Progress 01/15/10 to 01/14/14
Outputs
Target Audience: - The research community studying the interaction of Pseudomonas syringae and other bacteria with plants. - Scientists that are breeding plants for increased resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Funding for these studies in the 4 years have allowed for training of 4 graduate students, Anna Nelson, Julian Avila, Joel Gray, and Dongyin Su. These students were trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. They were also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. How have the results been disseminated to communities of interest? Over the past 4 years our studies on Adi3 cell death control and role in resistance to P. syringae have been disseminated to the general scientific public through nine publications in scientific journals and five abstract/poster/oral presentations at international plant science 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?
Our research for the total period of this grant was able to make significant advances in understanding how Adi3 controls cell death and how this cell death is regulated during resistance to P. syringae. We found that under normal conditions, Adi3 is found in the nucleus in order to suppress cell death and that Adi3 travels from the cell membrane to the nucleus via retrograde transport on endocytic vesicles. During the interaction of the tomato host resistance protein Pto with the P. syringae effector protein AvrPto, Adi3 is not allowed to enter the nucleus by retaining Adi3 in the endocytic vesicles. Thus, Adi3 can no longer suppress cell death and this leads to the cell death associated with resistance to P. syringae. We have also identified and characterized the interaction of Adi3 with several other proteins that lend insight into how Adi3 controls PCD in the presence and absence of P. syringae. We have found that Adi3 interacts with the protein Atg8h, which is involved in the regulation of autophagy. Autophagy is a process in which cellular contents (proteins, organelles) are recycled by engulfment in a membrane followed by transport to the vacuole where the items are broken down for reuse. Autophagy is induced as part of the cell death associated with the resistance responses to pathogens. We found that cell death control by Adi3 is also associated with autophagy through co-silencing of Adi3 and Atg8h using virus induced gene silencing. We have found that Adi3 interacts with the highly conserved eukaryotic SnRK1 protein complex, which is a major regulator of stress responses. Adi3 phosphorylates Gal83, the beta subunit of the SnRK1 complex. We have identified the Adi3 phosphorylation site on Gal83 as Ser26. We have also shown that overexpression of Gal83 is capable of suppressing cell death in a manner similar to Adi3. The phosphorylation of Gal83 by adi3 was shown to inhibit the kinase activity of the SnRK1 complex itself as well as control the cellular localization of the SnRK1 complex. Given the role of Adi3 in suppressing cell death and that a loss of Adi3 activity during the resistance response to P. syringae, we hypothesize that under non-pathogen conditions, Adi3 keeps SnRK1 inactive through phosphorylation. In response to P. syringae, Adi3 is inactivated and thus can no longer suppress SnRK1, which can then reallocate nutrients during the cell death associated with resistance to P. syringae. We have shown that Adi3 is ubiquitinated by AdBiL suggesting degradation of Adi3 as a possible mechanism for controlling cell death in response to P. syringae. We showed that Adi3 is indeed ubiquitinated by AdBiL and that Adi3 protein is degraded in a ubiquitin dependent manner. Additionally, we identified a region within the Adi3 protein sequence that may be a target for the degradation mediated by the proteasome or possibly other proteases such as metacaspases. Additional studies include the production of a mutant version of Adi3 that can specifically utilize bulky versions of ATP. We have shown that this version of Adi3 can phosphorylate a substrate, the beta-subunit of SnRK1, with bulky ATP to the same extent as the wild-type Adi3 using normal ATP. Thus, we can use the mutant version of Adi3 in an activity-based screen of tomato plant protein extracts to identify additional Adi3 substrates involved in cell death regulation during resistance to P. syringae. We have also identified an additional phosphorylation site within Adi3 that is important for phosphorylation of substrates. We have previously shown that the protein kinase Pdk1 is the activating kinase for Adi3. Pdk1 activates Adi3 