUB13 is a convergence point of defense and flowering signaling in plants. Plant Physiol 160, 28-37
• Li, W., Dai, L. & Wang, G.L. (2012). PUB13, a U-box/ARM E3 ligase, regulates plant defense, cell death, and flowering time. Plant Signal Behav 7, 898-900
• Li, W., Ahn, I.P., Ning, Y., Park, C.H., Zeng, L., Whitehill, J.G., Lu, H., Zhao, Q., Ding, B., Xie, Q., Zhou, J.M., Dai, L. & Wang, G.L. (2012). The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis. Plant Physiol 159, 239-50 Jiang N, Li Z, Wu J, Wang Y, Wu L, Wang Y, Wang D, Wen T, Liang Y, Sun P, Liu, J., Dai L, Wang Z, Luo M, Lui X & GL. (2012). Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson. Rice 5, 29 Ding, B., Bellizzi Mdel, R., Ning, Y., Meyers, B.C. & Wang, G.L. (2012). HDT701, a Histone H4 Deacetylase, Negatively Regulates Plant Innate Immunity by Modulating Histone H4 Acetylation of Defense-Related Genes in Rice. Plant Cell 24, 3783-94
• Chodavarapu, R.K., Feng, S., Ding, B., Simon, S.A., Lopez, D., Jia, Y., Wang, G.L., Meyers, B.C., Jacobsen, S.E. & Pellegrini, M. (2012). Transcriptome and methylome interactions in rice hybrids. Proc Natl Acad Sci U S A 109, 12040-5

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Plant disease is one of major limitations in crop production worldwide. Although chemicals are effective on disease control, repeated application in crop plants has damaged the environment and caused contamination of food. Use of host resistance has been proved to be the most economical and environmentally-sound way to control plant diseases. We focus on understanding the molecular basis of host defense to fungal pathogens using rice blast as the model. Rice blast, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. The results we obtained in 2011 are summarized below: 1. We found that the avirulence effector AvrPiz-t from the blast fungus Magnaporthe oryzae preferentially accumulates in the special structure called biotrophic interfacial complex (BIC), and is translocated into rice cells. Rice protoplast assays revealed that AvrPiz-t is degraded in rice cells via the 26S proteasome pathway and its six lysine residues are required for the degradation. Ectopic expression of AvrPiz-t in transgenic rice showed the suppression of chitin- or flg22-induced ROS generation, indicating its virulence function in suppression of the PAMP triggered immunity (PTI). Interaction analyses indicated that AvrPiz-t suppresses the E3 ligase activity of the rice RING E3 ligase APIP6 E3 in vitro, and in turn, APIP6 ubiquitinates AvrPiz-t in vitro and degrades APIP6 in vivo. Silencing of APIP6 in transgenic rice led to a significant reduction of flg22- and chitin-induced ROS generation and enhanced susceptibility against M. oryzae. Taken together, our results uncovered a novel mechanism in which a fungal effector targets a host E3 ligase for the suppression of PTI and the promotion of its virulence in plants. 2. We characterized the function of OsHDT701, a member of the plant-specific HD2 subfamily of HDACs, in rice innate immunity. Transcription of OsHDT701 is increased in the compatible reaction and decreased in the incompatible reaction after blast infection. Over-expression of OsHDT701 in transgenic rice leads to decreased levels of histone H4 acetylation and enhanced susceptibility to both M. oryzae and Xoo. In contrast, silencing of OsHDT701 in transgenic rice causes elevated levels of histone H4 acetylation and elevated transcription of pattern recognition receptor (PRR) and defense-related genes, increased generation of reactive oxygen species (ROS) after pathogen-associated molecular pattern (PAMP) elicitor treatment as well as to enhanced resistance to both M. oryzae and Xoo. Taken together, these results demonstrate that OsHDT701 negatively regulates innate immunity by modulating the levels of histone H4 acetylation of PRR and defense-related genes in rice. 3. Using molecular mapping techniques, we mapped two major resistance genes, designated Pi47 and Pi48, between RM206 and RM224 on chromosome 11, and between RM5364 and RM7102 on chromosome 12, respectively. The DNA markers linked to the new R genes identified in this study should be useful for further fine mapping, gene cloning, and marker-aided breeding of blast-resistant rice cultivars. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Rice growers, breeders, geneticists and extension professionals in the United States. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project should have broad impacts on plant disease control in rice and other cereal crops. Rice, the staple food for nearly half of the world population, continues to be the nutritional, financial, and social lifeline for many countries. Therefore, rice diseases that decrease yields or destroy entire harvests are major global problems. Rice blast is one of the most devastating diseases of this crop. The causal agent, the fungus M. oryzae, occurs in virtually all rice-growing areas with periodic epidemics affecting entire rice-growing regions. Moreover, the high variability of M. oryzae pathogenecity determinants has caused most resistant cultivars to be short-lived. Despite significant progress in isolating R genes in rice and Avr genes in M. oryzae, the intramolecular Avr protein function and the recognition between R and Avr proteins have not been well described. In addition, the timing of Avr protein secretion, recognition by the host, and subsequent downstream signaling are largely unknown. Cloning of Piz-t and AvrPiz-t genes provides an excellent opportunity for investigating recognition and defense activation mechanisms in this model pathosystem. The new results we obtained about the function of the histone H4 deacetylase gene OsHDT701 have demonstrated a role of epigenetic regulation in rice innate immunity. Although the importance of epigenetic regulations in disease resistance in Arabidopsis has been revealed, their function in crop plants is still unclear. Since rice is a model for cereals, We fully expect this research to advance the understanding of host plant resistance and spur the discovery of similar genes in other important cereals such as wheat and corn. Moreover, this project will provide extensive training opportunities for a postdoctoral fellow and a PhD student who might continue to work on this important disease in the future.

