Final Report Summary - PLANTPCDCONTROL (Control of Programmed Cell Death by Metacaspases in plants)
The IIF “Control of Programmed Cell Death by Metacaspases in plants (PlantPCDcontrol)” has allowed me to bring my line of research on plant programmed cell death, started as a postdoc in the USA, to my home country in Europe, Spain, establishing my own research group. During these 2 years of funding, PlantPCDcontrol has allowed me to move forward the frontiers of knowledge on the mechanisms governing HR and developmental plant programmed cell death (PCD), contributing to European research in this area. I have joined efforts with leading European teams that investigate different aspects of plant PCD with research that is complementary to theirs and have created a plant PCD network active with collaborations.
1.1 PROJECT OBJECTIVES
The main goal of this project was to understand the mode of action of type I metacaspases during PCD, with particular focus on the pathogen-triggered hypersensitive response cell death. For this we have used parallel genetics, biochemical, proteomic and cell biology approaches in the model plant Arabidopsis thaliana. The specific objectives of this project were the following:
A) Define the molecular mechanisms underlying HR cell death control by type I metacaspases in Arabidopsis.
B) Determine the role of AtMC3, an atypical type I metacaspase, during developmental and pathogen-triggered PCD in Arabidopsis.
1.2 WORK PERFORMED and MAIN RESULTS
A) Define the molecular mechanisms underlying HR cell death control by type I metacaspases in Arabidopsis.
In the past we have been able to identify a number of pathogen-triggered cell death regulators (Coll et al., 2010). Using immunoaffinity purification we have been able to isolate and characterize the first native, cell death-inducible complex: an AtMC1-containing supramolecular complexes that dynamically respond to PCD. In resting conditions, these complex contain a protease inhibitor that keeps AtMC1 repressed, preventing cell death to be initiated. After PCD is triggered, the AtMC1-containing complexes change composition, and bind, among other cell death regulators, a predicted downstream metacaspase. These results are a major breakthrough in biology, as for the first time evidence of such a cell death cascade in plants is provided, significantly moving forward the knowledge in plant defense pathways. These results are in preparation to be submitted. In addition, we have found a negative regulator of AtMC1, the suicide protease inhibitor AtSerpin1, which binds and inhibits AtMC1 activity. The inhibition of AtMC1 by AtSerpin1 resembles the mode of action of cowpox serpin CmrA blocking the immune caspase-1 in mammals, bringing forward extremely interesting evolutionary parallels between such distant systems as animals and plants. This work is under second revision in the high-impact plant journal New Phytologist (Lema et al., New Phytologist, last and corresponding author).
To complement protein complex data with the downstream proteolytic events taking place during pathogen-triggered cell death, we have analyzed the AtMC1 degradome. Defining which substrates do cell death proteases cleave (degradomes) is essential to mechanistically understand programmed cell death execution to be able to manipulate it. For this, we have used COFRADIC (COmbinedFRActionalDIagonal Chromatography), a proteomic technique successfully used to define in vivo caspasedegradomes. This part of our work has been done in collaboration with experts Drs. Frank Van Breusegem and Kris Gevaert (VIB, Ghent, Belgium). Preliminary data shows that metacaspase 1 cleaves the same downstream metacaspase previously identified as a complex member and also a transcription factor previously linked to plant defense (manuscript in preparation).
Together with Dr. Moritz Nowack (VIB, Ghent, Belgium) we have performed a programmed cell death transcriptomic meta-analysis. By putting together all publicly available raw transcriptome data involving plant cell death (developmental- or pathogen-triggered) we have been able to find core programmed cell death markers as well as specific cell death makers for different programs (developmental versus pathogen-triggered). These data have been published in a high-impact plant journal (Olvera-Carrillo et al., Plant Physiology, 2015).
Potentially interesting cell death regulators that we identify by our previously described proteomics, degradomics or transcriptomic approaches have been systematically characterized using genetics (obtention of mutants and transgenic plants) and biochemistry (interaction assays, substrate cleavage assays, functional characterization). Through this work, we have found an extremely interesting link between metacaspases and autophagy, a homeostatic cellular process that also regulates stress responses. This manuscript has just been published in the high-impact journal Cell Death & Differentiation (Coll et al., 2014, corresponding author).
B) Determine the role of AtMC3, an atypical type I metacaspase, during developmental and pathogen-triggered PCD in Arabidopsis.
AtMC3 homozygous tilling were crossed to different cell death mutant backgrounds: lsd1, lsd1 atmc1, lsd1 atmc2 and lsd1 atmc1 atmc2. The resulting homozygous mutant lines were assayed for cell death phenotypes using either BTH or Pto DC3000(avrRpm1). AtMC3 does not seem to act as a positive or negative regulator of cell death in response to pathogens.
In addition, the above mentioned genotypes were also assayed using the root vascular pathogen Ralstonia solanacearum, a soil-borne pathogen that has the ability to colonize the xylem of a very wide range of host plants (including Arabidopsis), causing bacterial wilt. AtMC3 mutants seemed more resistant to R. solanacearum infection. However, these mutants have shorter roots compared to Wt. Therefore, the defense results are difficult to interpret, because in shorter roots the pathogen has less surface to infect and from this it is difficult to conclude that AtMC3 has a role in root defense.
1.4 POTENTIAL IMPACT AND USE OF RESULTS
Beyond leading to several high-impact publications and establishing my niche in the field, this project has raised awareness among society on the importance of plant PCD research, through the organization of the First International Programmed Cell Death Conference in Plants (The Death of Plant Cells: from Proteases to Field Applications, www.bdebate.org/en/forum/death-plant-cells-proteases-field-applications) and the first Plant Proteostasis International Conference (Plant Proteostasis: towards a green-based industry, http://www.bdebate.org/en/forum/plant-proteostasis-towards-green-based-industry). We have also interested and attracted university students from Spain and abroad in plant PCD research. Finally, our results have attracted industry, and we are currently working with 3 national and 1 international companies to transfer the basic bench knowledge on plant PCD to new sustainable products for agriculture.
