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Proteolytic processing in plant stress signal transduction and responses to abiotic stress and pathogen attack

Periodic Reporting for period 4 - ProPlantStress (Proteolytic processing in plant stress signal transduction and responses to abiotic stress and pathogen attack)

Reporting period: 2019-12-01 to 2020-11-30

As sessile organisms, plants possess robust mechanisms to perceive environmental stress conditions and pathogen challenge and mount appropriate responses. Understanding the underlying molecular mechanisms how they do this will allow us to devise new methods for effective and sustainable crop protection and pest management and to breed or engineer more resilient and disease-resistant plants. This will help to satisfy increasing demands on agricultural productivity imposed by the transformation to a sustainable bio-based economy and the need to ensure food security for an increasing world population despite dramatically changing environmental conditions due to global climate change.

ProPlantStress focused on a specific biochemical mechanism termed proteolytic processing. Proteolysis is the hydrolysis of a peptide bond, resulting in protein cleavage and release of truncated protein forms and shorter peptides that have different interactions and activities than the unprocessed protein. Proteolysis is catalyzed by a family of enzymes called proteases. Individual proteases and proteolytic cleavages in specific proteins were known to be important for plant stress signaling and stress response pathways, but poorly understood on a system-wide level. Likewise, proteases are important effectors that pathogens use to manipulate plant immunity and determine the outcome of plant-microbe interactions, but their substrates in plants are incompletely understood.

ProPlantStress aimed to better understand which proteins are cut by which proteases under which conditions, how this changes the target protein function and what this means for a plant´s ability to cope with abiotic or biotic stress.
To identify proteolytic processing in living systems, one must not only be able to measure the abundance of peptides, but also to determine the termini, the start and end points of a protein. We had previously established “degradomics” methods to do so for hundreds to thousands of protein N termini (the start points) in biomedical samples using mass spectrometry.

We chose to work with the small plant Arabidopsis thaliana as a well-characterized model system with many established resources and practical advantages. However, the sample amounts required for existing degradomics methods were difficult to obtain with this small plants, for example in experiments that required sampling individual leaves at specific time points after manual infiltration with bacteria, or subcellular fractionations such as extracellular leaf apoplast fluid. We therefore developed a new method for N-termini enrichment, termed High-efficiency Undecanal-based N Termini EnRichment (HUNTER), which is much more sensitive, easier and faster to execute and even automatable. This method tremendously improved results and throughput in our project and has already found wider application in biomedical research. We have also established a new protease to improve protein sequence and proteome coverage and improved a method for protease specificity profiling.

Mass spectrometry not only allows us to identify protein termini, but also to compare their abundance in different samples. This can tell us which proteins are cut in plants exposed to stress compared to control conditions. We focused on high intensity light as an abiotic stress, which results in oxidative damages and impairs photosynthetic productivity under unfavorable environmental conditions, and challenge with the bacterium Pseudomonas syringae as a model pathogen. Similarly, we have applied these methods to define sequence specificity and in vivo substrates of selected proteases that literature data suggested to play important roles in these processes. In addition, we have collaborated with several plant science groups across Europe to teach visiting researchers in our methodology and to investigate protease function and proteolytic processes under different stress conditions and in additional plant-microbe interactions, including economically important crop species.
We achieved several important methodological advances to overcome challenges in plant protein N termini identification. We
1) developed (HUNTER), a new method for more sensitive detection of protein N termini even in microscale samples with less than 10 ug protein (published in MCP 2019)
2) developed software to improve annotation and facilitate interpretation of degradomics datasets (available at https://sourceforge.net/projects/manti/)
3) demonstrated that proteome-derived peptide libraries can be used in time series to determine kinetically favored protease substrates under different conditions (published in JBC 2020)
4) established legumain as a new protease for proteome digestion, enabling coverage of protein termini and post-translationally modified peptides undetectable in standard tryptic digests (published in Anal Chem 2020)

We applied our improved methods to study proteases and proteolysis in plants exposed to stress conditions and pathogen challenge. This accumulated the largest datasets on protein N termini in plants, which
1) provided experimental evidence for N-terminal sequence determinants regulating protein stability, co-translational N-terminal methionine processing, N-terminal acetylation and protein maturation after secretion or import in mitochondria or chloroplasts
2) identified changes in plastid proteostasis during short-term exposure to high intensity light and acclimation to high intensity fluctuating light (published in FiPS 2020)
3) revealed widespread proteolytic processing during virulent infection, which included many targets in the chloroplast already at an early time of infection
4) identified candidate substrates of selected plant and bacterial proteases with a role in plant immune signaling

In addition, we have applied our methods in collaborative work to
1) determine condition-dependent changes in the sequence specificity of Arabidopsis VPEs (JBC 2020)
2) assess the impact of mitochondrial DEG10 deletion (JXB 2019) and modulated abundance of chloroplast FtsH12 (JXB 2020) in vivo.
3) reveal new mechanisms how the fungus Ustilago maydis protease inhibitor targets maize papain-like cysteine proteases to promote infection (Nat Commun 2019).

Overall, ProPlantStress established degradomics in plant sciences and aggregated a vast resource of information on protein termini and stimulus-dependent proteolytic processing. This has provided new insights into the dynamic interactions in the plant protease web and revealed stress-induced changes in proteostasis on a proteome-wide scale.
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