Periodic Reporting for period 1 - EPILIPIN (Deciphering the role of oxylipins in the epigenetic mechanisms controlling plant immunization.)
Periodo di rendicontazione: 2018-01-16 al 2020-01-15
Reducing crop losses caused by pests and diseases is necessary in order to produce more food in less land, ensuring food security while avoiding the overexploitation of natural resources. As a result, international entities such are promoting the search for alternative strategies. Experts point to “plant immunization” or priming, as the safest and most effective approach. After a first infection plants respond more effectively against a second pathogen encounter, what is known as priming. This implies certain “memory” of the first stimulus in order to better respond to subsequent attacks in an adverse environment. Different priming processes have been described, being the first stimulus ‘remembered’ from short to long periods of time, and even transmitted to the next generation of plants. Unfortunately, deeper knowledge of the involved mechanisms is needed for its application in crop protection. In this context, the plant intracellular cascade occurring between the perception of the pathogen and the fingerprinting of the stress memory still uncertain. Epigenetic mechanisms and oxylipin pathways are involved in priming processes, but their specific contribution as well as the relationship between these two pathways had not been addressed to date. To fill this gap in our knowledge, we have carried out an ambitious project using the unique genetic and molecular tools available in the model plant Arabidopsis thaliana to analyse first the role of oxylipin natural compounds in priming processes and then, to study the relationship between both molecular pathways to delineate plant defence mechanisms and the memory of the stress.
Oxylipin production/signalling is required for transgenerational memory of the stress:
First we set up within generation (Systemic Acquired Resistance, SAR) and transgenerational (Transgenerational Acquired Resistance, TAR) priming analysis against the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), in the host laboratory. We found, that mutants altered in oxylipin production/signalling were not able to display TAR, unveiling an important role of oxylipin pathways in transgenerational memory of the stress.
Interaction between oxylipin and epigenetic pathways:
To determine the interaction of the oxylipin and epigenetic pathways, we analysed ability of DNA (de)methylation mutants in perceiving oxylipin compounds. As the DNA (de)methylation mutants were not affected in oxylipin perception, our results seem to position oxylipin signalling upstream the epigenetic fingerprint in the cascade mediating priming. In addition, mutants defective in oxylipin production/perception were altered in their response to compounds altering the epigenome, pointing to an alteration of the epigenetic landscape or epigenetic plasticity in the mentioned mutants.
Characterization of oxylipin-related genes in mobilizing the epigenetic machinery:
We designed Chop-PCR markers to perform a rough DNA methylation profile for the oxilipin related mutants in basal conditions and in response to infection. Our results supported the requirement of oxylipin production/signalling for the DNA methylation changes activated in response to pathogen attack. To validate those results, we took advantage of Ep5C gene, previously used in the reporter construct Ep5C::GUS to identify DNA methylation defective mutants associated with immune priming. We found a constitutive induction of the construct, when introgressed in the mentioned oxylipin related mutants, mimicking mutants defected in DNA methylation, and reinforcing the idea of oxylipin pathways mediating changes in DNA methylation associated with plant defence and priming.
Positioning mitochondrial stress in the signalling cascade:
During the project development, different results produced in the group in the context of other advances in the field, pointed to an important role of mitochondria in the oxilipin signalling pathway. Given that a) oxylipin signalling involves mitochondrial changes, b) oxylipin defective mutants are altered in mitochondrial proteins and c) the production of methyl-groups donors for epigenetic marks partially take place at the mitochondria; we developed a working model in which the signalling triggered by the pathogen recognition and mediated by oxylipins, would induce mitochondrial changes affecting the deposition of the epigenetic marks underlying the memory of the stress (priming). Accordingly, mitochondrial stress induced a very strong resistance phenotype against the biotrophic pathogen Hpa. The mentioned induction of mitochondrial stress was enough to trigger both SAR and TAR, therefore supporting our model.
In summary, our results demonstrate that specific oxylipins and related proteins are required for plant immune priming, the most promising target for the development of alternative, hopefully more sustainable, crop protection strategies, one of the priorities of the European Union. Beyond our first ambitions, the data obtained in the context of the last advances in the field, allowed us to place the mitochondria in a central position of the intracellular signalling cascades mediating epigenetic memory of the stress.
To maximize the impact in the scientific community, the outcomes of this project have been disseminated at 6 national and international meetings and 3 invited seminars. The results will be submitted to high impact journals, securing the high standard of the group, research centre and consolidating my position in the field. I have also participated in the host group and research centre seminars and continued my active contribution in Societies and scientific communities. Aware of the importance of communication, I took part in a broad variety of teaching and outreach activities, promoting the interest in science and the EU research investments by the general public; while engaging and better understanding their concerns and priorities. We will shortly assess the translation of some of the results to the development of sustainable food security strategies taking advantage of the centre´s infrastructures (CNB/CSIC´s Technology Transfer Departments).