by phosphorylating serine 539 and then Adi3 can phosphorylate substrates. We also know that Pdk1 phosphorylates one additional site on Adi3, but we did not know the role of this additional phosphorylation site. We have now identified this second Pdk1 phosphorylation site on Adi3 using mass spectrometry as serine 212. While phosphorylation of S212 does not affect the autophosphorylation of Adi3, it does contribute to the ability of Adi3 to phosphorylate substrate proteins. We also showed that phosphorylation of S539 and S212 by Pdk1 is sufficient for Adi3 to fully phosphorylate its substrates. Thus, we have identified all the important Pdk1 phosphorylation sites on Adi3 that are required for substrate phosphorylation and thus cell death control during pathogen resistance. Through collaborative studies we have also contributed research to the finding that the plant FLS2 receptor protein that recognizes P. syringae is ubiquitinated to direct its degradation and thus attenuate its function during resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Ek-Ramos MJ, Avila J, Cheng C, Martin GB, Devarenne TP (2010). The T-loop extension of the tomato protein kinase AvrPto-dependent Pto-interacting protein 3 (Adi3) directs nuclear localization for suppression of plant cell death. J. Biol. Chem. 285:17584-17594.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Devarenne TP (2011) The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h. Biochem. Biophys. Res. Commun. 412:699-703.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Lu D, Lin W, Wu S, Gao X, Cheng C, Avila J, Heese A, Devarenne TP, He P, Shan L (2011) Ubiquitination of the pattern-recognition receptor FLAGELLIN-SENSING 2 in plant innate immunity. Science. 332:1439-1442.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Nelson Dittrich AC, Devarenne TP (2012) An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification. Biochim Biophys Acta. 1824:269-273.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Nelson Dittrich AC, Devarenne TP (2012) Characterization of a PDK1 homologue from the moss Physcomitrella patens. Plant Physiol. 158:1018-1033.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Nelson Dittrich AC, Devarenne TP (2012) Perspectives in PDK1 evolution: insights from photosynthetic and non-photosynthetic organisms. Plant Sig & Behav. 7:642-649.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Avila J, Gregory OG, Su D, Deeter TA, Chen S, Silva-Sanchez C, Xu S, Martin GB, Devarenne TP (2012) The ?-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3. Plant Physiol. 159:1277-1290.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Avila J and Devarenne TP (2013) Ubiquitination of the tomato cell death suppressor Adi3 by the RING E3 ubiquitin ligase AdBiL. Biochem. Biophys. Res. Commun. 430:119-124.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Gray JW, Nelson Dittrich AC, Chen S, Avila J, Giavalisco P, Devarenne TP (2013) Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation. Biochim Biophys Acta - Proteins Proteom. 1834:1099-1106.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Avila J and Devarenne TP (2011) The ?-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3. Abstract for Plant Biology 2010, annual meeting of the American Society of Plant Biologists, Minneapolis, MN. August, 2011.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Nelson Dittrich AC and Devarenne TP (2011) 3-phosphoinositide-dependent protein kinase-1 (PDK1) in the moss Physcomitrella patens. Abstract for Plant Biology 2010, annual meeting of the American Society of Plant Biologists, Minneapolis, MN. August, 2011.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Avila J and Devarenne TP (2012) Cellular localization control of the SnRK1 ?-subunit Gal83 through phosphorylation by the cell death suppressor Adi3. Abstract for Plant Biology 2012, annual meeting of the American Society of Plant Biologists, Austin, TX. July 20-24, 2012.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Gray J and Devarenne TP (2012) Phosphorylation sites on the Arabidopsis AGC protein kinase AGC1-3 have opposing roles in cell death suppression. Abstract for Plant Biology 2012, annual meeting of the American Society of Plant Biologists, Austin, TX. July 20-24, 2012.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Su D and Devarenne TP (2013) Regulation of SnRK complex kinase activity by differential ?-subunit interaction and phosphorylation. Abstract for Plant Biology 2013, annual meeting of the American Society of Plant Biologists, Providence, RI. July 20-24, 2013.
