Overlapping functions and protein-protein interactions of LRR-extensins in Arabidopsis

Plant cell growth requires the coordinated expansion of the protoplast and the cell wall, which is controlled by an elaborate system of cell wall integrity (CWI) sensors linking the different cellular compartments.LRR-eXtensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (RapidALkalinizationFactor) peptide hormones that trigger diverse physiological processes related to cell growth. LRXs function in CWI sensing and in the case of LRX4 ofArabidopsis thaliana, this activity was shown to involve interaction with the transmembraneCatharanthusroseusReceptor-LikeKinase1-Like (CrRLK1L) proteinFERONIA (FER). Here, we demonstrate that binding of RALF1 and FER is common to most tested LRXs of vegetative tissue, including LRX1, the main LRX protein of root hairs. Consequently, anlrx1-lrx5quintuple mutant line develops shoot and root phenotypes reminiscent of thefer-4knock-out mutant. The previously observed membrane-association of LRXs, however, is FER-independent, suggesting that LRXs bind not only FER but also other membrane-localized proteins to establish a physical link between intra- and extracellular compartments. Despite evolutionary diversification of various LRX proteins, overexpression of several chimericLRXconstructs causes cross-complementation oflrxmutants, indicative of comparable functions among members of this protein family. Suppressors of the pollen-growth defects induced by mutations in the CrRLK1LsANXUR1/2also alleviatelrx1 lrx2-induced mutant root hair phenotypes. This suggests functional similarity of LRX-CrRLK1L signaling processes in very different cell types and indicates that LRX proteins are components of conserved processes regulating cell growth. Author summary Cell growth in plants requires the coordinated enlargement of the cell and the surrounding cell wall, which is regulated by an elaborate system of cell wall integrity sensors, proteins involved in the exchange of information between the cell and the cell wall. InArabidopsis thaliana, LRR-extensins (LRXs) are localized in the cell wall and bind RALF peptides, hormones that regulate cell growth-related processes. LRX4 also binds the plasma membrane-localized protein FERONIA (FER), thereby establishing a link between the cell and the cell wall. Here, we show that membrane association of LRX4 is not dependent on FER, suggesting that LRX4 binds other, so far unknown proteins. The LRR domain of several LRXs can bind to FER, consistent with the observation that mutations in multipleLRXgenes are required to recapitulate aferknock-out phenotype. Our results support the notion that LRX-FER interactions are key to proper cell growth.