Arabidopsis thaliana receptor-like protein AtRLP23 associates with the receptor-like kinase AtSOBIR1

Abstract

Plants employ a large number of receptors localizing to the cell surface to sense extracellular signals. Receptor-like proteins (RLPs) form an important group of such trans-membrane receptors, containing an extracellular domain which is involved in signal perception and a short cytoplasmic domain. In contrast to receptor-like kinases (RLKs), RLPs lack a cytoplasmic kinase domain. How intracellular signaling is triggered downstream of RLPs upon perception of an extracellular signal, is therefore still poorly understood. Recently, the RLK SOBIR1 (Suppressor Of BIR1–1) was identified as an essential regulatory RLK of various RLPs involved in plant immunity against fungal pathogens.¹ Given that SOBIR1 appears to be a crucial component of RLP-containing complexes, we aimed to identify additional proteins interacting with SOBIR1. Here, we report on the immunopurification of a functional Arabidopsis thaliana (At)SOBIR1-yellow fluorescent protein (YFP) fusion protein stably expressed in Arabidopsis, followed by mass-spectrometry to identify co-purifying proteins. Interestingly, and in line with various studies showing interaction between RLPs and SOBIR1, we discovered that AtSOBIR1 interacts with AtRLP23, an RLP of which the function is currently unknown.

Introduction

Receptor-like proteins (RLPs) are an important group of transmembrane receptors, frequently containing an extracellular leucine-rich repeat (LRR) domain, a transmembrane domain, and a short cytoplasmic domain.² RLPs play a vital role both in plant development and disease resistance. For example, Arabidopsis thaliana Too Many Mouths (TMM) and Clavata2 (CLV2) regulate stomatal patterning and meristem maintenance, respectively.^3,4 In addition, the tomato (Solanum lycopersicum, Sl) Cf proteins and Ve1 mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium spp., respectively.^5,6 Similarly, Arabidopsis AtRLP30 is required for resistance to the necrotrophic fungal pathogen Sclerotinia sclerotiorum.⁷ Because RLPs lack a kinase domain, it is hypothesized that they recruit one or more receptor-like kinases (RLKs) to activate downstream signaling. In agreement with this hypothesis, TMM interacts with the LRR-RLK Erecta,⁸ whereas CLV2 has been reported to be present in a complex with the transmembrane kinase CORYNE and the RLK CLV1 to activate downstream signaling.⁹

Recently, it was discovered that the 2 tomato homologs of the RLK AtSOBIR1 (SlSOBIR1 and SlSOBIR1-like) interact with, and are required for, the Cf proteins and Ve1 to mediate immune responses.¹ Furthermore, it was found that SOBIR1 homologs interact with a number of additional RLPs.¹ Remarkably, SOBIR1 homologs do not appear to interact with RLKs, suggesting that SOBIR1 is mostly required for RLP function.¹ Indeed, AtSOBIR1 was found to be required for AtRLP30-mediated resistance of Arabidopsis to S. sclerotiorum.⁷ In the same study, it was also shown that Atsobir1 mutants are not compromised in immune responses mediated by the RLK FLS2 (Flagellin Sensing-2), thereby strengthening the hypothesis that SOBIR1 is not involved in signaling mediated by RLKs.¹ Interestingly, just recently it was shown that the microbe-associated molecular pattern (MAMP) eMax (enigmatic MAMP of Xanthomonas) is perceived by the RLP AtReMAX (receptor of eMax) and that also this RLP requires AtSOBIR1 for its functionality.^10,11 Furthermore, also the Arabidopsis RLP RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), which recognizes fungal endopolygalacturonases, requires SOBIR1 for its function.¹² Together, these studies confirm a vital and possibly unique role for SOBIR1 in RLP-containing receptor complexes.

AtSOBIR1 was first identified as a suppressor of the Arabidopsis bir1–1 mutant.¹³ BIR1 (BAK1-interacting RLK-1) is an RLK that interacts with, and negatively regulates BAK1 (BRI1-Associated Kinase-1), which is a member of the SERK (Somatic Embryogenesis Receptor Kinase) family (SERK3).¹³ The bir1–1 mutant shows a constitutive defense response, which is suppressed by an additional mutation in AtSOBIR1.¹³ This suggests that AtSOBIR1 is a positive regulator of defense signaling. In accordance with this, overexpression of AtSOBIR1 in Arabidopsis constitutively activates defense responses and triggers cell death.¹³ Next to a role in plant innate immunity, AtSOBIR1 also plays an important role as a regulator of floral organ abscission.¹⁴ In this role, AtSOBIR1 is referred to as Evershed (EVR).¹⁴ Since SOBIR1 appears to interact specifically with RLPs, it is expected that yet unidentified RLPs may also play a role in floral organ abscission.¹⁵ Being such a crucial regulatory RLK for the function of RLPs,¹⁵ it is anticipated that more RLPs than the ones identified to date will interact with SOBIR1. In this study we investigated this hypothesis by immunopurifying AtSOBIR1 from Arabidopsis plants stably transformed with AtSOBIR1 fused to Yellow Fluorescent Protein (YFP) (AtSOBIR1-YFP).¹⁴ Subsequent mass-spectrometry-based analysis of the immunoprecipitate indeed revealed an additional Arabidopsis RLP to interact with AtSOBIR1.