First we set up within generation (Systemic Acquired Resistance, SAR) and transgenerational (Transgenerational Acquired Resistance, TAR) priming analysis against the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), in the host laboratory. We found, that mutants altered in oxylipin production/signalling were not able to display TAR, unveiling an important role of oxylipin pathways in transgenerational memory of the stress.
Interaction between oxylipin and epigenetic pathways:
To determine the interaction of the oxylipin and epigenetic pathways, we analysed ability of DNA (de)methylation mutants in perceiving oxylipin compounds. As the DNA (de)methylation mutants were not affected in oxylipin perception, our results seem to position oxylipin signalling upstream the epigenetic fingerprint in the cascade mediating priming. In addition, mutants defective in oxylipin production/perception were altered in their response to compounds altering the epigenome, pointing to an alteration of the epigenetic landscape or epigenetic plasticity in the mentioned mutants.
Characterization of oxylipin-related genes in mobilizing the epigenetic machinery:
We designed Chop-PCR markers to perform a rough DNA methylation profile for the oxilipin related mutants in basal conditions and in response to infection. Our results supported the requirement of oxylipin production/signalling for the DNA methylation changes activated in response to pathogen attack. To validate those results, we took advantage of Ep5C gene, previously used in the reporter construct Ep5C::GUS to identify DNA methylation defective mutants associated with immune priming. We found a constitutive induction of the construct, when introgressed in the mentioned oxylipin related mutants, mimicking mutants defected in DNA methylation, and reinforcing the idea of oxylipin pathways mediating changes in DNA methylation associated with plant defence and priming.
Positioning mitochondrial stress in the signalling cascade:
During the project development, different results produced in the group in the context of other advances in the field, pointed to an important role of mitochondria in the oxilipin signalling pathway. Given that a) oxylipin signalling involves mitochondrial changes, b) oxylipin defective mutants are altered in mitochondrial proteins and c) the production of methyl-groups donors for epigenetic marks partially take place at the mitochondria; we developed a working model in which the signalling triggered by the pathogen recognition and mediated by oxylipins, would induce mitochondrial changes affecting the deposition of the epigenetic marks underlying the memory of the stress (priming). Accordingly, mitochondrial stress induced a very strong resistance phenotype against the biotrophic pathogen Hpa. The mentioned induction of mitochondrial stress was enough to trigger both SAR and TAR, therefore supporting our model.
In summary, our results demonstrate that specific oxylipins and related proteins are required for plant immune priming, the most promising target for the development of alternative, hopefully more sustainable, crop protection strategies, one of the priorities of the European Union. Beyond our first ambitions, the data obtained in the context of the last advances in the field, allowed us to place the mitochondria in a central position of the intracellular signalling cascades mediating epigenetic memory of the stress.
To maximize the impact in the scientific community, the outcomes of this project have been disseminated at 6 national and international meetings and 3 invited seminars. The results will be submitted to high impact journals, securing the high standard of the group, research centre and consolidating my position in the field. I have also participated in the host group and research centre seminars and continued my active contribution in Societies and scientific communities. Aware of the importance of communication, I took part in a broad variety of teaching and outreach activities, promoting the interest in science and the EU research investments by the general public; while engaging and better understanding their concerns and priorities. We will shortly assess the translation of some of the results to the development of sustainable food security strategies taking advantage of the centre´s infrastructures (CNB/CSIC´s Technology Transfer Departments).
Our contribution represents a step forward in unravelling the epigenetic bases of plant immunity, a timely and relevant field. The results obtained allowed us to proposed a working model reinforcing the role of the oxylipins in priming processes, but also positioning the mitochondria as an interface, integrating external signals and coordinating the plant responses to environmental changes by modifying the plant epigenome. On the other hand, as oxylipins are natural compounds, the project has an important potential in delivering high interest results for the development of sustainable strategies in crop protection. Additionally, oxylipins play key roles in other organisms. For example, they are important components of the human lipidome. In humans, oxylipins has been demonstrated to be linked to aging as well as important diseases, including cardiovascular illnesses, Alzheimer or cancer; all of them associated to mitochondrial alterations and epigenetic changes. Therefore, the results obtained in this MSCA could importantly influence different disciplines, some of them with a direct impact in a big proportion of the society. Finally, the fellowship has supported me during my first steps as an independent researcher preparing me to undertake independent and mature research positions in the future.