Publications

• Venu, RC, Zhang Y, Weaver B, Carswell P, Mitchell TK, Meyers BC, Boehm MJ, Wang GL: Large Scale Identification of Genes Involved in Plant-Fungal Interactions Using Illumina's Sequencing-by-Synthesis Technology. Methods Mol Biol 2011, 722:167-178.
• Sun, X, Wang GL: Genome-Wide Identification, Characterization and Phylogenetic Analysis of the Rice LRR-Kinases. PLoS One 2011, 6(3):e16079.
• Venu, RC, Sreerekha MV, Nobuta K, Belo A, Ning Y, An G, Meyers BC, Wang GL: Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars. BMC Genomics 2011, 12(1):190.
• Xiao, YH, Pan Y, Luo LH, Zhang GL, Deng H, Dai LY, Liu XL, Tang WB, Chen LY, Wang GL: Quantitative trait loci associated with seed set under high temperature stress at the flowering stage in rice (Oryza sativa L.). Euphytica 2011, 178(3):331-338.
• Huang, H, Huang L, Feng G, Wang S, Wang Y, Liu J, Jiang N, Yan W, Xu L, Sun P et al: Molecular mapping of the new blast resistance genes Pi47 and Pi48 in the durably resistant local rice cultivar Xiangzi 3150. Phytopathology 2011.
• Ning, YS, Xie Q, Wang GL (2011). OsDIS1-mediated stress response pathway in rice, Plant Signaling & Behavior.6:1684-1686
• Shen, Y, Venu RC, Nobuta K, Wu X, Notibala V, Demirci C, Meyers BC, Wang GL, Ji G, Li QQ: Transcriptome dynamics through alternative polyadenylation in developmental and environmental responses in plants revealed by deep sequencing. Genome Res 2011, 21(9):1478-1486.
• Ning, Y, Jantasuriyarat C, Zhao Q, Zhang H, Chen S, Liu J, Liu L, Tang S, Park CH, Wang X et al: The SINA E3 Ligase OsDIS1 Negatively Regulates Drought Response in Rice. Plant Physiol 2011, 157(1):242-255.
• Liu, XL, Liu JL, Hu YJ, Ning Y, Jiang N, Wu J, Jeon JS, Xiao YH, Dai LY, Wang GL: Genetic Variation and Evolution of the Pi9 Blast Resistance Locus in the AA Genome Oryza Species. Journal of Plant Biology 2011, 54(5):294-302.
• La, H, Ding B, Mishra GP, Zhou B, Yang H, Bellizzi Mdel R, Chen S, Meyers BC, Peng Z, Zhu JK, Wang GL: A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice. Proc Natl Acad Sci U S A 2011, 108(37):15498-15503.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Plant diseases are a major limitation in crop production causing 10-20% yield loss annually. We focus on understanding the molecular basis of host defense to fungal pathogens using rice blast as the model. Rice blast disease, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. The following is the summary of our results obtained in 2010: 1. AvrPiz-t is degraded in rice protoplasts via the 26S proteosome pathway. To test if AvrPiz-t is poly-ubiquitinated by three E3 APIPs in vitro, we transfected the GFP-AvrPiz-t-HA construct into rice protoplasts with DMSO (control), 100 μM MG132 (inhibitor of 26S proteasome), and 300 nM BAF (inhibitor of vacuolar degradation system). Western blot analysis with anti-HA antibody detected no AvrPiz-t protein in rice protoplasts with the DMSO and BAF treatments. In contrast, a faint band was detected in the MG132-treated sample. Western blot analysis with anti-GFP antibody confirmed that AvrPiz-t was not degraded in the MG132-treated rice protoplasts, suggesting that AvrPiz-t is degraded through the 26S proteasome system, not through the vacuolar system. 2. AvrPiz-t is preferentially localized in BICs. In collaboration with Drs. Khang and Valent at KSU, we investigated the subcellular localization and secretion pattern of AvrPiz-t during infection. We made the AvrPiz-t:mCherry:NLS construct in which AvrPiz-t is fused with the mCherry and a nuclear localization signal fragments. The derived construct was transformed into blast strain 939-4. The transformed strain was inoculated in rice sheaths, and fluorescence was observed using a confocal microscopy. We found that AvrPiz-t:mCherry:NLS was preferentially localized in BICs. 3. Inducible expression of the AvrPiz-t gene leads to cell death in transgenic plants. To determine whether AvrPiz-t can trigger an HR reaction in rice cells, we made a construct in which the AvrPiz-t coding region lacking the signal peptide sequence is controlled by the estradiol-inducible XVE system and was transformed into Toride (Piz-t). After estradiol induction treatment, the leaves of AvrPiz-t-Toride transgenic plants exhibited necrosis-like cell death. Leaves of the wild type Nipponbare did not show any cell death after induction. These results indicate that AvrPiz-t is a cytoplasmic effector that is recognized by Piz-t directly or indirectly in rice cells. 4. Ectopic expression of AvrPiz-t in transgenic rice compromises the PTI. We recently obtained homozygous lines of the Avr-Piz-t OX construct under the maize ubiquitin promoter in NPB and determined their response to the PAMP elicitors flg22 and chitin. Reactive oxygen species (ROS) generation after the treatment with both elicitors was monitored using luminol chemi-luminescence assay. Strikingly, the ROS response of the AvrPiz-t plants was completely abolished in the flg22 treatment and was over 50% reduced in the chitin treatment 10-12 min after the treatment. These results indicate that AvrPiz-t has virulence function and might target the rice PTI during infection. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The information from this project will be useful for plant pathologists to understand the molecular basis of plant-microbe interactions and rice breeders to select broad-spectrum resistant cultivars PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The project should have broad impacts on plant disease control in rice and other cereal crops. Rice, the stable food for nearly 50% of the world's population, continues to be the nutritional, financial, and social lifeline for many countries. Therefore, rice diseases that decrease yields or destroy entire harvests are major global problems. Rice blast is one of the most devastating diseases of this crop. The causal agent, the fungus M. oryzae, occurs in virtually all rice-growing areas with periodic epidemics affecting entire rice-growing regions. Moreover, the high variability of M. oryzae pathogenecity determinants has caused most resistant cultivars to be short-lived. Despite significant progress in isolating R genes in rice and Avr genes in M. oryzae, the intramolecular Avr protein function and the recognition between R and Avr proteins have not been well described. In addition, the timing of Avr protein secretion, recognition by the host, and subsequent downstream signaling are largely unknown. Pi-ta/Avr-Pita are the only R/Avr pair in rice blast shown to bind directly to each other but our understanding of how Avr-Pita and Pi-ta interact in rice cells remains unclear. Cloning of Piz-t and AvrPiz-t genes provides an excellent opportunity for investigating recognition and defense activation mechanisms in this model pathosystem. While this research is focused on rice, the insights gained with regard to Avr gene function and host protein association will have broad impacts. We fully expect this research to advance the understanding of host plant resistance and spur the discovery of similar genes in other important cereals such as wheat and corn. Moreover, this project will provide extensive training opportunities for a postdoctoral fellow and a PhD student who might continue to work on this important disease in the future.

Publications

• Venu, R.C., Sheshu MadhaV, M., Sreerekha, M. V., Nobuta, K., Zhang, Y., Carswell, P., Boehm, M., Meyers, B.C., Korth, K., Wang, G.L. (2010). Deep and Comparative Transcriptome Analysis of Rice Plants Infested by the Beet Armyworm (Spodoptera exigua) and Water Weevil (Lissorhoptrus oryzophilus). Rice. 3:22-35.
• Dai, L., Wu, J., Li, X., Wang, X., Liu, X., Jantasuriyarat, C., Kudrna, D., Yu, Y., Wing, R.A., Han, B., Zhou, B., Wang, G.L. (2010). Genomic structure and evolution of the Pi2/9 locus in wild rice species. Theor Appl Genet. 121:295-309