Results

We immunopurified AtSOBIR1-YFP from transgenic Arabidopsis plants, expressing AtSOBIR1-YFP under control of the endogenous AtSOBIR1 promoter,¹⁴ and included wild-type Arabidopsis Col-0 as a negative control. AtSOBIR1-YFP was immunopurified using GFP_TrapA affinity beads,¹⁶ which also show high affinity for YFP, followed by a tryptic on-bead digestion of the total immunoprecipitate. The digest was analyzed by mass-spectrometry¹⁶ to reveal peptides derived from co-purifying proteins. Among the identified peptides, a significant proportion originated from AtSOBIR1-YFP itself, confirming that we had successfully purified this RLK (Table 1). Interestingly, besides the various AtSOBIR1-YFP-derived peptides, we also identified one peptide matching an Arabidopsis RLP with high significance. We found this peptide to match AtRLP23 (Table 1; Figure 1), an RLP to which no function has been assigned to date.² This result suggests that AtSOBIR1 is capable of interacting with additional RLPs, in addition to the ones identified so far. No peptides of RLKs were present among the digested immunoprecipitate, again strengthening our hypothesis that AtSOBIR1 does not directly interact with RLKs.¹⁵

Table 1. Sequences and Protein Lynx scores of peptides, identified by mass-spectrometry, specifically matching AtSOBIR1 and AtRLP23 upon tryptic digestion of an immunoprecipitate of SOBIR-YFP from transgenic Arabidopsis plants.

Protein name	Peptide sequence	Protein Lynx scores
AtSOBIR1	AEDLAFLENEEALASLEIIGR	56.1
AtSOBIR1	AEDVAFLENEE	33.3
AtSOBIR1	GSLQDILTDVQAGNQELMWPAR	57.4
AtSOBIR1	KAEDLAFLENEEALASLEIIGR	68.2
AtSOBIR1	LMDQGFDEQMLLVLK	88.5
AtSOBIR1	SLQDILTDVQ	28.1
AtSOBIR1	VEWLDIDSSDLK	73.9
AtRLP23	NNNLEGSIPDALCDGASLR	67.6

Figure 1. Match of the identified peptide on the sequence of AtRLP23. The identified peptide is underlined and projected in bold.

To confirm the interaction of AtSOBIR1 with AtRLP23, we performed co-immunoprecipitation experiments. For this, we amplified the AtRLP23 coding sequence from Arabidopsis cDNA and C-terminally epitope-tagged it with the sequence encoding eGFP by inserting the AtRLP23 fragment into a binary vector for transient Agrobacterium-mediated expression assays in Nicotiana benthamiana.¹⁷ Subsequently, AtRLP23-eGFP and AtSOBIR1-Myc¹ were transiently co-expressed in N. benthamiana leaves. As a positive control for showing interaction, we also transiently co-expressed Cf-4-eGFP¹⁶ and AtSOBIR1-Myc,¹ whereas as a negative control we included the SlFLS2-GFP¹⁸ and AtSOBIR1-Myc combination.¹ Total proteins were extracted from the infiltrated N. benthamiana leaves and eGFP-tagged fusion proteins were immunoprecipitated using GFP_TrapA affinity beads. Using an anti-GFP antibody the purified eGFP-tagged fusion proteins were detected on western blot, whereas co-purifying proteins were detected with an anti-Myc antibody. We were able to successfully purify AtRLP23-eGFP, Cf-4-eGFP, and SlFLS2-GFP (Fig. 2, upper panel). Interestingly, AtSOBIR1-Myc co-purified with AtRLP23-eGFP and with the positive control Cf-4-eGFP, whereas as expected AtSOBIR1-Myc did not co-purify with the negative control SlFLS2-GFP (Fig. 2, middle panel). Together, these results indicate interaction between AtRLP23-eGFP and AtSOBIR1-Myc, thereby confirming the mass-spectrometry data showing that AtRLP23 is present in the immunoprecipitate upon purification of AtSOBIR1-YFP.

Figure 2.AtSOBIR1 associates with AtRLP23 and tomato Cf-4, but not with SlFLS2. AtRLP23-eGFP, Cf-4-eGFP, and SlFLS2-GFP were co-expressed with SlSOBIR1-Myc in N. benthamiana. Total proteins were extracted and subjected to immunopurification using GFP_TrapA affinity beads. Total proteins (Input) and immunopurified proteins (IP) were subjected to SDS-PAGE and blotted to PVDFmembrane. Blots were subsequently incubated (IB) with αGFP antibody to detect the immunopurified eGFP fusion proteins (upper panel) and incubated with αMyc antibody to detect co-purifying AtSOBIR1-Myc (middle panel). Coomassie-stained blots show the 50 kDa Rubisco band present in the input samples to confirm equal loading (lower panel).

