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Identification of cell wall ligands for the root hair growth regulator ERULUS.

Plant cell growth ultimately relies on controlled loosening/strengthening of the cell wall, yielding a matrix that is not too stiff, yet not too loose. Plants have evolved specific mechanisms to sense and control cell wall composition and rigidity, in the form of cell wall binding receptor proteins. In this regard, the Catharanthus roseus Receptor-like Kinase 1-like (CrRLK1L) family of plant proteins has gained major attention during recent years. Several CrRLK1L proteins control cell growth through sensing of the cell wall status during expansion. Nevertheless, the mechanisms that lie at the basis of CrRLK1L-mediated signaling remain poorly understood. In an effort to further characterize CrRLK1L-functioning we identified ERULUS (ERU), a key regulator of root hair cell expansion, and a putative sensor of cell wall rigidity. ERU loss-of-function root hairs are short an stunted, and exhibit drastic defects in cell wall composition, pectin (a cell wall polysaccharide) modification and dynamics. The degree of pectin methylesterification directly relates to the degree of cell wall rigidity. Hence, pectin modification needs to be sensed and adjusted continuously to facilitate maintained cell growth. The ERU extracellular domain is highly similar to that of the CrRLK1Ls BUDDHAS PAPER SEAL 1/2 (BUPS1/2), which directly bind pectin in vitro. More so, ERU control of root hair growth involves the CrRLK1L FERONIA (FER), which also directly binds pectin. Together, these data provide a strong case for a direct ERU-cell wall interaction. Here, we propose to use a broad-spectrum carbohydrate array to screen for ERU-specific cell wall ligands. This method has previously been used to identify/characterize antibody specificity, carbohydrate binding modules and cell wall modifying enzymes. The procedure involves cloning and heterologous expression of a His6- and GST-tagged ERU extracellular protein domain, and subsequent array analysis using a bacterial lysate containing the soluble recombinant protein. The protocol is low-risk, simple and affordable (no protein upscaling and purification). Moreover, the tagged protein construct can be used for several downstream applications aimed at identifying protein-protein and protein-cell wall interactions. This experiment will be performed during a 2 months research stay, and will bring new expertise to the IMPRES group. Moreover, the outcome of this experiment is pivotal to our ongoing research and crucial to the understanding of cell wall sensing during plant cell growth.
Date:1 Apr 2019  →  30 Mar 2020
Disciplines:Plant cell and molecular biology