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Rice blast disease, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. In 2009, we made significant progress in understanding the molecular basis of rice blast resistance in the following four areas: 1) Identification of AvrPiz-t interacting proteins (APIPs): To search for the host targets of AvrPiz-t, we performed yeast-two-hybrid (Y2H) screens using AvrPiz-t as the bait. A total of 12 AvrPiz-t interacting proteins (APIPs) were identified. Interestingly, four APIPs (APIP2, 6, 8, and 10) are involved in the ubiqintination-mediated protein degradation pathway. This finding is significant because evidence increasingly suggests that pathogen effectors may target the ubiqintination-mediated protein degradation systems in the host to interfere with the defense responses and, as far as we know, none of the cloned fungal Avr protein have been reported as having this function. 2) Cellular localization of AvrPiz-t and APIPs. To determine the cellular localization of AvrPiz-t in rice cells, we fused the ORF, minus the signal peptide sequence, with the GFP reporter gene and cloned the fragment behind the 35S promoter for expression in rice protoplasts. A weak GFP fluorescent signal was evident throughout the cell with enhanced florescence in the nucleus. To determine the cellular localization of APIPs 2, 6, and 10 in rice cells and to evaluate co-localization with AvrPiz-t, we cloned the full-length ORF of each gene and fused them to the GFP reporter gene at their C-terminus, and the fused fragment is under the control of the 35S promoter. At 24 h after transfection of rice protoplasts, weak GFP signals were similarly evident throughout the cell with enhanced florescence in the nucleus. 3) Evaluation of E3 ligase activity of APIPs. Bioinformatics analysis indicated that APIP2, 6, and 10 contain a RING finger domain and are putative ubiquitin E3 ligases. To test their E3 ligase activity in vitro, full-length APIP 2, 6, and 10 cDNA fragments were expressed in E. coli as a fusion with the GST tag. Each was purified by affinity chromatography and combined with the wheat E1 and 2 Arabidopsis E2s. Ubiquitination activity was observed for each purified MBP-APIP 2, 6, and 10 protein in the presence of both E2s, indicating that these three APIPs posses E3 ligase activity. 4) AvrPiz-t interfers with ubiquitination and is ubiquitinated by the E3 ligases. When the MBP-AvrPiz-t was included in the E3 ligase assays of APIP2, 6, and 10, western blot analysis with anti-ubiqiutin antibody showed that the E3 activity of all three APIPs was significantly reduced, suggesting that AvrPiz-t interferes with ubiquitination. Surprisingly, many high molecular weight bands appeared in all three E3 ligase reactions in the presence of the MBP-AvrPiz-t recombinant protein while no signal was detected in the absence of the MBP-AvrPiz-t protein when anti-MBP antibody was used in the western blot analysis, indicating that the MBP-AvrPiz-t is ubiquitinated by the APIP E3 ligases in vitro. These exciting findings have not been previously reported for any fungal plant pathogen. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The ongoing project has broad impacts on both food security and the training of the next generation of plant scientists. First, rice is unarguably the most important food crop for the developing world, and is a model plant for cereal crops. Rice blast is the major limit to stable rice production in many countries. The information gained from this project will be critical to the development of novel approaches to control this important disease. Since rice is a model plant for cereals, the novel discovery will lead to better understanding of the defense signaling networks in both monocot plants such as wheat and corn. Second, this project was designed to enhance the scientific skills of postdocs and students. The proposed research involves state-of-the-art techniques such as protein-protein interaction, protein purification, and ubiquitination assays that will provide students and postdocs with advanced skills. Additionally, the project includes a mentoring plan designed specifically to broaden the training beyond the laboratory bench so that students and postdocs acquire all the skills necessary to become effective researchers.

Publications

• Chen, S. M. Gowda, R. C. Venu, P. Songkumarn, C. H. Park, M. Bellizzi, D. Ebbole, G. L. Wang. 2009. Isolation and functional analysis of putative effectors from M. oryzae using integrated genomic approaches. In Advances in Genetics, Genomics and Control of Rice Blast Disease, Eds. G.L. Wang and B. Valent, Springer, pp 93-103.
• Qu S, Jeon JS, Ouwerkerk PB, Bellizzi M, Leach J, Ronald P, Wang GL. 2009. Construction and application of efficient Ac-Ds transposon tagging vectors in rice. J Integr Plant Biol. 51(11):982-92.
• Chen S, Songkumarn P, Liu J, and Wang GL. 2009. A Versatile Zero Background T-vector System for Gene Cloning and Functional Genomics. Plant Physiology, 150(3):1111-21. Bruce M, Hess A, Bai J, Mauleon R, Diaz MG, Sugiyama N, Bordeos A, Wang GL, Leung H, Leach JE. 2009. Detection of genomic deletions in rice using oligonucleotide microarrays. BMC Genomics. 25;10:129.
• Gray J, Bevan M, Brutnell T, Buell CR, Cone K, Hake S, Jackson D, Kellogg E, Lawrence C, McCouch S, Mockler T, Moose S, Paterson A, Peterson T, Rokshar D, Souza GM, Springer N, Stein N, Timmermans M, Wang GL, Grotewold E. 2009. A recommendation for naming transcription factor proteins in the grasses. Plant Physiol. 149(1):4-6.
• Li W, Wang B, Wu J, Lu G, Hu Y, Zhang X, Zhang Z, Zhao Q, Feng Q, Zhang H, Wang Z, Wang G, Han B, Wang Z, Zhou B. 2009. The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t. Mol Plant Microbe Interact. 22(4):411-20.
• Lee SK, Song MY, Seo YS, Kim HK, Ko S, Cao PJ, Suh JP, Yi G, Roh JH, Lee S, An G, Hahn TR, Wang GL, Ronald P, Jeon JS. 2009. Rice Pi5-Mediated Resistance to Magnaporthe oryzae Requires the Presence of Two Coiled-Coil-Nucleotide-Binding-Leucine-Rich Repeat Genes. Genetics. 181(4):1627-38.
• Zhou, B., G. L. Wang. 2009. Functional and evolutionary 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, pp 127-135.
• La H.G. and Wang G.L. 2009: The DNA glycosylase/lyase DNG701 is involved in DNA demethylation, Tos17 transposition, seedling growth and seed development in rice, Presented at 11th Annual Plant Molecular Biology & Biotechnology Program Symposium. Columbus, OH. April 24-25
• Yang H.M., Madhav S.M. and Wang G.L. 2009. Towards map-based cloning of the broad-spectrum resistance gene Pi40(t) to rice blast, Presented at The 4th Annual International Scholar Research Exposition at OSU, Columbus OH. Nov. 19
• Songkumarn P., Chen S, Gowda M, Venu RC, Songkumarn P, Bellizzi M, Ebbole D, Mitchell T and Wang GL. 2009. Isolation and functional analysis of novel secreted proteins in Magnaporthe oryzae. Presented at 11th Annual Plant Molecular Biology & Biotechnology Program Symposium. Columbus, OH. April 24-25
• Chen S, Gowda M, Venu RC, Songkumarn P, Bellizzi M, Ebbole D, Mitchell T and Wang GL. 2009. In-planta Expressed Secreted Proteins From M. oryzae Are Enriched For Extracellular Effectors That Induce Cell Death In Rice. Presented at 11th Annual Plant Molecular Biology & Biotechnology Program Symposium. Columbus, OH. April 24-25.
• Chen S, Gowda M, Venu RC, Songkumarn P, Bellizzi M, Ebbole D, Mitchell T and Wang GL. 2009. In-planta Expressed Secreted Proteins From M. oryzae Are Enriched For Extracellular Effectors That Induce Cell Death In Rice. Presented at XIV international Congress on Molecular Plant-Microbe Interactions, Quebec City, Canada, July 19-23.
• Rohila JS, Liu W, Chen S, Wang GL, Yang Y. 2009. A small family of NPP1-like proteins from Magnaporthe oryzae elicits cell death in monocot as well as dicot plants. Presented at XIV international Congress on Molecular Plant-Microbe Interactions, Quebec City, Canada, July 19-23.
• Songbiao Chen , Pattavipha Songkumarn , Jianli Liu , Guo-Liang Wang. 2009. A Zero Background Versatile T-Vector System For Gene Cloning And Functional Genomics. Presented at the XVIII Plant and Animal Genome Conference, Town & Country Convention Center, San Diego, CA. Jan. 10-14
• Ding B. and Wang GL. 2009. AGO706, A Rice Argonaute Gene Negatively Regulating Resistance To Magnaporthe oryzae And Xanthomonas oryzae pv. oryzae Pathogens. Presented at the XVIII Plant and Animal Genome Conference, Town & Country Convention Center, San Diego, CA. Jan. 10-14

