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

Descrizione del progetto

Districare funzioni ridondanti o multiple nelle interazioni geniche complesse

La compartimentazione è un principio organizzativo unificante negli organismi viventi, e si verifica a tutti i livelli, dagli organuli e le cellule ai tessuti, agli organi e all’intero organismo. Nell’endoderma delle radici delle piante superiori, le bande di Caspary, come le giunzioni strette nelle cellule epiteliali animali, forniscono una barriera alla diffusione extracellulare all’interno delle radici delle piante. In un certo senso, si tratta di deviazioni che costringono l’acqua e le sostanze nutritive ad entrare nelle cellule attraverso le proteine di trasporto della membrana plasmatica. Negli ultimi dieci anni, si sono compiuti progressi significativi nell’identificazione delle proteine necessarie per la produzione delle bande di Caspary. Tuttavia, i progressi sono stati bloccati in gran parte a causa dell’esistenza di più geni con la stessa funzione (ridondanza) e di geni con funzioni multiple. Il progetto Wall-E, finanziato dall’UE, sta sviluppando un innovativo processo di screening per identificare set di geni minimamente sufficienti utilizzando la banda di Caspary come sistema modello, con possibili applicazioni in altri processi e organismi.

Obiettivo

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.