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

In plants, the root endodermis functions as a barrier, allowing the selective uptake of nutrients and water.

The barrier is formed by cell wall impregnations called Casparian strips (CS), produced by differentiating endodermal cells.

In the last decade, pioneering work on the endodermis has identified numerous players involved in CS formation, including transmembrane proteins, peroxidases, dirigent-like proteins, lignin polymerising enzymes, laccases and super-oxide dismutases.

However, the forward and reverse genetic approaches used to uncover these proteins are slowly coming to a standstill due to their limitations when faced with gene redundancy or genes with a broader range of activities, causing pleiotropy or lethality.

I now propose to use CS formation as a model in order to pioneer a combinatorial, gain-of-function screen, aiming to define a minimally sufficient gene set for the assembly of a CS.

I will attempt to reconstitute a CS in the endodermis of the myb36 mutant – a master regulator of endodermal differentiation - by activation screening for genes within the MYB36-dependent gene set.

Firstly, I will introduce a “core machinery” for CS formation by expressing important known players and assessing CS formation and stability (WP1).

Secondly, I will test the most recently developed second generation CRISPR activator systems for their efficiency in activating genes of interest in the endodermis (WP2).

Thirdly, using the CRISPR activator technology, I will screen for novel genes that will improve the formation, stability and functionality of the CS (WP3).

Identifying new genes involved in CS formation through a combinatorial, gain-of-function approach represents a novel way to genetically elucidate molecular mechanisms and could become a model for other cellular and developmental processes in Arabidopsis or other organisms.