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Rice blast disease, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. To elucidate the function of effector proteins from M. oryzae in pathogenesis and interaction with the host, we have performed RL-SAGE and MPSS approaches to study the gene expression profiles of M. oryzae during the interaction. The RL-SAGE and MPSS analyses identified 3,441 and 3,004 annotated M. oryzae genes, respectively. Among them, 217 in-planta expressed putative secreted protein genes of M. oryzae were identified. Using multiple approaches, 38 in-planta expressed secreted protein genes have been isolated and tested, and six novel proteins that induce rice cell death were identified. On the other hand, expression of these genes without signal peptide sequence in rice protoplasts did not induce cell death, suggesting the proteins are extracellular effectors that activate cell death from the outside of the host cells. The effectors are highly diverse in their sequences. While four effectors have several sequential homologs from other microorganisms, two effectors do not share any significant similarity with all known proteins. Interestingly, four of the six proteins also induced cell death when they were transiently expressed in non-host Nicotiana benthamiana, suggesting that extracellular effectors from M. oryzae may induce cell death in host and non-host plants through both shared and distinct mechanisms. In addition, we isolated the avirulence gene AvrPiz-t from rice blast by a map-based cloning strategy. Eight interacting proteins were identified from the yeast two hybrid screens. To investigate the role of programmed cell death in the defense response to pathogen invasion, we characterized the lesion mimic mutant spl11 and cloned the Spl11 gene via a map-based cloning strategy. The predicted SPL11 protein contains a U-box domain and an armadillo (ARM) repeat domain. In searching for other components involved in the Spl11-mediated signaling pathway, eight different SPL11-interacting proteins (SPINs) were identified in yeast two-hybrid screenings. In-vitro GST binding assay confirmed the interaction between SPL11 and SPIN1, SPIN3 and SPIN6. RNAi and overexpression lines of these genes are being characterized for their alterations in defense responses to rice pathogens. Interestingly, we identified a T-DNA insertion mutant of the Arabidopsis Spl11 ortholog which displays accelerated cell death and stunted growth phenotypes. The Arabidopsis mutant, named Atspl11, conferred enhanced resistance to the bacterial pathogens Pseudomonas syringae pv. maculicola and Xanthomonas campestris pv. campestris, and the fungal pathogen Golovinomyces cichoracearum. RT-PCR analysis indicated that the PR1 gene is highly induced and the PDF1.2 gene is highly suppressed in the mutant. In vitro E3 ubiquitination assay showed that AtSPL11 has ubiquitin ligase activity. These results demonstrate that the function of the Spl11 gene in suppressing plant cell death and activating defense response is conserved between monocots and dicots. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The information generated in the project are useful for scientists who study host resistance and plant-microbe interactions. In addition, the genes identified in the project can be used for rice breeders to select resistant rice cultivars to pathogens. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Rice is not only the most important food crop in the developing world and but also a model monocot plant for functional genomics research. Both disease resistance and flowering are important agronomically important traits in rice production. Effective manipulation of the two traits will enhance rice yield stability. We identified a gene controlling both disease resistance and flowering regulation in rice and Arabidopsis. The novel discovery will lead to better understanding of the signaling networks in both monocots and dicots. Fungal diseases cause tremendous yield losses in crop production every year worldwide. How a fungal pathogen manipulates the host defense and causes diseases is not well understood. We identified a novel avirulence protein from the fungal pathogen Magnaporthe oryzae and its host targets in rice. The availability of the pair of avirulence and resistance proteins provides the excellent material for in-depth understanding the molecular events underlying fungal and plant interactions. We found that six effector proteins induce cell death in rice protoplasts. Functional analysis of these genes in pathogenicity and interaction with the host proteins will provide new insights into the molecular events underlying the rice and rice blast interactions.

Publications

• Wang W, Yang X, Tangchaiburana S, Ndeh R, Markham JE, Tsegaye Y, Dunn TM, Wang GL, Bellizzi M, Parsons JF, Morrissey D, Bravo JE, Lynch DV, Xiao S. 2008. An Inositolphosphorylceramide Synthase Is Involved in Regulation of Plant Programmed Cell Death Associated with Defense in Arabidopsis. The Plant Cell, 20(6):1456-69.
• Andrea Bela, Kan Nobuta, R. C. Venu, Prakash E. Janardhanan, Guo-liang Wang and Blake C. Meyers. 2008. Transposable Element Regulation in Rice and Arabidopsis : Diverse Patterns of Active Expression and siRNA-mediated Silencing. Tropical Plant Biology. 1:72-84.
• Bu-Jun Shi and Wang, G.L. 2008. Comparative study of genes expressed from rice fungus-resistant and susceptible lines during interactions with Magnaporthe oryzae. Gene, 427:80-85.
• Li-Rong 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, 1:800-815.
• Vega-Sanchez ME, Zeng L, Chen S, Leung H, Wang GL. 2008. SPIN1, a K Homology Domain Protein Negatively Regulated and Ubiquitinated by the E3 Ubiquitin Ligase SPL11, Is Involved in Flowering Time Control in Rice. The Plant Cell, 1456-1469.
• Wu C, Bordeos A, Madamba MR, Baraoidan M, Ramos M, Wang GL, Leach JE, Leung H. 2008. Rice lesion mimic mutants with enhanced resistance to diseases. Mol Genet Genomics. 279(6):605-19.
• Pattavipha Songkumarn, Songbiao Chen, Malali Gowda, Venu Reddyvari Channarayappa and Guo Liang Wang. 2008.Isolation and functional analysis of novel secreted proteins in Magnaporthe oryzae. The 2008 APS Centennial Meeting in Minneapolis, Minnesota, Jul 26-30, 2008.
• Miguel E. Vega-Sanchez, Lirong Zeng, Songbiao Chen, Hei Leung, and Guo-Liang Wang. 2008.The rice E3 ligase SPL11 ubiquitinates SPIN1, an RNA/DNA binding protein involved in flowering time control. Annual meeting of American Society of Plant Biologist, Merida, Mexico, June 26-July 1, 2008
• Park CH, Jung K, Phetsom J, Babu MR, Bruce M, Mauleon R, Bordeos A, Bellizzi M, Leach J, Leung H, Ronald P and Wang GL. 2008. Expression Characterization Of Rice Defense Mutants Using Whole Genome Expression Profiling. Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA.
• Park CH, Jung K, Phetsom J, Babu MR, Bruce M, Mauleon R, Bordeos A, Bellizzi M, Leach J, Leung H, Ronald P and Wang GL. 2008. Characterization Of 10 Rice Defense Mutants Using Whole Genome Expression Profiling. ISRFG. Nov. 10-12, 2008, Jeju, Korea.
• Kan Nobuta, Cheng Lu, R.C. Venu, Giriprasad Sridhara, Mayumi Nakano, Richi Guputa, Roli Shrivastava, Prakash Janardhanan, Kevin McCormick, Guo-liang Wang, Pam J. Green, Blake C. Meyers. 2008.Expression Profiling Using Signature-Based Sequencing Technologies. Plant & Animal Genomes XVI Conference Jan. 12-16, 2008, San Diego, CA.
• Songbiao Chen, Pattavipha Songkumarn, Malali Gowda, Venu Reddyvari-Channarayappa, Chan Ho Park, Maria Bellizzi, Daniel Ebbole, Guo-Liang Wang. 2008. Large-Scale Isolation And Analysis Of Putative Effectors From M. oryzae Using Integrated Genomics Approaches. Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA.
• Bo Ding, Maria Billizzi, Guo-Liang Wang. 2008.Expression Analysis Of Chromatin-Related Genes In Response To Pathogen Infection In Rice Plant & Animal Genomes XVI Conference Jan. 10-14, 2008, San Diego, CA..
• Honggui La, Bo Ding, Bo Zhou, Maria del Rosario Bellizzi, Guosheng Li, Blake Meyers, Zhaohua Peng and Guo-Liang Wang.2008. DNG701, encoding a putative DNA glycosylase, is involved in DNA demethylation and plays an important roles in rice development and growth Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA.
• Gautam S. Shirsekar, Miguel E. Vega-Sanchez, Alicia Bordeos, Marietta Baraoidan, Qi Sun, Hei Leung and Guo-Liang Wang. 2008.Identifying the components in Spl11-mediated defense pathway and determining the relationship between Spl11 and other defense signaling genes in ric at APS Centennial Meeting held at Minneapolis in July 26-30, 2008.
• R.C.Venu, Kan Nobuta, M.V. Sreerekha, Andre Belo, Huameng Li, Eric Stahlberg, Blake C. Meyers, Guo-Liang Wang. 2008.Expression profiling of rice in response to Magnaporthe oryzae and Xanthomonas oryzae infections using Massively Parallel Signature Sequencing. Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA.
• Kan Nobuta, Cheng Lu, R.C. Venu, Giriprasad Sridhara, Mayumi Nakano, Richi Guputa, Roli Shrivastava, Prakash Janardhanan, Kevin McCormick, Guo-liang Wang , Pam J. Green, Blake C. Meyers. 2008.Expression Profiling Using Signature-Based Sequencing Technologies. Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA
• R.C.Venu, Kan Nobuta, M.V. Sreerekha, Andre Belo, Huameng Li, Eric Stahlberg, Blake C. Meyers, Guo-Liang Wang. 2008.Expression profiling of rice in response to Magnaporthe oryzae and Xanthomonas oryzae infections using Massively Parallel Signature Sequencing. PMBB meeting, The Ohio State University, Columbus, April 11-12, 2008.
• Zhaohua Peng, Pamela Ronald, Guo-Liang Wang.2008. Identification Of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics And Yield In Rice (Oryza sativa), Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA.
• Kan Nobuta, Cheng Lu, R. C. Venu, Andrea Bele, Mayumi Nakano, Richi Guputa, Roli Shrivastava, Gayathri Mahalingam, Prakash Janardhanan, Kevin McCormick, Emanuele De Paoli1, Monica Accerbi, Linda Rymarquis, Marcelo German, Mario Arteaga-Vasquez, Vicki L Chandler, Guo-liang Wang, Pamela J Green, Blake C Meyers. 2008.Signature-Based Transcriptional Profiling Of mRNA And Small RNA In Plants. Plant & Animal Genomes XVI Conference, Jan. 12-16, 2008, San Diego, CA.

