Roles of SnRK1, ADK, and APT1 in the Cellular Stress Response and Antiviral Defense Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Gireesha T. Mohannath, M.S. Plant Cellular and Molecular Biology Graduate Program The Ohio State University 2010 Dissertation Committee: Dr. David M. Bisaro, Advisor Dr. Erich Grotewold Dr. Venkat Gopalan Dr. Jyan-Chyun Jang Copyright by Gireesha T. Mohannath 2010 ABSTRACT Members of the SNF1/AMPK/SnRK1 family of kinases are highly conserved. Representatives include SNF1 kinase (sucrose non-fermenting 1) in yeast, SnRK1 (SNF1-related kinase 1) in plants, and AMPK (AMP-activated protein kinase) in animals. These Ser/Thr kinases play a central role in the regulation of metabolism. In response to nutritional and environmental stresses that deplete ATP, they turn off energy-consuming biosynthetic pathways and turn on alternative ATP-generating systems as part of the cellular stress response (CSR). However, the mechanisms that activate these enzyme complexes are not completely understood. These kinases function as heterotrimeric complexes. The catalytic subunit consists of an N-terminal kinase domain with an activation loop that contains a conserved threonine residue which must be phosphorylated for activity. Following phosphorylation by upstream kinase(s), 5'-AMP is known to allosterically stimulate AMPK activity and to inhibit its inactivation due to dephosphorylation of subunit at Thr172 by protein phosphatase 2C (PP2C). Direct allosteric stimulation of the SnRK1 complex by 5'-AMP has yet to be demonstrated. However, 5'-AMP has been shown to suppress dephosphorylation of SnRK1 by PP2C. Adenosine kinase (ADK) phosphorylates adenosine to 5'-AMP. ADK and SnRK1 play key roles in antiviral defense, and geminivirus AL2 and L2 proteins inactivate both ii kinases. Because ADK generates 5'-AMP that is known to activate CSR, this study hypothesizes that ADK contributes to rapid activation of the CSR. In support of this hypothesis, we showed that ADK and SnRK1 form a complex in vivo. This study also demonstrates an increase in SnRK1 activity in transgenic Arabidopsis plants over-expressing ADK, and a 4-6 fold in vitro stimulation of ADK by SnRK1. Interestingly, ADK stimulation does not require SnRK1 kinase activity. We conclude that SnRK1 and ADK mutually stimulate each other to rapidly activate CSR. SnRK1 phosphorylates ADK in vitro, and we speculate that this phosphorylation might be playing a role in negative regulation of the SnRK1-ADK complex. Adenine phosphoribosyl transferase 1 (APT1) also generates 5'-AMP. We hypothesize that APT1 might also play a role in activating CSR. In vitro and in vivo data is presented in this thesis to demonstrate interaction between SnRK1 and APT1. Also, SnRK1 is shown to phosphorylate APT1 in vitro. We speculate that SnRK1, ADK, and APT1 might form a ternary complex that could play critical roles in detecting and responding to cellular stress. We also show that four different geminivirus proteins, AL2, L2, AV2, and C1 interact with SnRK1, ADK, and APT1, and discuss the possible consequences of these interactions. Finally, we hypothesize that, as an antiviral defense, SnRK1 might play a role in regulating protein synthesis, and in support of this idea we observed phosphorylation of translation initiation factors eIF-2 and eIF-(iso)4E by SnRK1 in vitro. Future experiments have been proposed to confirm this hypothesis. iii Taken together, the findings from this study further our understanding on the potential roles of SnRK1, in conjunction with ADK and APT1, in sensing and mediating responses to various kinds of biotic and abiotic stresses. iv Dedicated to my family and teachers v ACKNOWLEDGEMENTS Mathrudevobhava, Pithrudevobhava, and Gurudevobhava, (The mother, the father, and the teacher to be revered as God) I begin my acknowledgements by expressing the deepest gratitude to my advisor, Dr. David Bisaro, for his support, critical comments, and the invaluable knowledge I received from him during my tenure in his lab. I will be forever grateful to him for encouraging me to be innovative and exploratory. He greatly helped me to become a better scientist. I am sincerely grateful to my committee members Drs. Erich Grotewold, Venkat Gopalan and J.C. Jang for their time, unwavering support, guidance and advice. I have been fortunate to have these accomplished scientists as my committee members as their input into my research was very useful and has been highly valued. From bottom of my heart I thank my current and former lab colleagues Dr. Kenn Buckley, Dr. Cody Buchmann, Dr. Priya Raja, and Jamie Wolf, for their consistent moral and technical support throughout my tenure in the lab. Specifically, I cannot