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

Project description

Untangling redundant or multiple functions in complex gene interactions

Compartmentalisation is a unifying organisational principle in living organisms, and it occurs on all levels, from organelles and cells to tissues, organs and the entire organism. In the endodermis of roots in higher plants, Casparian strips, like the tight junctions in animal epithelial cells, provide an extracellular diffusion barrier within plant roots. They are a detour in a way, forcing water and nutrients to enter the cells via plasma membrane transport proteins. Over the last decade, significant progress has been made in identifying the proteins needed to make the Casparian strips. However, this progress has stalled largely due to the existence of multiple genes with the same function (redundancy) and genes with multiple functions. The EU-funded Wall-E project is developing an innovative screening process to identify minimally sufficient gene sets using the Casparian strip as a model system, with possible application in other processes and organisms.

Objective

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.

Fields of science (EuroSciVoc)

Keywords

Programme(s)

Topic(s)

Funding Scheme

MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)

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Call for proposal

(opens in new window) H2020-MSCA-IF-2019

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Coordinator

UNIVERSITE DE LAUSANNE
Net EU contribution
€ 191 149,44
Address
QUARTIER UNIL CENTRE - BATIMENT UNICENTRE
1015 LAUSANNE
Switzerland
Region
Schweiz/Suisse/Svizzera Région lémanique Vaud
Activity type
Higher or Secondary Education Establishments
Links
Total cost
€ 191 149,44