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.
