Understanding plant immunity through the identification of immunogenic cell death indicators and the characterization of plant metacaspases in Arabidopsis thaliana.

Abstract

Losses to plant pathogens pose a major threat to food security, bringing about serious economic and societal burdens across the globe. with around 20-40% of crop production lost to plant diseases. In the face of climate change, rapidly evolving pathogens can easily overcome resistance provided by traditional pesticides. Consequently, a thorough understanding of the plant immune system is of paramount importance to breed disease-resistant crops._x000D_ As a strategy to counteract pathogen invasion, infected plant cells elicit type of regulated cell death known as the hypersensitive response (HR). Tight regulation of HR is critical for confinement of the immune response exclusively to the pathogen ingress site. However, our understanding of how cell death zonation is achieved and how by-stander cells (neighbouring cells) respond to infection remains fragmentary. In the first chapter of my PhD thesis, I explored how HR is spatiotemporally regulated at the transcriptional level in the plant model Arabidopsis thaliana. These results allowed us to identify bona fide transcriptional indicators of HR. Moreover, we provide for the community a fluorescent reporter transgenic line that displays a strong spatiotemporally resolved signal specifically in cells destined to undergo HR. Use of this reporter line for specific and-or high-throughput techniques involving single-cell “omics” will enable further dissection of the spatial aspect of plant immunity. _x000D_ Over the last decade, accumulating evidence suggest that plant proteases play crucial roles during HR. In the second chapter of my PhD, I attempt to unravel the role of Arabidopsis metacaspase 1 (AtMC1), a type of cysteine protease, in plant immunity. Although originally described as a positive regulator of HR in young plants, adult plants lacking AtMC1 exhibit constitutive activation of immune responses under basal conditions, thus acting as a negative regulator of plant immunity. We report that mutating the catalytic cysteine of the protease causes severe autoimmunity. Through a combination of genetic, biochemical and cell biology experiments we show that catalytically inactive AtMC1 acts as a sticky docking site for immune-related components including immune receptors, possibly preventing their timely turnover. Based on this data and previous literature, we infer that AtMC1 might directly or indirectly control the homeostasis of immune receptors. Therefore, interfering with the wild-type function of the protease has negative impacts on plant growth._x000D_ Finally, I actively participated in a second line of research in which we try to understand the function of AtMC1 upon proteotoxic stress. AtMC1 is dynamically recruited to highly conserved cytoplasmic condensates, known as stress granules, regulating senescence (Chapter 3). To biochemically characterize this function, we removed certain domains predicted to be high aggregation-prone and successfully expressed and isolated the protease. This major step forward allowed us to prove that AtMC1 exhibits a strong and evolutionary conserved capacity to clear protein aggregates, including those formed by pathological protein forms that cause a diversity of life-threatening pathologies in humans. The implementation of recombinant proteins with high aggregate-clearance activity may open new avenues for therapeutic intervention in diseases caused by misfolded proteins._x000D_ In the last five years, the plant science community has particularly witnessed a quantum leap in our understanding of the plant immune system thanks to mechanistic studies on plant immune receptors and signalling pathways regulating and leading to HR. Leveraging this knowledge to engineer disease resistance in staples and economically important crops will be a priority in the years to come. I hope that the works herein and conclusions drawn from this thesis can contribute to future endeavours to achieve such ambitious goals.

Date of Award	24 Mar 2023
Original language	English
Supervisor	Núria Sánchez Coll (Director)