Functional Interactomics

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 the function of the eIF2B complex in the TOR-related regulation of protein production (see below), and the function of new regulators 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 maize.
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 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 of targets selected from our networks. 
Regulation of translation upon nutrient signaling
Plant growth is supported through the promotion of protein synthesis. In plants, inactive TOR leads to a decrease in the global amount of polysomes, suggesting an important role for the TOR kinase in translation regulation. In analogy to the mammalian systems, plant TOR phosphorylates and concomitantly activates the S6 kinase 1 (S6K1), thereby orchestrating ribosome biogenesis via RPS6 and reinitiation at 5’ UTR upstream open reading frames (uORF) through eIF3h modification. Furthermore, our proposed TOR signalling network reveals a tight link with proteins involved in translation (figure). Because of the increased importance of TOR in nutrient and stress response, and the intertwining of TOR signalling with translation regulation, this project focuses on uncovering the biological role of the unique phosphorylation of the translation initiation complex eIF2B by TOR, in relation to nutrient availability. The existence of a nutrient–TOR–eIF2B axis could potentially serve as a useful approach for targeted engineering strategies in obtaining growth efficient plants.
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. On the one hand, the most interesting genotypes obtained in Arabidopsis are validated in maize. On the other hand, we develop our network and combinatorial CRISPR technology straight into maize to increase yield under low-nitrogen supply by the engineering of the TOR/SnRK1 signaling pathway. 
Plant Interactomics Facility
We offer the plant research community and companies access to our protein complex purification platform.
Purifications under research service agreement are performed based on well-established protocols for complex purification from either the Arabidopsis cell suspension cultures PSB-D/L, seedlings or isolated plant tissues. We have recently been able to show that our complex purification technology performs equally well in crop species such as corn and rice. We have extensive experience with tandem affinity purification (TAP) for the isolation of stable protein complexes at high purity, whereas for more short-term interactions, we apply pull-downs using GFP-containing tags, or a more efficient protocol based on the Protein A/G moiety present in our TAP tags. More recently, we have developed Turbo-ID protocols and an approach to enrich for unstable protein interactions by means of protein cross-linking.

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Gene targeted insertion of AP-MS tags in plant cells through CRISPR/Cas9
Affinity purification coupled to mass spectrometry (AP-MS) generally relies on the expression of ectopic DNA, eg. a gene of interest fused with an affinity tag, either by overexpressing or by complementation of a mutant. This last approach can be challenging in crops, due to a lack of mutant libraries in combination with laborious transformation and regeneration protocols. In our group, a technique is being developed to circumvent this problem through the precise insertion of a tag in the plant genome itself using homologous recombination (HR). The programmable nuclease Cas9 (DNA or protein) is delivered to plant cells or calli, along with a gRNA for the gene of interest and a donor template. The donor template comprises an AP-MS tag flanked by arms that are homologous to the targeted genomic region. The double stranded break (DSB) generated by the Cas9 nuclease is subsequently repaired via HR using the donor template provided (figure). 
The proof of concept for this technology has been established in the model species Arabidopsis thaliana and we are transferring it to Zea mays.



Debbie Rombaut - Left - Went to the Plant Genome Editing group in PSB
Bernard Cannoot - Left - Went to the Systems Biology of Yield group in PSB
Caroline Matthijs - Left - Went to Sanofi
Michiel Bontinck - Left - Went to Tech transfer office at VIB-HQ
Nienke Besbrugge - Left - Went to Biogazelle
Chao Han - Left - Went to Shangdong University (Qingdao), China
Maarten Dedecker - Left - Went to Sanofi
Leen Vercruysse - Left - Went to Foodcoach.Gent
Aurine Verkest - Left - Went to Arche Consulting
Gert Van Isterdael - Left - Went to VIB core facility at IRC
Jan Geerinck - Left - Went to Biotalys
Yelle Buffel - Left - Went to Oleum Olieslagerij
Sandy Neirynck - Left - Went to a bakery in Cambodja
Hilde Stals - Left - Went to Argenx
Annelies De Clercq - Left - Went to Patent office De Clercq & Partners