Wall-E · Make it, don’t break it: a reconstitution screen for Casparian strip formation in the root endodermis

Make it, don’t break it: a reconstitution screen for Casparian strip formation in the root endodermis

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.

Data: CORDIS, © European Union