Periodic Reporting for period 1 - Wall-E (Make it, don’t break it: a reconstitution screen for Casparian strip formation in the root endodermis)
Periodo di rendicontazione: 2021-08-01 al 2023-07-31
Due to the ever-growing world population and climate change, food security has turned into a global concern and scientist all over the world are working hard to find innovative ways to tackle this problem. The optimization of water-and-nutrient use efficiency of crops has become a top priority to face with modern-day challenges such as drought and soil erosion. Therefore, understanding the molecular mechanisms by which plant roots absorb essential nutrients and water is crucial to increase our current food productivity to meet global demand. In plants, the root endodermis functions as a specialized cell layer allowing for the selective uptake of nutrients and water from the soil, whilst blocking the absorption of unwanted or toxic compounds through the roots. This is achieved by the formation of an apoplastic diffusion barrier around endodermal cells called the casparian strip (CS). Although many proteins have been characterized in CS formation and stability, one could argue that we only unravelled the tip of the iceberg. The transcription factor MYB36 regulates the expression of a great number of endodermal differentiation genes. Interestingly, myb36 loss-of-function leads to a complete absence of CSs and MYB36 ectopic expression is sufficient to induce CS formation in cortical cells. In this project I proposed a novel, bottom up, combinatorial gain-of-function approach that should deliver fundamental advances in our understanding of CS formation. Conceptually, the objective of this project is to attempt to reconstitute or assemble a CS by expressing all of its minimally required components. More specifically, I will try to reconstitute a CS in the endodermis of the myb36 mutant by screening for genes that enable a myb36 endodermis to regain a CS. To do so, I will first define a probable “core machinery” responsible for CS formation by co-expressing important known players and assessing CS formation and stability (Work Package (WP)1). Next, using the CRISPR activator technology, I will screen for novel genes that will improve the formation and stability of a functional CS by specifically activating genes-of-interest in the endodermis (WP2 and WP3). To achieve my goal, I pursued the following specific objectives:
Objective 1: Identify and characterize the “core machinery” required for CS formation
Objective 2: Test and further develop different CRISPR/Cas9 activator systems in the endodermis
Objective 3: Identify genes involved in CS formation through a CRISPR activator-based, combinatorial reconstitution screen.
Objective 1: Identify and characterize the “core machinery” required for CS formation
Objective 2: Test and further develop different CRISPR/Cas9 activator systems in the endodermis
Objective 3: Identify genes involved in CS formation through a CRISPR activator-based, combinatorial reconstitution screen.
WORK PACKAGE I: In this first work package, I aimed at identifying and establishing the “core machinery” in myb36 plants which are completely devoid of casparian strips. A multigene construct expressing CASP1, CASP3, CASP5, PER64 and ESB1 (all essential genes for CS formation) was generated and transformed into the myb36 mutant background. A stable line expressing all five genes was selected and fully characterized. qPCRs were performed to assess gene expression levels and CASP1-GFP fusion protein localization was analyzed during endodermal differentiation. Although these 5 essential genes were successfully co-expressed, they were, as expected, insufficient to properly localize CASP1 and establish a functional CS. This stable line serves as a solid base for carrying out the gain of function screen proposed in work package III.
WORK PACKAGE II: In this second work package I aimed at testing, developing and implementing the state of the art CRISPR activator technologies developed by other labs. As such, the CRISPR Act2.0 CRISPR-TV and CRISPR-Suntag activation systems were carefully examined and characterized. In my hands, among the three systems, the CRISPR-Suntag system clearly outperformed the others in terms of gene activation potential. The activation was validated using various genes (LOVE1, GPAT2, GPAT3, 4CL3, CHS, CHI, PAL1, C4H, F3H, FLS1 and TT7). The CRISPR Suntag activation system allows for co-activation of at least 7 genes (tested so far), rendering it ideal for carrying out the gain-of-function screen proposed in work package III. As such, the CRISPR Suntag activation system was stably introduced into our “core machinery” line and its activation potential was validated using the LOVE1 transcriptional reporter. The data generated in WP2 will be disseminated as a technical paper and should provide essential information regarding the efficient implementation of the CRISPR activation technologies in plants and its use in synthetic biology approaches.
