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

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Research Article

Aline Herger, 

Shibu Gupta, 

Gabor Kadler, 

Christina Maria Franck, 

Aurélien Boisson-Dernier, 

Christoph Ringli


Published: June 19, 2020

?This is an uncorrected proof.

AbstractPlant 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 (Rapid ALkalinization Factor) peptide hormones that trigger diverse physiological processes related to cell growth. LRXs function in CWI sensing and in the case of LRX4 of Arabidopsis thaliana, this activity was shown to involve interaction with the transmembrane Catharanthus roseus Receptor-Like Kinase1-Like (CrRLK1L) protein FERONIA (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, an lrx1-lrx5 quintuple mutant line develops shoot and root phenotypes reminiscent of the fer-4 knock-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 chimeric LRX constructs causes cross-complementation of lrx mutants, indicative of comparable functions among members of this protein family. Suppressors of the pollen-growth defects induced by mutations in the CrRLK1Ls ANXUR1/2 also alleviate lrx1 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. In Arabidopsis 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 multiple LRX genes are required to recapitulate a fer knock-out phenotype. Our results support the notion that LRX-FER interactions are key to proper cell growth.

Citation: Herger A, Gupta S, Kadler G, Franck CM, Boisson-Dernier A, Ringli C (2020) Overlapping functions and protein-protein interactions of LRR-extensins in Arabidopsis. PLoS Genet 16(6):
e1008847. Gloria K. Muday, Wake Forest University, UNITED STATESReceived: July 29, 2019; Accepted: May 11, 2020; Published: June 19, 2020Copyright: © 2020 Herger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the manuscript and its Supporting Information files.Funding: This work was supported by the Swiss National Science Foundation (grant Nr. 31003A_166577) to CR and in part by a grant from the University of Cologne Centre of Excellence in Plant Sciences to ABD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.

