We develop technology to map protein interaction networks, protein complexes, and their posttranslational regulation. These tools are applied to better understand carbon and nitrogen signaling in relation to plant growth, stress resilience and crop yield, with the aim to adapt to or mitigate climate change. Biological functions are generally accomplished by short- and long-term molecular interactions. In our research group, we run the Plant Interactomics Facility of PSB where we isolate protein complexes and map protein interaction networks based on AP-MS and proximity labeling. Based on this technology, we explore and study the TOR/SnRK1 regulatory pathway that links central metabolism with plant growth. Therefore we have mapped the protein interaction networks comprising the target of rapamycin (TOR) and SNF1-related (SnRK1) kinases with the aim to understand how these networks regulate plant growth and stress resilience in relation to carbon and nitrogen availability.
Nutrient signaling and Plant Growth
The TOR/SnRK1 pathway has been shown to be involved in yield and stress tolerance in plants (figure). The TOR kinase is a central regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. The plant SnRK1 kinase is a highly conserved cellular fuel gauge acting antagonistic to the TOR kinase. We are studying upstream regulators and downstream targets of TOR and SnRK1 activity we recently isolated (see below). On the other hand, we are translating our network data into beneficial phenotypes. Because of the complex wiring of these networks, we make higher order mutants in Arabidopsis through a pooled combinatorial CRISPR library approach. We screen for plants that show enhanced growth, stress resilience or nitrogen use efficiency. To boost combinatorial CRISPR screening for crops, we are also evaluating the potential of cell cultures as a fast pre-screening tool before going to plants. Finally, we transfer our technology into crops.
TOR/SnRK1 signaling in response to nitrogen
Increasing nitrogen fertilizer applications and the selection of high-yield crops have sustained our growing world population. However, the future challenge is to restrict fertilizer use, while maintaining crop yield to prevent further land clearing for farming. To facilitate crop growth with less N, we want to upregulate nitrogen utilization efficiency (NUtE) in plants through a better understanding of the TOR/SnRK1 crosstalk in response to the nitrogen status. In addition to the downstream targets, we study how the TOR and SnRK1 kinases are upstream regulated in response to the nitrogen state (figure). Our network data are translated into plants with better nitrogen efficiency by targeted gene modification, and combinatorial CRISPR screening. Besides, we translate our findings to economically relevant crops.
Functional analysis of the PPI networks around TOR and SnRK1
Although the TOR pathway is conserved across eukaryotes, plants developed unique adaptations to this pathway to cope with their autotrophic and sessile nature. We generated for the first time a comprehensive TOR signaling network in plants (figure), elucidating both evolutionarily conserved as well as novel plant-specific links, covering a broad range of biological processes such as protein and nucleotide biosynthesis, autophagy, auxin signaling, chloroplast development, lipid metabolism, and senescence.
In energy-depleting stress conditions, SnRK1 stimulates catabolic reactions while repressing energy-consuming anabolic processes, shifting the balance from growth to survival. We map and study the TOR and SnRK1 regulatory network by combining quantitative AP-MS, proximity labeling, PTM and cross linking MS analyses. In parallel, we are performing functional analyses based on new interesting regulators or downstream targets of TOR and SnRK1 identified in our networks. Because of the key roles played by the TOR and SnRK1 kinases in plant growth and survival, their underlying networks have great potential towards the optimization of plant growth and stress tolerance. To rewire these networks at multiple levels, we are generating combinatorial CRISPR libraries which will enable simultaneous knock-out, overexpression, or baseediting of targets selected from our networks.
In energy-depleting stress conditions, SnRK1 stimulates catabolic reactions while repressing energy-consuming anabolic processes, shifting the balance from growth to survival. We map and study the TOR and SnRK1 regulatory network by combining quantitative AP-MS, proximity labeling, PTM and cross linking MS analyses. In parallel, we are performing functional analyses based on new interesting regulators or downstream targets of TOR and SnRK1 identified in our networks. Because of the key roles played by the TOR and SnRK1 kinases in plant growth and survival, their underlying networks have great potential towards the optimization of plant growth and stress tolerance. To rewire these networks at multiple levels, we are generating combinatorial CRISPR libraries which will enable simultaneous knock-out, overexpression, or baseediting of targets selected from our networks.