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2012
Citation:
ACTIVITY AND REGULATION OF AGC KINASES FROM Physcomitrella patens AND TOMATO
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2012
Citation:
UNDERSTANDING POSTRANSLATIONAL MODIFICATIONS INVOLVED IN ADI3 PROGRAMMED CELL DEATH SIGNALING
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2013
Citation:
NON-ACTIVATION LOOP PHOSPHORYLATION AND DOWNSTREAM SIGNALING OF AGC1-3, THE ARABIDOPSIS THALIANA HOMOLOGUE OF THE TOMATO CELL DEATH SUPPRESSOR, ADI3
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Progress 01/15/12 to 01/14/13
Outputs
OUTPUTS: We have previously shown that the tomato serine/threonine protein kinase Adi3 functions as a suppressor of cell death and is involved in regulation of cell death associated with resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). In the past year we have made progress in studying the proteins that Adi3 interacts with to suppress cell death and how these interactions may be affected to bring about cell death associated with Pst resistance. We have found that Adi3 interacts with the SnRK1 protein complex, which is known to regulate gene expression and enzymatic activity associated with metabolism pathways. This complex also regulates the redistribution of carbon resources during the plant response to pathogen attack. In this manner, pathogen access to valuable sources of carbon can be limited by movement away from the site of attack. Another protein that we found to interact with Adi3 is the ubiquitin E3 ligase termed AdBiL. Ubiquitin is a small protein that can be added to target proteins to regulate the degradation, cell localization, or activity of the target protein. An E3 ligase is the enzyme that adds the ubiquitin to the target protein. We have also engineered a mutant version of Adi3 that can be used for identification of substrates involved in plant cell death. This version of Adi3 contains a mutation that allows Adi3 to specifically utilize bulky ATP analogs that can not be used by non-mutated kinases. Thus, the mutant Adi3 can be use to specifically phosphorylate substrates from a protein extract. Our studies on Adi3 interacting proteins have been disseminated to the general scientific public through three publications in scientific journals and abstract/poster presentations at scientific meetings. PARTICIPANTS: PI Timothy P. Devarenne Graduate student Julian Avila Graduate student Joel Gray Graduate student Anna C. Nelson Dittrich Collaborators: Greg Martin, Scientist, Boyce Thompson Institute for Plant Research, Professor, Department of Plant Pathology and Plant-Microbe Biology, Cornell University; Sixue Chen, Department of Biology, Interdisciplinary Center for Biotechnology Research, University of Florida. TARGET AUDIENCES: - The research community studying the interaction of Pseudomonas syringae with plants. - Scientists that are breeding plants for increased resistance to pathogens PROJECT MODIFICATIONS: There have been no modifications at this time.
Impacts
The focus of these studies is to determine how host cell death is induced during the resistance response to the tomato pathogen Pseudomonas syringae pv. tomato (Pst). The research for this study is is geared toward determining signaling pathways involved in regulating plant PCD and how these pathways are manipulated during plant-pathogen interactions. Understanding these cellular mechanisms will help lead to the development of plants with enhanced resistance to pathogens. Given the role of Aid3 in controlling cell death associated with resistance to Pst, our findings of Adi3 interacting proteins adds additional valuable information to how host cell death is regulated in response to pathogens. This information has thus generated host target genes that can be manipulated through engineering to produce plants that will not only offer better pathogen resistance, but also increased life span and improved overall plant health. In the current reporting period (2012) we have found that Adi3 phosphorylates the beta-subunit of the SnRK1 complex. This phosphorylation event was shown to inhibit the kinase activity of the SnRK1 complex itself. Given the role of Adi3 in suppressing cell death and that a loss of Adi3 activity during the resistance response to Pst, we hypothesize that under non-pathogen conditions, Adi3 keeps SnRK1 inactive through phosphorylation. In response to Pst, Adi3 is inactivated and thus can no longer suppress SnRK1, which can then reallocate nutrients during the cell death associated with resistance to Pst. We also found that the ubiquitin E3 ligase AdBiL can ubiquitinate Adi3, which leads to proteasomal-dependent degradation of Adi3. Thus, a loss of Adi3 through degradation during the resistance response to Pst would lead to the induction of cell death through a loss of Adi3 cell death suppression. Additional outcomes include the production of a mutant version of Adi3 that can specifically utilize bulky versions of ATP. We have shown that this version of Adi3 can phosphorylate a substrate, the beta-subunit of SnRK1, with bulky ATP to the same extent as the wild-type Adi3 using normal ATP. Thus, we can use the mutant version of Adi3 in an activity-based screen of tomato plant protein extracts to identify additional Adi3 substrates involved in cell death regulation during resistance to Pst. All three of these findings have helped to achieve the project goals of gaining a better understanding of how Adi3 functions in regulating cell death associated with resistance to Pst.