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

Outputs
1). The SPL11-interacting protein 1 (SPIN1) is monoubiquitinated but not targeted for degradation by SPL11. SPL11 was shown to possess E3 ubiquitin ligase activity and interact with SPIN1 both in vitro and in vivo, suggesting that SPL11 might target SPIN1 for ubiquitination and/or degradation. In-vitro E3 ligase activity assay showed that SPIN1 was monoubiquitinated when SPL11 was present in the reaction. To test whether the oligoubiquitination of SPIN1 by SPL11 in vitro leads to the degradation of SPIN1, a cycloheximide (CHX)-chase experiment was carried out in rice protoplasts overexpressing Spin1 and Spl11. SPIN1 protein levels accumulated to similar levels before and after addition of CHX, even when SPL11 was overexpressed. These results indicate that ubiquitinated-SPIN1 protein is stable and not targeted for proteolysis by SPL11. 2). SPIN1 has both RNA and DNA binding activities in vitro. Our bioinformatic analyses indicated that SPIN1 is a member of the STAR family of RNA binding proteins. To confirm its biochemical function, recombinant SPIN1 protein was incubated in vitro with ribohomopolymer-bound beads representing poly A, poly U, poly G and poly C, as well as single and double stranded calf thymus DNA. Western blot analysis following incubation and washing of beads revealed that recombinant SPIN1- bound to all RNA and DNA polymers tested. As a control, GST did not show any RNA or DNA binding activity. These results confirmed that SPIN1 has RNA/DNA binding activity in vitro. 3). Overexpression of Spin1 causes late flowering under short day conditions by repression of Hd3a expression. To functionally characterize Spin1, RNAi and overexpression transgenic lines were generated in the rice japonica cultivar Nipponbare (NPB). Over 20 RNAi lines silencing Spin1 and 13 lines overexpressing the gene with an N-terminal TAP (tandem affinity purification) tag were generated. Examination of Spin1-TAP lines revealed that they showed delayed flowering time compared to the RNAi and NPB plants. The delayed flowering phenotype correlated with the overexpression of the TAP-SPIN1 protein in T4 transgenic lines, and was due to the overexpression of the Spin1 gene, since transgenic lines overexpressing the TAP tag alone in the Kitaake background had no differences in flowering time compared to non-transformed plants. 4). Mapping and cloning of suppressor genes of spl11 lesion mimic mutant. In order to map genes involved in suppression of spl11 lesion mimic phenotype from suppressor lines 963, 1902, 2143 and 6455, crosses of these lines were made with TP309spl11 in the 2006-2007 season. Trueness of F1 was determined using RAPD-PCR, where random primers showing polymorphism between respective parents involved in that particular cross were used. F2 population is currently being grown in the greenhouse. Mapping will be done using PCR with Simple Sequence Repeat (SSR) markers in coming months.

Impacts
Rice is the most important food crop for the half of the world population. It is recently becoming a model monocot plant for functional genomics research due to its small genome size, extensive genetic map, available genome sequence, and relative ease of transformation. Successful characterization of the genes involved in cell death and disease resistance as carried out in this project will lead to a better understanding of the molecular basis of the broad-spectrum resistance in rice. Some of the findings about the function of these genes are unique and novel to most crop plants. Therefore, our on-going experiments will greatly advance understanding of the function of ubiquitination related genes in disease resistance in rice and other crop plants in the US. The information obtained from this project will facilitate the design of new plant resistance genes with desired specificities for effective control of plant diseases and significant reduction of the use of environmentally damaging pesticides in the US.