WORK PACKAGE III: This work package consists of executing the gain of function combinatorial reconstitution screen. RNA-seq experiments have revealed about 100-150 high confidence genes as genuine MYB36 downstream targets, which potentially could be involved in CS formation. The list of genes was further narrowed down to 60 candidates by closer examination and generation of reporter lines. To activate genes of interest, two sgRNAs were designed for every gene in the high confidence list of MYB36 downstream targets. Because many genes might only exert their function upon presence of another protein partner, I will employ a combinatorial approach in which multiple genes will be activated at once. Using vector systems designed in the Geldner lab, up to 9 or 18 independent gRNAs can be simultaneously expressed and screened against in T1. I will constrain the combinations to be tested by a set of reasonable assumptions, effectively clustering the gene pool of 60 genes into smaller clusters of genes whose potential combinatorial activities can be tested (based on predicted localisation, predicted common pathway functions, predicted interaction potential, etc). Any cluster below 18 genes could be tested as a whole “functional unit” by transforming constructs carrying the appropriate sgRNAs and assessing their ability to improve CS formation by evaluating CASP1-GFP localisation and stability. Currently, gRNAS have been designed for all 60 candidate genes, cloned into expression vectors and the reconstitution screen is about to be initiated.
WORK PACKAGE II: In this second work package I aimed at testing, developing and implementing the state of the art CRISPR activator technologies developed by other labs. As such, the CRISPR Act2.0 CRISPR-TV and CRISPR-Suntag activation systems were carefully examined and characterized. In my hands, among the three systems, the CRISPR-Suntag system clearly outperformed the others in terms of gene activation potential. The activation was validated using various genes (LOVE1, GPAT2, GPAT3, 4CL3, CHS, CHI, PAL1, C4H, F3H, FLS1 and TT7). The CRISPR Suntag activation system allows for co-activation of at least 7 genes (tested so far), rendering it ideal for carrying out the gain-of-function screen proposed in work package III. As such, the CRISPR Suntag activation system was stably introduced into our “core machinery” line and its activation potential was validated using the LOVE1 transcriptional reporter. The data generated in WP2 will be disseminated as a technical paper and should provide essential information regarding the efficient implementation of the CRISPR activation technologies in plants and its use in synthetic biology approaches.
WORK PACKAGE III: This work package consists of executing the gain of function combinatorial reconstitution screen. RNA-seq experiments have revealed about 100-150 high confidence genes as genuine MYB36 downstream targets, which potentially could be involved in CS formation. The list of genes was further narrowed down to 60 candidates by closer examination and generation of reporter lines. To activate genes of interest, two sgRNAs were designed for every gene in the high confidence list of MYB36 downstream targets. Because many genes might only exert their function upon presence of another protein partner, I will employ a combinatorial approach in which multiple genes will be activated at once. Using vector systems designed in the Geldner lab, up to 9 or 18 independent gRNAs can be simultaneously expressed and screened against in T1. I will constrain the combinations to be tested by a set of reasonable assumptions, effectively clustering the gene pool of 60 genes into smaller clusters of genes whose potential combinatorial activities can be tested (based on predicted localisation, predicted common pathway functions, predicted interaction potential, etc). Any cluster below 18 genes could be tested as a whole “functional unit” by transforming constructs carrying the appropriate sgRNAs and assessing their ability to improve CS formation by evaluating CASP1-GFP localisation and stability. Currently, gRNAS have been designed for all 60 candidate genes, cloned into expression vectors and the reconstitution screen is about to be initiated.
I believe that the combined realization of these objectives will provide us with crucial information on how CS are established and stabilized. This project can be seen as a proof-of-principle, asking: Can we identify a minimal gene set for a complex secondary structure via such a combinatorial, gain-of-function screening approach in plants? Moreover, the validation, implementation, and the further improvement of the recently developed CRISPR activation system will be welcomed with open arms in the field of synthetic biology. The current data should shortly be published as a technical paper and provide a powerful resource to the scientific community. Moreover, multiple collaborations have been initiated, further increasing the impact of our work.