forget the technical help and advice I received from Drs. Buckley and Buchmann during my early days as a graduate student. I am immensely thankful to Veena Patil (my wife), Dr. Youn Lee, and Allie Varner for helping me in my project. I highly value their contributions to my projects. I would also like to acknowledge the other current and past members of the Bisaro lab who have been helpful in various ways: Xiaojuan Yang, Dr. vi Hui Wang, Dr. Lin Hui, Dr. Shaheen Asad, Dr. Mohammed Mubeen, Isaac Heard, Sizhun Lee, Jeff Ostler, Brad Sanville, and other undergraduate students. Definitely I won‘t forget the help and the various constructive conversations I had with students, postdocs, and faculty members from different departments especially from Molecular Genetics/Plant Cellular and Molecular Biology (PCMB). I am also thankful to current PCMB staff: Eduardo Acosta, Rene Reese, Laurel Shannon, and Joan Leonard for their help. A special acknowledgement to Dr. Biao Ding for his permission to use his confocal and fluorescent microscopes. Also, my sincere thanks to the staff of the biotechnology center: Melinda Parker, Diane Furtney, Dave Long, Scott Hines, Joe Takayama and MCDB secretary, Jan Zinich for all of their help, and I offer my sincere acknowledgements to ABRC for their Arabidopsis mutants and DNA clones. I owe a great debt of gratitude to all of my family members: My mother, father, brother, sister, and their family members for their kind and compassionate support throughout my career. Without their support I wound not have made it U.S. for my education. Although for my efforts I am the only person who receives Ph.D., my wife is an exemplary partner in all of my efforts and I share with her whatever I receive. It would have been impossible to pursue my doctoral studies but for my wife‘s moral support, tolerance, understanding, and help. Many of my teachers, friends and relatives, in conjunction with my family, gave me the strength to propel myself forward through the most difficult and trying times of my graduate career. I don‘t know how I would have managed without their support. vii I am extremely grateful to PCMB and CLSE for funding me every quarter throughout my Ph.D. years by employing me as a graduate teaching associate. I think because of their support I became a better teacher and received ‗Molecular Genetics Teaching Award‘. Finally, I bow with reverence to The Ohio State University. Go Bucks! viii VITA 1993 – 1997………………. B.S., Agricultural Sciences, UAS, Dharwad, India 1997 – 1999………………. M.S., Genetics & Plant Breeding, UAS, Bangalore, India 1999 – 2003………………. Research Associate, UAS, Bangalore, India 2003 – present……………... Graduate Teaching and Research Associate, the Ohio State University. (UAS: University of Agricultural Sciences) PUBLICATIONS Research Publications: 1. Buchmann, R.C., Asad, S., Wolf, J.N., Mohannath, G., and Bisaro, D.M. (2009). Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation. J. Virol. 83(10): 5005-5013) (featured in the “Spotlight” section of the journal) 2. Girish, T.N., Gireesha, T.M. Vaishali, M.G., Hanamareddy, B.G., and Hittalmani, S. (2006). Response of a new IR50/Moroberekan recombinant inbred population of rice (Oryza sativa L.) from an indica x japonica cross for growth and yield traits under aerobic conditions. Euphytica 152: 149-161. 3. Nadaradjan, S., Gireesha, T. M., Sheshashayee, M. S., Shankar, A. G., Prasad, T. G., and Udayakumar, M. (2003). Progress in genetic polymorphism studies via molecular markers in groundnut (Arachis hypogaea L.) - A review. J. Plant Biol. 30 (3): 285 – 292. ix 4. Toorchi, M., Shashidhar, H.E., Gireesha, T.M., and Hittalmani, S. (2003). Performance of backcross transgressant doubled haploid lines of rice under contrasting moisture regimes: Yield components and Marker heterozygosity. Crop Science 43:1448-1456. 5. Toorchi, M., Shashidhar, H.E., Hittalmani, S., and Gireesha, T.M. (2002). Rice root morphology under contrasting moisture regimes and contribution of molecular marker heterozygosity. Euphytica 126(2): 251-257. 6. Gireesha, T. M., Shashidhar, H. E., and Hittalmani, S. (2000), Genetics of root morphology and related traits in an indica-indica based mapping populations of rice (Oryza sativa L.). Res. on Crops 1(2): 208-215. FIELDS OF STUDY Major Field: Plant Cellular and Molecular Biology x TABLE OF CONTENTS Abstract ..................................................................... ……………………………………..ii Dedication ............................................................................................................................v Acknowledgements .........................................................................................................