Publications

• 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
• Kan Nobuta, Venu Reddyvari-Channarayappa, Cheng Lu, Andre Belo, Kalyan Vemaraju, Pam Green, Guo-liang Wang, and Blake C. Meyers. 2007. An Expression Atlas of Rice mRNA and Small RNA, Nature Biotechnology, 25:473-477.
• Liangying Dai, Xionglun Liu, Yinghui Xiao, GL Wang. 2007 Recent advances in cloning and characterization of disease resistance genes in rice. Journal of Integrative Plant Biology. 49 (1): 112-119.
• Gowda M, Reddyvarichannarayappa Venu, Jia Y, Stahlberg E, Pampanwar V, Soderlund C, GL Wang (2007). Use of RL-SAGE Analysis to Identify Novel Fungal and Plant Genes Involved in Host-Pathogen Interactions. Methods Mol Biol. 2006;354:131-44.
• Jo Y.-K., G.-L. Wang, and M. J. Boehm. 2007. Expression Analysis of Rice Defense-Related Genes in Turfgrass in Response to Magnaporthe grisea, Phytopathology, Phytopathology 97:170-178.
• Malali GOWDA, RC. VENU, Huameng LI, Chatchawan JANTASURIYARAT, Songbiao CHEN, Maria BELLIZZI, Vishal PAMPANWAR, HyeRan KIM, Ralph A. DEAN, Eric STAHLBERG, Rod WIN, Cari SODERLUN, Guo-Liang WANG. 2007. Magnaporthe grisea Infection Triggers RNA Variation and Antisense Transcript Expression in Rice. 13th ISMPMI, Sorrento, Italy, July 21-27, 2007
• Songbiao Chen and Guo-Liang Wang. 2007. Development Of A Highly Efficient Protoplast-Based Gene Expression System For Functional Genomics And Proteomics Assays In Rice. 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. 2007. 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.
• 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. 2007. 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.
• Eric A Stahlberg, Venu Reddyvary Channaraya, Jeff Doak, Daniel P Dougherty, Malali Gowda, Quinn Li, Wolfgang Sadee , Dave Strenski, Guo-liang Wang. 2007. The Ohio Biosciences Library: High-Performance Computing Technologies For High-Throughput Comparative Bioinformatics, 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. 2007. The Rice Innate Immunity Interaction Map. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA.
• Kan Nobuta, R.C. Venu, Kalyan Vemeraju, Mayumi Nakano, Kevin McCormick, Maria Bellizzi , Guo-liang Wang, Blake C Meyers. 2007. Deep Transcriptional Profiling Of Rice Using Signature Sequencing. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA
• R.C. Venu, Kan Nobuta, Kalyan Vemaraju, Maria Bellizzi, Eric Stahlberg, Blake Meyers, Guo-Liang Wang. 2007. Expression Patterns Of Transcription Factors In The Cold, Drought And Salt Treated Rice Plants. 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. 2007. Abstract presented at the Plant and Animal Genome meeting, January 13-17, 2007, San Diego, CA
• Honggui La, Bo Zhou, Maria Bellizzi, Zhaohua Peng, and Guo-Liang Wang. 2007. DNG701, encoding a putative DNA glycosylase, is involved in DNA demethylation and plays an important role in rice development and growth. Presented at the meeting of Epigenetics: Regulation of Chromatin Structure in Development and Disease, April 11-16, 2007, Breckenridge, Colorado.
• Chen S, Vega-Sanchez M, Songkumarn P, Wang GL. 2007. Development of a highly efficient protoplast-based gene expression system for functional genomics and proteomics assays in rice. Abstract. PMBB SYMPOSIUM 2007, March 30-March 31, 2007, The Ohio State University, Museum of Biological Diversity, Columbus, OH.
• Chen S, Gowda M, Venu RC, Songkumarn P, Park CH, Bellizzi M, Ebbole D, Wang GL 2007. Large-scale Isolation and Functional Analysis of Putative Effectors from M. oryzae Using Integrated Genomics Approaches. Oral presentation. The 4th international rice blast conference, October 9-October 14, 2007, Changsha, China.

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

Outputs
Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is the most destructive fugal disease of rice. To better understand the mechanism underlying the resistance mediated by major resistance genes, we isolated the Pi9 gene using a map-based cloning strategy in 2005. In 2006, we cloned another two alleles, Pi2 and Piz-t, using both map-based cloning and homology cloning methods. The main findings are summarized below: 1) We first mapped the gene on chromosome six using the Pi9 linked markers. A BAC contig in the region was constructed using overlapping BAC clones containing the linked markers. High-resolution mapping and sequencing identified the Pi2 candidate genes. Rice transformation experiments and resistance analysis of transgenic lines confirmed that Nbs4-Pi2 is the Pi2 gene. It is a member of a gene cluster comprising nine gene members (named Nbs1-Pi2 to Nbs9-Pi2) and encodes a protein with an NBS and LRR domain. The Piz-t gene, a Pi2 allele in the rice cultivar Toride 1, was isolated based on the Pi2 sequence information. Complementation tests confirmed that the family member Nbs4-Piz-t is the Piz-t gene. Sequence comparison revealed that only eight amino acid changes, which are confined within three consecutive LRRs, differentiate Piz-t from Pi2. Of the eight variants, only one locates within the xxLxLxx motif, which is thought to be critical for the resistance specificity determinant of the LRR-containing R genes. A reciprocal exchange of the single amino acid between Pi2 and Piz-t did not convert the resistance specificity to each other rather abolished the function of both resistance proteins. These results indicate that the single amino acid in the xxLxLxx motif may not be sufficient to determine the resistance specificities of the Pi2 and Piz-t genes. 2) The Pi2/9 locus contains at least four resistance specificities to M. grisea and belongs to a gene complex comprised of multiple genes that encode highly homologous NBS/LRR proteins. To investigate the genetic events involved in the evolution of the Pi2/9 locus, we analyzed the Pi2/9 locus at the inter- and intra-locus levels in five rice cultivars. The NBS-LRR genes in the five cultivars belong to the same phylogenetic clade among rice NBS-LRR genes and all have a phase-2 intron at the N-terminus. However, the paralogues within each haplotype show a significant sequence divergence and their N-terminal intron and 5' regulatory regions are very different. On the contrary, the orthologues from different haplotypes are highly similar, indicating an obvious orthologous relationship has been maintained during the evolution of the Pi2/9 locus. These results suggest that sequence diversification in 5' regulatory region and N-terminal intron of the paralogues may have led to suppression of meiotic recombination between the paralogues within each haplotype, facilitating the maintenance of the orthologous relationship among rice cultivars. Our observations provide valuable insight into the genomic dynamics and evolutionary mechanism of an NBS-LRR resistance gene complex in rice.

Impacts
Rice blast is well documented as the leading cause of yield loss of rice worldwide. All parts of the plant, including roots, are subject to attack, but an infection that occurs immediately below the panicle is most destructive, virtually ensuring a complete loss of rice. M. grisea is known for its high variability in field conditions, which causes frequent loss of host resistance. The annual yield loss due to the disease is about $5.0 billion dollars worldwide. Cloning and characterization of the Pi2, Pi9 and Piz-t genes by our lab has provided new insights about the molecular mechanism underlying their broad-spectrum resistance. Markers linked to the genes can be used for marker-aided selection in rice breeding programs.

Publications

• Zhou B, M. Dolan, H. Sakai, and G.-L. Wang. 2007. The Genomic Dynamics and Evolutionary Mechanism of the Pi2/9 Locus in Rice. Mol. Plant-Microbe Interact., MPMI 20:63-71.
• Zhou B, Q Qu, G Liu, M Dolan, H Sakai, G Lu, M Bellizzi, GL Wang (2006). The eight amino acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea, Mol. Plant-Microbe Interact.19(11):1216-28.
• Qu SH, Liu GF, Zhou B, Bellizzi M, Zeng LR, Dai LY, Han H and Wang GL. 2006. The Broad-Spectrum Blast Resistance Gene Pi9 Encodes an NBS-LRR Protein and is a Member of a Multigene Family in Rice. Genetics 172: 1901-1914.