IntroductionThe plant cell wall is a complex network of interwoven polysaccharides and structural proteins that supports plant structure and protects the cell from biotic and abiotic stresses [1]. Importantly, it serves as a shape-determining structure that resists the internal turgor pressure emanating from the vacuole. Cell growth requires a tightly regulated expansion of the cell wall, generally accompanied by the concomitant biosynthesis of new cell wall material that is integrated into the expanding cell wall [2]. The signal transduction machinery required for coordinating the intra- and extracellular processes involves a number of transmembrane proteins at the plasma membrane to connect the different cellular compartments [3]. Among these, the Catharanthus roseus Receptor-Like Kinase 1-Like protein (CrRLK1L) THESEUS1 (THE1) acts as a Cell Wall Integrity (CWI) sensor that perceives reduced cellulose content in the cell wall and induces compensatory changes in cell wall composition to restrain growth [4]. Several members of the CrRLK1L family are involved in cell growth processes [5,6,7,8,9]. FERONIA (FER) is required for successful pollen tube reception during the fertilization process involving local disintegration of the cell wall to release sperm cells [10]. The extracellular domain of FER has been demonstrated to bind pectin, a major component of cell walls [11]. This interaction could contribute to the function of FER during CWI sensing and perception of mechanical stresses [11,12,13]. Several CrRLK1L receptors have been demonstrated to bind Rapid ALkalinization Factor (RALF) peptides that are involved in the alkalinization of the extracellular matrix, the change of Ca2+ fluxes and the modulation of cell growth and response to pathogens [14,15,16,17,18,19,20,21]. Hence, CrRLK1L proteins appear to have multiple functions, suggesting that their activity is at the nexus of different cellular processes.
RALF1 was identified as a ligand of FER and a number of additional proteins are involved in the RALF1-FER triggered signaling process, either as signaling components such as ROP2, ROPGEF, ABI2, RIPK [22,23,24,25], as co-receptors such as BAK1 and LLG1/2 [22,26,27], or as targets of the FER-dependent pathway, such as AHA2 [15]. The receptor-like cytoplasmic kinase MARIS (MRI) and the phosphatase ATUNIS1 (AUN1) were identified as downstream components of signaling activities induced by the pollen-expressed ANXUR1 (ANX1) and ANX2, the closest homologs of FER [6,28,29]. Both AUN1 and MRI also influence root hair growth, indicating that they function downstream of a CrRLK1L protein in root hairs and MRI has been demonstrated to function downstream of FER [28]. Apart from this observation, it remains unclear to what extent the signaling components are shared among the different CrRLK1Ls.
LRXs (LRR-eXtensins) are extracellular proteins involved in cell wall formation and cell growth. They consist of an N-terminal (NT) domain and a Leucine-rich repeat (LRR) of 11 repeats, followed by a short Cys-rich domain (CRD) that serves as a linker to the C-terminal extensin domain (Fig 1A) [30]. The extensin domain contains Ser-Hypn repetitive sequences that are characteristic for hydroxyproline-rich glycoproteins [31,32] and appears to serve in anchoring the protein in the extracellular matrix [33,34]. The LRX family of Arabidopsis consists of eleven members, which can be grouped according to three specific expression patterns. LRX1/2 are predominantly expressed in root hairs, LRX3/4/5 in the main root and the shoot, and LRX8/9/10/11 in pollen. Mutations in these genes cause cell wall perturbation and cell growth defects in the respective cell types [33,35,36,37,38,39]. lrx1 mutants develop deformed root hairs that are swollen, branched, and frequently burst [33]. This phenotype is strongly aggravated in the lrx1 lrx2 double mutant that is virtually root hair-less [35]. The lrx345 triple mutant shows defects in vacuole development, monitoring of cell wall modifications, and sensitivity to salt stress that are reminiscent of the fer-4 knock-out mutant, and LRX4 physically interacts with FER [40,41]. Therefore, these results suggest that LRXs and FER function in a common process. The N-terminal moiety with the NT- and LRR-domains has been shown to associate with the membrane fraction [37], indicating a function of LRXs in linking the cell wall with the plasma membrane by binding of a membrane-localized interaction partner.
Fig 1. Membrane association of variants of LRX proteins.(A) LRX proteins consist of a signal peptide for export of the protein (light brown), an NT-terminal domain (purple) of unknown function, an leucine-rich repeat (LRR) domain (yellow), a Cys-rich hinge region (CRD), and a C-terminal extensin domain (green), with Ser-Hypn repeats typical of hydroxyproline-rich glycoproteins, for insolubilization of the protein in the cell wall. The different deletion constructs used in this study are listed, with”Δ” indicating deleted domains. In the LRX1 construct, a cMyc tag was introduced between the NT- and the LRR-domain, which does not interfere with protein function and allows for immuno-detection of LRX1. (B and C) Immunoblots of total extracts (left) and membrane fractions (right) of transgenic Arabidopsis expressing LRX4 constructs as indicated. AHAs are transmembrane proteins used as plasma membrane marker proteins. PEPC is the cytosolic PEP carboxylase used as a cytosolic marker. proteins were recently identified as high-affinity binding sites for RALF peptides, with the binding spectrum differing among the LRXs. LRX8 of pollen tubes was shown to physically interact with RALF4 [42], while in vegetative tissues, the root/shoot-expressed LRX3, LRX4, and LRX5 were reported to bind RALF1,22,23,24, and 31 [40,41]. Whether and how the binding of RALFs and FER to LRXs influences the interaction of these proteins remains to be investigated. It is also unknown to what extent the different LRXs are functionally similar and whether they share FER as a common interaction partner.
Here, we demonstrate that the root hair-expressed LRX1 binds FER and RALF1, like most of the LRX proteins that function in vegetative tissues. Together with the fer-like phenotype of higher-order lrx mutants, this suggests that the LRX-FER module is conserved in vegetative tissues. Cross-complementation experiments of lrx mutants with overexpression constructs encoding chimeric proteins with the NT-LRR domains of different LRXs fused to the extensin domain of LRX1 indicate that most but not all LRX NT-LRR domains exert similar functions. The membrane association of LRXs, however, is independent of binding to FER, suggesting that additional binding activities of LRX proteins are relevant for their activity in the regulation of cell growth and CWI maintenance.