To obtain more information about a possible co-expression of AtRLP23 and AtSOBIR1 in Arabidopsis leaves, thereby providing an explanation why originally AtRLP23 was found to co-purify with AtSOBIR1, an expression analysis was performed by using the Arabidopsis electronic fluorescent pictographic (eFP) browser of the Bio-Analytic Resource (BAR) website (http://bar.utoronto.ca/).^19.On this website, the gene expression profiles based on microarray data obtained from different tissues at various developmental stages of Arabidopsis are combined. Interestingly, AtRLP23 and AtSOBIR1 show co-expression in various tissues. In particular, AtRLP23 and AtSOBIR1 show relatively high co-expression levels in rosette leaf tissue, whereas they show a relatively low expression in seeds and flowers at various developmental stages (Fig. 3). Furthermore, AtRLP1 (AtReMAX),¹⁰ AtRLP30,⁷ and AtRLP42 (RBPG1),¹² do not show this particular co-expression with AtSOBIR1 in rosette leaf tissue (Fig. 3).

Figure 3. Expression profiles of AtSOBIR1 and AtRLP23, AtRLP1, AtRLP30, and AtRLP42 in different tissues at different developmental stages of Arabidopsis, as obtained from the Bio-Analytic Resource (BAR) website. The log2-transformed expression level is color coded (red for relatively high expression and green for relatively low expression, as compared with the median expression level of the gene across all tissues indicated), as shown by the color bar at the bottom. Black indicates no difference in the expression levels between tissues or developmental stages. The highest and lowest log2 signal values are 4.54 and -4.41, respectively.

Discussion

Being an essential regulatory RLK for the function of various RLPs,^1,15 it is expected that SOBIR1 is able to associate with a large set of different RLPs. Here we report that, in addition to the SOBIR1-interacting RLPs that were recently published,^1,7,10-12 also AtRLP23 interacts with SOBIR1.

Previously it was shown that SOBIR1 associates with different RLPs involved in plant defense as well as in development.¹ In addition, it was found that SOBIR1 does not interact with any of the RLKs that were tested, including SlFLS2, AtCLV1, and SlSERK1 and SlSERK3 (SlBAK1).¹ Recently, AtRLP30 was identified as the receptor recognizing a novel proteinaceous elicitor, referred to as SCFE1, for Sclerotinia Culture Filtrate Elicitor-1.⁷ In addition, it was found that AtSOBIR1 is required for SCFE1-triggered immunity in Arabidopsis.⁷ Intriguingly, in the same study it was shown that sobir1 Arabidopsis mutants are not compromised in immune responses mediated by the RLK FLS2.⁷ These results suggest that AtSOBIR1 is specially involved in RLP, but not in RLK function. Our results strengthen this hypothesis because we only found an RLP, AtRLP23, to interact with AtSOBIR1, whereas we did not identify peptides originating from RLKs in our mass-spectrometry data.

The role of the regulatory RLK SOBIR1 in RLP-containing complexes might be similar to the function of the well-studied regulatory RLK BAK1 in RLK-containing complexes.^15,20-22 BAK1 interacts in a ligand-dependent manner with various RLKs involved in defense and development,^20-22 whereas SOBIR1 interacts with RLPs independently of the presence of the ligand matching the interacting RLP.¹ Since there currently is no ligand known for AtRLP23, it remains unknown whether interaction of SOBIR1 with this RLP is ligand-dependent, however, given previous results this is most likely not the case. Both AtBAK1 and AtSOBIR1 have been shown to be involved in Ve1- and AtRLP30-mediated innate immunity.^1,7,23 In addition, SERK1 has been reported to be involved in Cf-4-mediated immunity against C. fulvum.²³ It remains to be elucidated whether additional RLKs, such as BAK1 and/or other members of the SERK family, interact with RLP/SOBIR1 complexes upon ligand perception.

AtRLP23 is a typical RLP containing an extracellular LRR-domain, a transmembrane domain, and a short cytoplasmic domain.² The expression observed at the BAR website¹⁹ shows that AtRLP23 and AtSOBIR1 are predominantly expressed in leaf tissue at all developmental stages. We used Arabidopsis leaf tissue to immunopurify AtSOBIR1-YFP with the aim to identify SOBIR1-interacting proteins. Probably because of the strong co-expression pattern of AtSOBIR1 and AtRLP23, and the relatively high expression of AtRLP23 as compared with the other AtRLPs of which the expression was studied (Fig. 3), AtRLP23 was identified as an AtSOBIR1 interactor whereas the other RLPs (AtRLP1, AtRLP30, and AtRLP42), which have also been shown to require AtSOBIR1 for their functionality, were not identified in the immunoprecipitate.

SOBIR1 appears to be a crucial regulatory RLK for various RLPs.^1,15 However, the exact role of SOBIR1 in the different RLP-containing complexes is unknown. In future experiments, the identification of (downstream) interactors of RLP/SOBIR1 complexes and the determination of the phosphorylation status of the kinase domain of SOBIR1, before and after ligand perception by the interacting RLP, will be the central theme. Eventually, this should help to elucidate the exact role that SOBIR1 plays in plant innate immunity and development. (Table S1)