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

Outputs
A. Cloning of the Pi2 and Piz-t genes that confer broad-spectrum resistance to Magnaporthe grisea: To understand the molecular basis of broad-spectrum resistance to M. grisea, we first cloned the broad-spectrum resistance gene Pi9 via a map-based cloning strategy. Recently, we cloned the Pi2 and Piz-t genes using multi-faceted approaches. First, we mapped the Pi2 gene in a gene cluster that is allelic to the Pi9 locus and contains nine NBS-LRR gene homologs named Nbs1-Pi2 to Nbs9-Pi2. Second, we characterized the susceptible mutants derived from the Pi2 isogenic line C101A51 and found that the loss of the resistance of the mutants resulted from a two-nucleotide deletion in the Nbs4-Pi2 coding region, indicating that Nbs4-Pi2 is the single candidate for the Pi2 gene. Finally, with gene complementation tests, we confirmed that Nbs4-Pi2 is Pi2. Using PCR primers derived from the Pi9 and Pi2 loci, two candidate genes were amplified from the Piz-t donor line. Complementation experiments indicated that Nbs4-Piz-t is Piz-t. Pi2 and Piz-t encode a 1,032-amino acid and a 1,033-amino acid protein product, respectively. B. Eight amino acid differences likely determine the resistance specificity of the Pi2 and Piz-t genes. Comparison between the protein products of the Pi2 and Piz-t genes revealed a striking finding, i.e., that only eight amino acid residues distinguish Pi2 from Piz-t in three locations in the overall protein sequence. One region contains six variants and one amino acid insertion in Piz-t compared with Pi2. The other two locations carry a single amino acid change each. The three locations were confined within three consecutive LRRs: LRR11-LRR13, suggesting that the distinctive resistance specificities conferred by Pi2 and Piz-t are probably attributed to the variation in their LRR domain. Only a single amino acid change (838Arg in Pi2 and 839Ser in Piz-t) is within the xxLxLxx motif in LRR13, whereas the other seven are located outside the xxLxLxx motif in LRR11 and LRR12. C. Intra- and inter-haplotype comparative analyses of the Pi2/9/zt locus. Comparative analysis of the sequences in the five different rice haplotypes showed that the gene members at the corresponding genomic positions from different haplotypes are extremely conserved in their sequence and structure, suggesting an obvious orthologous relationship at the Pi2/9/zt locus. In contrast, we found that a considerable sequence variation exists within a single haplotype at the same locus. The orthologues show extreme sequence similarity (over 98% similarity in nucleotides overall) not only in the coding regions but also in the non-coding regions. The same case was observed in other orthologues/alleles at the Pi2/9/zt locus. The highly conserved feature of the genic regions was extended to most of the intergenic regions at the locus. These findings indicate that the Pi2/9/zt locus has maintained an obvious orthologous/allelic relationship during the course of evolution, in which sequence and structure are more conserved between the orthologues in different rice haplotypes than that between the paralogues within a single haplotype.

Impacts
Rice is not only the most important food crop in the developing world but is also a model monocot plant for functional genomics research due to its small genome size, extensive genetic map, available genome sequence, and relative ease of transformation. Successful characterization of the Pi9, Pi2 and Piz-t genes to rice blast in the project will lead to a better understanding of the molecular basis of the broad-spectrum resistance in rice. Some of the findings about the Pi9, Pi2, and Piz-t loci are unique and novel to most crop plants. Therefore, our on-going experiments will greatly advance understanding of the function of NBS-LRR genes in disease resistance in rice and other crop plants in the US. At present, plant breeders are restricted to the resistance genes available in germplasm collections. The information obtained from this project will facilitate the design of new plant resistance genes with desired specificities for effective control of plant diseases and significant reduction of the use of environmentally damaging pesticides in the US.

Publications

• Z. Wang, L.Dai, Z. Jiang, W. Peng, L. Zhang, GL. Wang, D. Xie. (2005) GmCOI1, a soybean F-box protein gene, shows ability to mediate jasmonate-regulated plant defense and fertility in Arabidopsis. Molecular Plant-Microbe Interactions, 18:1285-1295.
• Gu K, Yang B, Tian D, Wu L, Wang D, Sreekala C, Yang F, Chu Z, Wang GL, White F, Yin Z. (2005) Type III effector-induced R gene expression triggers disease resistance in rice. Nature, 435:1122-1125.
• Jantasuriyarat C, Gowda M, Haller K, Hatfield J, Lu G, Stahlberg S, Zhou B, Li H, Kim HR, Yu YS, Dean RA, Wing RA, Soderlund C and GL Wang (2005). Large-scale identification of ESTs involved in rice and rice blast (Magnaporthe grisea) interaction. Plant Physiology, 138(1):105-15.
• Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MR, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat VJ, Bruskiewich R, Wang GL, Leach J, Khush G, Leung H. (2005) Chemical- and Irradiation-induced Mutants of Indica Rice IR64 for Forward and Reverse Genetics. Plant Mol Biol. 2005 59(1):85-97.

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

Outputs
A. Molecular analysis of the broad-spectrum resistance gene Pi9. To understand the molecular basis of broad-spectrum resistance to rice blast, we cloned the broad-spectrum resistance gene Pi9, which was introgressed from the wild rice Oryza minuta. After sequencing a 76 kb fragment in the contig, six resistance candidate genes with nucleotide binding sites (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 confirmed that NBS2 is Pi9. cDNA clone of the NBS2 gene has been recently cloned and detection of its expression pattern during infection was detected. A manuscript describing the Pi9 cloning and its expression analysis is in revision for publication in Plant Journal. The cloned resistance gene Pi9 not only provides us with a starting point to dissect disease resistance pathway in rice but also an excellent tool to engineer broad-spectrum resistant rice cultivars. B. Lesion mimic mutation and disease resistance. Lesion mimic mutations are characterized by formation of necrotic lesions in the absence of biotic and abiotic stresses. These mutants are excellent materials for studying the relationship between programmed cell death or hypersensitive response (HR) and disease resistance without pathogen inoculation. In 2004, we cloned the Spl11 gene via a map-based cloning strategy. Sequence analysis indicated that Spl11 encodes a novel U-box and ARM repeat-containing protein. To the best of our knowledge, this is the first demonstration of the U-box and ARM repeat gene's involvement in cell death and defense signaling pathway. The paper on the gene cloning and characterization of the Spl11 gene was published in Plant Cell C. Large-scale identification of defense response genes involved in the interaction between rice and Magnaporthe grisea. To have a deeper analysis of the gene expression during rice and blast interaction, we have made 6 LongSAGE libraries using RNA isolated from infected and uninfected leaf tissues. Comparison analysis of the unique tags in each library revealed that transcriptomes of resistant and susceptible ractions are completely different with each other and with that of the uninoculated control. The matching rate of the unique tags in the resistant and susceptible libraries to both EST and genomic sequences was significantly lower than that of unique tags from the control library, suggesting a possible RNA editing mechanism on rice transcripts during the rice blast infection. Interestingly, a total of 5,222 antiseses tags were obtained when matching to the TIGR EST collection. Among them, majority of the tags were highly suppressed and few are highly induced in the infected libraries.

Impacts
Rice is not only the most important food crop in the world but also has become a model monocot plant for functional genomics research due to its small genome size, extensive genetic map, available genome sequence, and relative ease of transformation. Comparative mapping studies have shown that most cereal genomes share both a similar gene content and long stretches of co-linear gene order. Successful characterization of interested genes in the project will lead to a better understanding of the molecular basis of disease resistance in rice. The new information from this project may lead to the development of novel strategies to develop highly and durably resistant varieties of rice and crop plants in the US.

Publications

• Zeng LR, Qu S, Bordeos A, Yang C, Baraoidan M, Yan H, Xie Q, Nahm BH, Leung H, and GL Wang. (2004). Spl11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/ARM Repeat Protein Endowed with E3 Ubiquitin Ligase Activity. Plant Cell, 16, 2795-2808
• Gowda M, Jantasuriyarat C, Dean R., Wang GL (2004) A robust-longSAGE method for large-scale gene discovery and transcriptome profiling. Plant Physiology, 134:890-7.
• 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.