Publications
- Nelson Dittrich AC, Devarenne TP 2012 An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification. Biochim Biophys Acta. 1824:269-273. Avila J, Gregory OG, Su D, Deeter TA, Chen S, Silva-Sanchez C, Xu S, Martin GB, Devarenne TP (2012) The -subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3. Plant Physiol. 159:1277-1290.
- Avila J and Devarenne TP (2012) Cellular localization control of the SnRK1 -subunit Gal83 through phosphorylation by the cell death suppressor Adi3. Abstract for Plant Biology 2012, annual meeting of the American Society of Plant Biologists, Austin, TX. July 20-24, 2012.
- Gray J and Devarenne TP (2012) Phosphorylation sites on the Arabidopsis AGC protein kinase AGC1-3 have opposing roles in cell death suppression. Abstract for Plant Biology 2012, annual meeting of the American Society of Plant Biologists, Austin, TX. July 20-24, 2012.
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Progress 01/15/11 to 01/14/12
Outputs
OUTPUTS: We have previously shown that the tomato serine/threonine protein kinase Adi3 functions as a suppressor of cell death and is involved in regulation of cell death associated with resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). In the past year we have made progress in studying the the proteins that Adi3 interacts with to suppress cell death and how these interactions may be affected to bring about cell death associated with Pst resistance. We have found that Adi3 interacts with the protein Atg8h, which is involved in the regulation of autophagy. Autophagy is a process in which cellular contents (proteins, organelles) are recycled by engulfment in a membrane followed by transport to the vacuole where the items are broken down for reuse. Autophagy is induced as part of the cell death associated with the resistance responses to pathogens. Thus, we are studying the significance of the Adi3/Atg8h interaction. We have also engineered a mutant version of Adi3 that can be used for identification of substrates involved in plant cell death. This version of Adi3 contains a mutation that allows Adi3 to specifically utilize bulky ATP analogs that can not be used by non-mutated kinases. Thus, the mutant Adi3 can be use to specifically phosphorylate substrates from a protein extract. Through collaborative studies we have also contributed research to the finding that the plant FLS2 receptor protein that recognizes Pst is ubiquitinated to direct its degradation and thus attenuate its function during resistance. These studies have been reported in three publications. PARTICIPANTS: PI Timothy P. Devarenne Graduate student Julian Avila Graduate student Anna Nelson Dittrich Collaborators Ping He, Assistant Professor, Department of Biochemistry & Biophysics, Texas A&M University; Libo Shan, Assistant Professor, Department of Plant Pathology & Microbiology. TARGET AUDIENCES: The research community studying the interaction of Pseudomonas syringae with plants. Scientists that are breeding plants for increased resistance to pathogens. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The focus of this grant is to study how host cell death is induced during the resistance response to the tomato pathogen Pseudomonas syringae pv. tomato. The research for this study is is geared toward determining signaling pathways involved in regulating plant PCD and how these pathways are manipulated during plant-pathogen interactions. Understanding these cellular mechanisms will help lead to the development of plants with enhanced resistance to pathogens. Given the role of Aid3 in controlling cell death associated with resistance to Pst, our findings of Adi3 interacting proteins adds additional valuable information to how host cell death is regulated in response to pathogens. This information has thus generated host target genes that can be manipulated through engineering to produce plants that will not only offer better pathogen resistance, but also increased life span and improved plant health overall.