1.1 PROJECT OBJECTIVES
The main goal of this project was to understand the mode of action of type I metacaspases during PCD, with particular focus on the pathogen-triggered hypersensitive response cell death. For this we have used parallel genetics, biochemical, proteomic and cell biology approaches in the model plant Arabidopsis thaliana. The specific objectives of this project were the following:
A) Define the molecular mechanisms underlying HR cell death control by type I metacaspases in Arabidopsis.
B) Determine the role of AtMC3, an atypical type I metacaspase, during developmental and pathogen-triggered PCD in Arabidopsis.
1.2 WORK PERFORMED and MAIN RESULTS
A) Define the molecular mechanisms underlying HR cell death control by type I metacaspases in Arabidopsis.
In the past we have been able to identify a number of pathogen-triggered cell death regulators (Coll et al., 2010). Using immunoaffinity purification we have been able to isolate and characterize the first native, cell death-inducible complex: an AtMC1-containing supramolecular complexes that dynamically respond to PCD. In resting conditions, these complex contain a protease inhibitor that keeps AtMC1 repressed, preventing cell death to be initiated. After PCD is triggered, the AtMC1-containing complexes change composition, and bind, among other cell death regulators, a predicted downstream metacaspase. These results are a major breakthrough in biology, as for the first time evidence of such a cell death cascade in plants is provided, significantly moving forward the knowledge in plant defense pathways. These results are in preparation to be submitted. In addition, we have found a negative regulator of AtMC1, the suicide protease inhibitor AtSerpin1, which binds and inhibits AtMC1 activity. The inhibition of AtMC1 by AtSerpin1 resembles the mode of action of cowpox serpin CmrA blocking the immune caspase-1 in mammals, bringing forward extremely interesting evolutionary parallels between such distant systems as animals and plants. This work is under second revision in the high-impact plant journal New Phytologist (Lema et al., New Phytologist, last and corresponding author).
To complement protein complex data with the downstream proteolytic events taking place during pathogen-triggered cell death, we have analyzed the AtMC1 degradome. Defining which substrates do cell death proteases cleave (degradomes) is essential to mechanistically understand programmed cell death execution to be able to manipulate it. For this, we have used COFRADIC (COmbinedFRActionalDIagonal Chromatography), a proteomic technique successfully used to define in vivo caspasedegradomes. This part of our work has been done in collaboration with experts Drs. Frank Van Breusegem and Kris Gevaert (VIB, Ghent, Belgium). Preliminary data shows that metacaspase 1 cleaves the same downstream metacaspase previously identified as a complex member and also a transcription factor previously linked to plant defense (manuscript in preparation).
Together with Dr. Moritz Nowack (VIB, Ghent, Belgium) we have performed a programmed cell death transcriptomic meta-analysis. By putting together all publicly available raw transcriptome data involving plant cell death (developmental- or pathogen-triggered) we have been able to find core programmed cell death markers as well as specific cell death makers for different programs (developmental versus pathogen-triggered). These data have been published in a high-impact plant journal (Olvera-Carrillo et al., Plant Physiology, 2015).
Potentially interesting cell death regulators that we identify by our previously described proteomics, degradomics or transcriptomic approaches have been systematically characterized using genetics (obtention of mutants and transgenic plants) and biochemistry (interaction assays, substrate cleavage assays, functional characterization). Through this work, we have found an extremely interesting link between metacaspases and autophagy, a homeostatic cellular process that also regulates stress responses. This manuscript has just been published in the high-impact journal Cell Death & Differentiation (Coll et al., 2014, corresponding author).
B) Determine the role of AtMC3, an atypical type I metacaspase, during developmental and pathogen-triggered PCD in Arabidopsis.
AtMC3 homozygous tilling were crossed to different cell death mutant backgrounds: lsd1, lsd1 atmc1, lsd1 atmc2 and lsd1 atmc1 atmc2. The resulting homozygous mutant lines were assayed for cell death phenotypes using either BTH or Pto DC3000(avrRpm1). AtMC3 does not seem to act as a positive or negative regulator of cell death in response to pathogens.
In addition, the above mentioned genotypes were also assayed using the root vascular pathogen Ralstonia solanacearum, a soil-borne pathogen that has the ability to colonize the xylem of a very wide range of host plants (including Arabidopsis), causing bacterial wilt. AtMC3 mutants seemed more resistant to R. solanacearum infection. However, these mutants have shorter roots compared to Wt. Therefore, the defense results are difficult to interpret, because in shorter roots the pathogen has less surface to infect and from this it is difficult to conclude that AtMC3 has a role in root defense.
1.4 POTENTIAL IMPACT AND USE OF RESULTS
Beyond leading to several high-impact publications and establishing my niche in the field, this project has raised awareness among society on the importance of plant PCD research, through the organization of the First International Programmed Cell Death Conference in Plants (The Death of Plant Cells: from Proteases to Field Applications, www.bdebate.org/en/forum/death-plant-cells-proteases-field-applications) and the first Plant Proteostasis International Conference (Plant Proteostasis: towards a green-based industry, http://www.bdebate.org/en/forum/plant-proteostasis-towards-green-based-industry). We have also interested and attracted university students from Spain and abroad in plant PCD research. Finally, our results have attracted industry, and we are currently working with 3 national and 1 international companies to transfer the basic bench knowledge on plant PCD to new sustainable products for agriculture.