The membrane association of LRX4 is independent of FER
We have previously shown that apoplastic LRX proteins associate with the plasma membrane and LRX4 binds to the transmembrane CrRLK1L FER [37,41]. This property of LRXs was investigated in more detail. LRX constructs lacking the extensin coding sequence (LRX4ΔE) were used in this and in later experiments to prevent insolubilization of the LRX protein in the cell wall. To determine the protein domain(s) necessary for membrane-association, LRX4ΔE-FLAG (coding for LRX4 missing the extensin domain), LRX4ΔLRRΔE-FLAG (coding for LRX4 missing the LRR- and the extensin domain), and LRX4ΔNTΔE-FLAG (coding for LRX4 missing the NT- and the extensin domain) (Fig 1A) were expressed under the CaMV35S (35S) promoter in transgenic Arabidopsis. The calculated molecular weight of all recombinant proteins are shown in S1 Table. Membrane fractions of the different transgenic lines were prepared from 10 days-old seedlings and tested for presence of the recombinant proteins. As controls, antibodies against plasma membrane-localized (α-AHA) and cytoplasmic (α-PEPC) proteins were used to confirm successful enrichment of the membrane fraction. As shown in Fig 1B, all proteins were present in the total fraction. LRX4ΔE-FLAG and LRX4ΔNTΔE-FLAG were also detected in the membrane fraction, but not LRX4ΔLRRΔE-FLAG. Hence, membrane association of LRX4 depends on the presence of its LRR domain that also binds FER [41], which raises the question whether membrane association of LRX4 depends on its interaction with the plasma membrane-localized FER. We investigated this using a wild type and fer-4 mutant line producing an LRX4ΔE-Citrine fusion protein. As for LRX4ΔE-FLAG, also LRX4ΔE-Citrine was detected in the membrane fraction. This localization, however, was not dependent on FER, as it was also observed in the fer-4 mutant background (Fig 1C). Since the LRR domain is unlikely to directly interact with membranes, membrane association of LRX4 appears to involve other proteins.

Multiple LRXs of vegetative tissue interact with FER
The root hair-localized LRX1 is so far the best characterized LRX protein and the lrx1 root hair mutant represents a convenient genetic system for analyses of LRX protein function [33,34,43,44]. It was previously reported that LRX4 and FER interact [41], but since FER was reported to be necessary for CWI maintenance in growing root hairs [23,28], it was of interest to test whether LRX1 also interacts with FER. To this end, constructs encoding LRX1ΔE-FLAG and the extracellular domain of FER fused to citrine (FERECD-Citrine) under the 35S promoter were expressed in tobacco for Co-IP experiments. LRX1ΔE showed interaction with FERECD (Fig 2A), which was confirmed in a yeast-two-hybrid (Y2H) experiment (S1 Fig). The Y2H experiments were extended to other LRXs of vegetative tissues, namely the LRR domains of LRX2, LRX3, LRX4, and LRX5. LRX2, LRX4, and LRX5 showed interaction with FERECD (S1 Fig), confirming the previous finding of LRX4 interacting with FERECD [41]. Due to technical problems with LRX3 LRR expression in the Y2H system, we conducted Co-IP experiments with LRX3ΔE and FERECD, which revealed interaction of the two proteins (Fig 2B). Thus, the LRR domains of all LRXs of vegetative tissues that were tested so far show interaction with FERECD.
Fig 2. LRX interaction with FER and RALF1.Immunoblots of Co-IP experiments with proteins expressed in N. benthamiana. (A and B) Both LRX1ΔE and LRX3ΔE show interaction with FERECD. Addition of synthetic RALF1 peptide (50, 100, 200, 1000 nM) to extracts does not influence the interaction of LRX1ΔE with FERECD. (C) LRX1ΔE shows binding to RALF1. Antibodies used for IP and subsequent detection by immunoblotting are indicated. RALF1-FLAG and FERECD-Citrine run slower than according to their molecular weight of 9.5 and 75.7 kDa, respectively, whereas LRX1ΔE runs higher than expected (54 kDa).

