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Control of programmed cell death by metacaspases in plants

Final Report Summary - PCDMC (Control of programmed cell death by metacaspases in plants)

The CIG “Control of Programmed Cell Death by Metacaspases in plants (PCDMC)” 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, PCDMC 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 project also aimed at using a translational research approach, applying the knowledge obtained in Arabidopsis into melon, an agronomically important horticultural crop. 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.
C) Determine the role of type I metacaspases in melon during HR cell death triggered by economically relevant pathogens in this crop species.

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. The manuscript of this work will be soon submitted to a major multidisciplinary journal (last authorship), further increasing the relevance of this field of research and its applicability to other species.
To complement protein complex data with the downstream proteolytic events taking place during pathogen-triggered cell death, we have analyzed the AtMC1degradome. 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 (see above proteomics section) and also a transcription factor previously linked to plant defense.
Together with Dr. Moritz Nowack (VIB, Ghent, Belgium) we are performing a programmed cell death transcriptomic meta-analysis. We are putting together all publicly available raw transcriptome data involving plant cell death (developmental- or pathogen-triggered) and analyzing it to find core programmed cell death markers as well as specific cell death makers for different programs (developmental versus pathogen-triggered). This approach has yield a very much sought set of robust and reliable HR cell death markers, which will be useful for different sectors: basic researchers, breeders and industry (manuscript in preparation together with Dr. Nowack).
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 publish 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.
atmc3G175E tilling mutant has been complemented with HA-tagged AtMC3 under the control of its native promoter (pAtMC3::AtMC3-HA). Work on atmc3A269T has been abandoned due to the low penetrancy of this mutation. It has not been possible to complement atmc3G183E mutant. This is in fact very intersting, as it opens the possibility that G183E may be a gain-of-function or dominant mutation. To verify this, we have transformed Arabidopsis wild-type Col-0 plants with a G183E mutated version of AtMC3. We have tested the effect of these mutations on immune responses, testing them against the array of pathogens that we have available in the lab, and have found that AtMC3 may have a role in defense. We have verified AtMC3 vascular expression using GUS assays Our collaborator in the Centre for Plant Biotechnology and Genomics (CBGP, Dr. Miguel Moreno-Risueño) is currently analyzing the interaction of AtMC3 with known vascular and meristematic developmental regulators using yeast-two-hybrid. This approach will set the basis to understand the role of AtMC3 in root development in a tissue- or cell-specific manner.

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 organisation 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). 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 2 national and 1 international companies to transfer the basic bench knowledge on plant PCD to new sustainable products for agriculture.