Progress 01/01/03 to 12/31/03

Outputs
A. Molecular analysis of the broad-spectrum resistance gene Pi9(t): Rice blast, caused by the fungus Magnaporthe grisea, is the most devastating disease of rice worldwide. To understand the molecular basis of broad-spectrum resistance to rice blast, we cloned the resistance gene Pi9(t). After sequencing a 76 kb fragment in the locus, six resistance candidate genes with nucleotide binding sites (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 confirmed that NBS2 is Pi9(t). cDNA clone of the NBS2 gene has been recently cloned and detection of its expression pattern during infection is progress. The cloned resistance gene Pi9(t) not only provides us with a starting point to dissect disease resistance pathway but also an excellent tool to engineer new resistant cultivars. B. MAP kinase pathway and disease resistance: The activation of the mitogen-activated protein (MAP) kinases by different environmental stresses has been previously observed in several dicot plant species. To study the temporal and spatial expression patterns of BWMK1, its promoter and GUS fusion construct was made and transformed into rice. GUS expression during different developmental stages and under different stress conditions was monitored. Results showed that BWMK1 is induced by cold, drought, heat and jasmonic acid but suppressed by salicylic acid. To identify the function of BWMK1 in disease resistance, over- and under-expression transgenic lines were produced. Interestingly, under-expression lines conferred enhanced resistance to both rice blast and bacterial blight pathogens, suggesting a negative role of BWMK1 in disease resistance in rice. C. Large-scale identification of defense response genes involved in the interaction between rice and rice blast: (Funded by NSF-Plant Genome Program): Host resistance is the most effective way to control the rice blast disease. To understand the molecular basis of host resistance to rice blast, an EST sequencing approach is being used to profile gene expression at early infection stages. Using RNA isolated from blast infected-tissues, 8 cDNA libraries were constructed. About 7,000 cDNA clones from each library were randomly picked and were sequenced from both ends. Sequences from a total of 69,137 ESTs were generated and submitted to the Genbank. Clustering and assembly of these ESTs resulted in a total of 22,857 unique sequences. To have a deeper analysis of the gene expression during rice and blast interaction, we have made 6 LongSAGE libraries using RNA isolated from infected and uninfected leaf tissues. Preliminary analysis indicated that about 67 % of the tags matched to rice ESTs exactly in the NCBI database and the other 33% did not match any known or predicted rice genes. These results indicate the LongSAGE method is useful in gene expression analysis and many previously uncharacterized rice defense genes could be identified.

Impacts
Rice, one of the most important food crops, is also 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 important pathogens. This study will lead to identification of novel genes governing disease resistance and creation of transgenic rice lines with enhanced disease resistance. Some of the mutants with high level of resistance generated from the project can be directly used in rice breeding programs. PCR primers based on the sequences of important defense genes can be used in marker-aided selection to accelerate disease resistance breeding in the US and other countries.

Publications

• Gu K, Tian D, Yang F, Wu L, Chellamma S, Wang D, Wang GL, Yin Z (2003). Fine mapping of Xa31, a new rice resistance gene introgressed from wild species Oryza minuta and conferring broad-spectrum resistance to Xanthomonas oryzae pv oryzae. Theoretical and Applied Genetics, Theoretical and Applied Genetics, In press.
• Wang GL, Wu C, Zeng L, He C, Baraoidan M, William CE, Ronald P, Leung H (2003) Isolation and characterization of rice mutants compromised in the Xa21-mediated resistance to Xamthomonas oryzae pv oryzae. Theoretical and Applied Genetics, in press
• Lu G, Jantasuriyarat C, Zhou B, Wang GL (2003). Isolation and characterization of novel defense response genes involved in compatible and incompatible interactions between rice and Magnaporthe grisea. Theoretical and Applied Genetics, in press.
• Qu S, Coaker G, Francis D, Zhou B, Wang GL (2003) Development of a new transformation-competent artificial chromosome (TAC) vector and construction of tomato and rice TAC libraries. Molecular Breeding, in press.
• Jeon JS, Chen D, Yi GH, Wang GL, Ronald PC (2003). Genetic and physical mapping of Pi5(t), a locus associated with broad-spectrum resistance to rice blast. Molecular Genetics and Genomics, 269(2):280-289.

Progress 01/01/02 to 12/31/02

Outputs
We have made significant progress in the following 4 projects in 2002: 1. LongSAGE Approach to a Comprehensive Expression Profiling of Rice and Magnaporthe grisea Interaction. LongSAGE is a new strategy for deep transcriptome analysis and genome annotation. To analyze the gene expression profiles during rice and blast interaction, six LongSAGE libraries are being made using RNA isolated from blast-infected and uninfected leaf tissues. In the first LongSAGE library, 7000 clones with an average insert size of 800 bp randomly picked are being sequenced. Analysis of these libraries will provide a comprehensive expression profiling during rice and rice blast interaction and identify many novel defense genes in rice. 2. Large-scale identification of defense response genes involved in the interaction between rice and Magnaporthe grisea. To identify defense genes up or down-regulated in the rice and blast interaction, a large-scale EST sequencing approach has been initiated. Eight cDNA libraries reflecting different aspects of the disease resistance were constructed. Up to now, six libraries have been sequenced, and three were analyzed and posted at AGI database. This large collection of rice defense ESTs will be a valuable resource for functional genomics in rice and other cereals. 3. Cloning and characterization of the broad-spectrum resistance gene Pi2(t) in rice. Rice blast, caused by the fungal pathogen Magnaporthe grisea, is the most devastating disease affecting rice production worldwide. Pi2(t) is one of the major genes with broad-spectrum resistance to the rice blast. To fine-map the gene on chromosome 6, DNA was extracted from over 500 susceptible F2 and F3 plants. Five primers pairs, based on the genomic sequence at the Pi9(t) region, were used in the screen. No recombinants between NBS2 and NBS4 were found in all screened plants. Five BAC clones spanning the Pi2(t) region were identified. At least six of NBS/LRR homologous genes were identified after partially sequencing the Pi2(t) BAC contig. All NBS/LRR genes are being transformed into a susceptible rice variety for complementation test. 4. Positional cloning and functional characterization of the rice lesion mimic gene, Spl11 To gain insight into the molecular mechanism underlying programmed cell death and disease resistance in rice, molecular isolation of the Spl11 gene and global gene expression profiling of the spl11 mutant are attempted in this study. High resolution genetic and physical mapping further narrow the gene down to a 40-kb DNA. Six genes are predicted in the 40-kb region by bioinformatic programs. Determining the sequence change of the mutant at the candidate gene loci and complementation test of the candidate genes are in progress. To have a better understanding of the impact of the spl11 mutation on rice global gene expression, transcriptome of the spl11 mutant and IR68 was performed using a 24,000-gene rice genechip. High percentage of known defense-related genes are induced significantly upon the appearance of lesions on the leaf, inferring the close connection of Spl11 to disease defense in rice.

Impacts
1. Identification and characterization of defense response genes to rice blast will provide us with insight in molecular mechanism of disease resistance in rice and other monocot plants. 2. Successful cloning of the Pi2 gene will lead to engineering of broad-spectrum resistant rice cultivars to rice blast.

Publications

• Lirong Zeng, Zhongchao Yin, Jun Chen, Hei Leung, Guo-Liang Wang.(2002) Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160 kb DNA segment of the rice genome, Molecular Genetics and Genomics, 268:253-261.
• Guifu Liu, Guodong Lu, Lirong Zeng, G. L. Wang. (2002) The two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), are physically linked on rice chromosome 6. Molecular Genetics and Genomics, 267:472-480.
• Zhai WX, Wang WM, Zhou YL, Li XB, Zheng XW, Zhang Q, G.L.Wang GL, Zhu LH. (2002) Breeding bacterial blight resistant hybrid rice with the cloned bacterial resistance gene Xa21. Molecular Breeding, 8(4):285-293.