RALF1 binds with high affinity to LRX1, LRX4, and LRX5
LRX4 has been shown to bind RALF1, a peptide hormone that also interacts with FER [15,41]. Here, we tested binding of RALF1 to LRX1. Transient expression of LRX1ΔE-HA and RALF1-FLAG in N. benthamiana followed by Co-IP and immunoblotting showed interaction of the two proteins (Fig 2C). This was confirmed by Y2H, where under selective conditions, yeast cells grew effectively in the presence of the two proteins (S2 Fig).
The kinetics of the interaction of LRX proteins with RALF1 were tested with Biolayer Interferometry (BLITZ). The LRXΔE-FLAG proteins of LRX1, LRX3, LRX4, and LRX5 used for this experiment were expressed transiently in tobacco. Accumulation of all proteins to comparable levels was confirmed by immunoblotting prior to BLITZ analysis. For RALF1, in vitro synthesized peptide was used. Biological activity of the peptide was tested and confirmed in root growth assays where application of RALF1 effectively inhibited root growth of Arabidopsis seedlings (S3 Fig). The BLITZ analysis revealed a dissociation constant Kd of around 5 nM for the interaction of LRX1, LRX4, and LRX5 with RALF1 (S4 Fig), whereas LRX3 showed a much lower affinity (Table 1). While the LRXΔE-FLAG had to be soluble in order to get purified prior to application on the sensor (for details, see Materials and Methods), it cannot be completely ruled out that LRX3ΔE-FLAG has a reduced solubility in this experiment that would provide an alternative explanation for the observed high Kd.
In a next step, the dynamics of LRX-RALF1-FER interaction was investigated with a focus on LRX1. To this end, extracts used to show Co-IP of LRX1ΔE and FERECD were supplemented with increasing concentrations of RALF1 peptide. The addition of RALF1, however, did not affect the interaction between LRX1ΔE and FERECD (Fig 2A), suggesting that RALF1 does not affect LRX1-FER interaction.

The lrx12345 quintuple mutant mimics the fer-4 mutant phenotype
Mutations in FER such as fer-4 exhibit both root and shoot phenotypes [23,40,41]. The results described above suggest that the five LRX proteins produced in vegetative tissues exert overlapping functions. Since a double mutant for the root hair-expressed LRX1 and LRX2 displays a root hair phenotype comparable to the knock-out mutant fer-4 [23,35], and the lrx345 triple mutant develops a shoot phenotype that is reminiscent of fer-4 [40,41], we anticipated that an lrx12345 quintuple mutant would be globally similar to fer-4. The lrx1 lrx2 mutant was crossed with the lrx345 triple mutant and an lrx12345 quintuple mutant was identified in the segregating F2 population of this cross based on a root hair-less phenotype and delayed shoot growth with an increase in anthocyanin content. Indeed, the lrx12345 quintuple mutant shows fer-4 like phenotypes in the root and shoot at the seedling stage and increased accumulation of anthocyanin compared to the wild type (Fig 3A). fer-4 seedlings grown in a vertical orientation display reduced gravitropic growth of the root [45]. This growth defect was assessed in the wild type, fer-4, and different lrx mutant combinations by assessing the vertical growth index [46]. For quantification, the ratio between the absolute root length and the progression of the root along the gravity vector, the arccos of α, was used as illustrated in Fig 3B. The quintuple lrx12345 mutant develops an agravitropic response comparable to fer-4 (Fig 3C). Altogether, our results indicate that the LRXs of vegetative tissues interact with and are relevant for several activities of FER.
Fig 3. fer-4 and lrx12345 mutants show comparable phenotypes.(A) Seedlings were grown for 5 days on half-strength MS for analysis of root hair formation (bottom) and another 5 days for analysis of shoot development (top). (B) Quantification of gravitropic response by the root growth index. (C) Increasing agravitropy by accumulation of lrx mutations, represented by the angle α as shown in (B). Error bars represent SEM. Different letters above bars indicate significant differences (T-test, n>20, P LRX1ΔNTΔE> LRX1ΔLRRΔE. Taken together, this indicates that both the LRR- and the NT-domains are required but neither is sufficient to induce the dominant-negative effect on root hair development.
Fig 4. Both the LRR and NT domains are required for function of LRX1.(A) Root-hair phenotype of wild type seedlings (Col) or transgenic Col lines expressing LRX1ΔE, LRX1ΔLRRΔE, or LRX1ΔNTΔE (for protein structures, see Fig 1A). The dominant-negative effect induced by LRX1ΔE depends on both the LRR and the NT domains. A representative example of several independent transgenic lines is shown. (B) Immunoblots using the anti-cMyc antibody 9E10 detecting the proteins encoded by the transgenic lines shown in (A). (C) In the absence of the NT domain (LRX1ΔNT) complementation of lrx1 gives variable phenotypes (upper lane) and no complementation of lrx1 lrx2 is observed. An immunoblot showing LRX1ΔNT accumulation in the different lines is shown in S5 Fig. Bar=0.5 mm (A, C). a complementary approach, we tested whether the NT-domain is required for the function of the full-length LRX1. To this end, the lrx1 and lrx1 lrx2 mutants developing intermediate and strong root hair defects, respectively [35], were transformed with the constructs LRX1:LRX1 and LRX1:LRX1ΔNT. Unlike the full-length LRX1 which complements the lrx1 mutant [33,34], complementation with LRX1:LRX1ΔNT produced inconsistent results. A number of independent T2 families were analyzed, some showing complementation of lrx1, i.e. wild type-like root hair formation, whereas others developed a stronger defect comparable to the lrx1 lrx2 double mutant (Fig 4C). Detection of LRX1ΔNT by immunoblotting revealed no correlation between protein abundance and root hair formation (S5 Fig). In lrx1 lrx2 mutants, the full-length LRX1:LRX1 induced wild type-like root hairs whereas LRX1:LRX1ΔNT consistently failed to reestablish root hair development (Fig 4C) despite accumulation of the transgene-encoded protein (S5 Fig). Hence, the absence of the NT-domain does not completely abolish protein activity but interferes with the function of LRX1.