Publications
- Nelson Dittrich AC, Devarenne TP (2012) An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification. Biochim Biophys Acta. DOI:10.1016/j.bbapap.2011.10.004. (In Press. Published online October 17, 2011).
- Devarenne TP (2011) The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h. Biochem. Biophys. Res. Commun. 412:699-703. Lu D, Lin W, Wu S, Gao X, Cheng C, Avila J, Heese A, Devarenne TP, He P, Shan L (2011) Ubiquitination of the pattern-recognition receptor FLAGELLIN-SENSING 2 in plant innate immunity. Science. 332:1439-1442.
- Avila J and Devarenne TP (2011) The β-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3. Abstract for Plant Biology 2010, annual meeting of the American Society of Plant Biologists, Minneapolis, MN. August, 2011.
- Nelson Dittrich AC and Devarenne TP (2011) 3-phosphoinositide-dependent protein kinase-1 (PDK1) in the moss Physcomitrella patens. Abstract for Plant Biology 2010, annual meeting of the American Society of Plant Biologists, Minneapolis, MN. August, 2011.
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Progress 01/15/10 to 01/14/11
Outputs
OUTPUTS: We have previously shown that the tomato serine/threonine protein kinase Adi3 functions as a suppressor of cell death and is involved in regulation of cell death associated with resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). In the past year we have made progress in studying the the proteins that Adi3 interacts with to suppress cell death and how these interactions may be affected to bring about cell death associated with Pst resistance. We have found that Adi3 interacts with the highly conserved eukaryotic SnRK1 protein complex, which is a major regulator of stress responses. Adi3 phosphorylates Gal83, the beta subunit of the SnRK1 complex. We have identified the Adi3 phosphorylation site on Gal83 as Ser26. We have also shown that Gal83 is a functional SnRK1 beta subunit since it can complement a yeast Gal83 knockout and restore yeast growth on sucrose as well as invertase activity. We have also shown that overexpression of Gal83 is capable of suppressing cell death in a manner similar to Adi3. We have initiated studies to see how Adi3 phosphorylation of Adi3 affects Gal83 cell death control and how the presence of Pst may interrupt this interaction to bring about cell death. We have also been studying the interaction of Adi3 with the E3 ubiquitin ligase Dnp2. We have shown that Adi3 is ubiquitinated by Dnp2 suggesting degradation of Adi3 as a possible mechanism for controlling cell death in response to Pst. PARTICIPANTS: Tim Devarenne TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The focus of this grant is to study how host cell death is induced during the resistance response to the tomato pathogen Pseudomonas syringae pv. tomato. We have previously identified the tomato protein kinase Adi3 as a suppressor of host cell death that interacts with the tomato resistance protein Pto and the Pst effector protein AvrPto. Under normal conditions, Adi3 is found in the nucleus in order to suppress cell death. In the presence of Pto/AvrPto, Adi3 is not allowed to enter the nucleus and can no longer suppress cell death, leading to the cell death associated with resistance to Pst. Thus, our research is geared toward determining signaling pathways involved in regulating plant PCD and how these pathways are manipulated during plant-pathogen interactions. Understanding these cellular mechanisms will help lead to the development of plants with enhanced resistance to pathogens.
Publications
- Ek-Ramos, M.J., Avila, J., Cheng, C., Martin, G.B., Devarenne, T.P. 2010. The T-loop extension of the tomato protein kinase AvrPto-dependent Pto-interacting protein 3 (Adi3) directs nuclear localization for suppression of plant cell death. J. Biol. Chem. 285:17584-17594.
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