Functional equivalence among different LRX proteins
Analyses performed so far suggested that most vegetatively-expressed LRX proteins have comparable functions with the possible exception of LRX3 that appears to poorly bind RALF1. To compare in planta the function and activity of LRX genes expressed in different tissues, trans-complementation experiments were performed. To this end, the genomic coding sequence of LRX1 including the cMyc-tag (Fig 1A) was cloned into an overexpression cassette containing the 35S promoter and the resulting 35S:LRX1 construct was used for transformation of the lrx345 triple mutant. Several independent homozygous transgenic lines were identified and characterized. Semi-quantitative RT-PCR confirmed expression of the transgene in the lines (S6A Fig). For assessment of the complementation of the lrx345 phenotype, alterations in plant growth and physiology were used as parameters. lrx345 mutants are smaller than the wild type both at the seedling stage and at later stages when grown in soil [36]. This phenotype is alleviated in the 35S:LRX1 transgenic lines (Fig 5A, S7A Fig). The increased anthocyanin accumulation in lrx345 mutant seedlings compared to the wild type is significantly reduced in transgenic lines (Fig 5B), and the salt-hypersensitivity of shoot and root growth in the lrx345 triple mutant [40] was also alleviated in the transgenic lines (Fig 5C and 5D). Hence, the 35S:LRX1 construct can largely rescue the lrx345 mutant phenotypes.
Fig 5. Functional redundancy among LRX proteins.(A-C) Complementation of the lrx345 triple mutant with the 35S:LRX1 (LRX1oE) construct. Representative examples are shown. (A) Seedling shoots after 7 days of growth on half-strength MS (upper lane) and plants grown in soil (lower lane). (B) Anthocyanin accumulation in 12-days-old seedlings is increased in the lrx345. (C and D) The lrx345 mutant seedlings grown in the presence of 100 mM NaCl show increased salt sensitivity, resulting in shorter roots (C) and impairment of shoot growth (D) compared to control Col. These phenotypes are alleviated by LRX1 overexpression. Error bars represent SEM; different letters above the graphs indicate significant differences (T-test, N>20, P
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