New insights into receptor-like protein functions in Arabidopsis

ABSTRACT

Receptor-like proteins (RLPs) are implicated in plant development and immunity. Genome-wide sequence analysis identified fifty-seven RLPs in Arabidopsis. However, only a few AtRLPs have been functionally characterized. The major problems in determing the biological roles for AtRLP genes are the lack of suitable screening conditions and the high-degree of functional redundancy. In order to unravel the functions of AtRLP genes, recently we undertook a systematically functional analysis of AtRLP genes using transcriptional profiling and overexpression. Our findings indicate that most AtRLP genes are differentially expressed upon various conditions, and the expression of single AtRLP gene is often perturbed by multiple stimuli. Transgenic Arabidopsis plants overexpressing AtRLP genes were generated. Our study presents an overview of biological processes in which AtRLP genes possibly are involved, and provides a valuable resource for further investigations into the biological roles of AtRLP genes. In this article, we elaborate our findings and propose further strategies concerning the function of unknown AtRLP genes.

KEYWORDS:

• Arabidopsis thaliana
• overexpression
• redundancy
• receptor-like protein
• transcriptional profiling

Plants exploit cell-surface receptors to sense and respond to both external and internal signals in a sophisticated way. Receptor-like proteins (RLPs), one of the most important groups of such cell-surface receptors, have been shown to be implicated in plant growth and development as well as in pathogen defense.¹ The CLAVATA2 (CLV2) and its ortholog FASCIATED EAR2 were shown to play important roles in the regulation of stem cell homeostasis in Arabidopsis and maize, respectively.^2,3 The TOO MANY MOUTHS (TMM) is involved in controlling stomatal patterning and distribution.⁴ In addition, it has been found that RLPs are important players in disease resistance. The RLP disease resistance genes include the tomato Cf and Ve genes that provide resistance against Cladosporium fulvum and Verticillium spp., respectively,^5,6 LeEIX genes that combat against the ethylene-inducing xylanase produced from Trichoderma biocontrol fungi,⁷ apple HcrVf genes that confer resistance to the scab fungus Venturia inaequalis,⁸ the LepR3 and Rlm2 genes that render race-specific resistance to the fungal pathogen Leptosphaeria maculans.^9,10

Genome-wide sequence analyses identified a total of 57 AtRLPs in Arabidopsis.^11,12 Although the biological functions of most AtRLP genes still remain unknown, a number of AtRLP genes have been assigned functions. In addition to CLV2 and TMM, the functionally characterized AtRLP proteins comprise AtRLP1/ReMAX that mediates recognition of eMAX,¹³ AtRLP3/RFO that involves in resistance to the vascular wilt fungus Fusarium oxysporum,¹⁴ AtRLP23 that perceives a conserved fragment present in most necrosis and ethylene-inducing peptide (NEP) 1-like proteins and subsequently activate the immunity,¹⁵ AtRLP30 that implicates in resistance against Pseudomonas syringae pv. phaseolicola,¹² AtRLP42/RBPG1 that confers resistance to endopolygalacturonases,¹⁶ AtRLP44 that activates brassinosteroid signaling through interaction BAK1,¹⁷ and AtRLP51/SNC2 and AtRLP55 that implicate in basal defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000.¹⁸

Previously we assembled a genome-wide collection of T-DNA insertion lines for AtRLP genes to elucidate their functions in regard to Arabidopsis growth and development, and sensitivity to various stresses including a wide range of pathogens.¹² However, only few new phenotypes linked with mutations in AtRLP genes were found.¹² Additionally, RNA interference (RNAi) to knockdown the expression of several AtRLP genes simultaneously was performed, while no biological functions could be assigned to additional AtRLP genes.¹ It is likely that the lack of identification of biological roles for most AtRLP genes may be caused by functional redundancy. To gain further insights into the biological functions of AtRLPs, several AtRLPs that are closely-related with CLV2 were identified.¹⁹ Of the CLV2 homologues exmined, AtRLP2 and AtRLP12, when driven by the endogenous promoter of CLV2, could rescue the clv2 meristem defects.¹⁹ However, no phenotypic alterations were observed for different combinations of the T-DNA insertion mutants of AtRLP2, AtRLP12 and CLV2.¹⁹ These results highlighted the existence of both functional redundancy and functional divergence among AtRLP proteins.

Despite these achievements on the studies of AtRLP genes, only a few AtRLPs have been characterized to date. Our previous study implied that the functional specificity of AtRLP genes, at least in part, was determined at the level of their transcriptional regulation.¹⁹ In addition, the fact that phenotypic alterations only be visible under suitable conditions makes studies on the function of AtRLP genes even more difficult. To obtain a more precise understanding of the transcriptional regulation of AtRLP genes, in a recent study we undertook a systematic analysis of the expression patterns of AtRLP genes with respect to a broad range of conditions including abiotic/biotic stresses and hormones.²⁰ Our findings identified potential biological processes in which AtRLP genes may be involved based on the observation that many AtRLP genes are responsive to abiotic and biotic stresses, as well as to hormones.²⁰ The expression of some AtRLP genes were perturbed under hormone treatments,²⁰ suggesting that these AtRLP genes functioned in secondary signaling events that were incurred by respective receptor. Multiple AtRLP genes are differentially expressed under one specific condition, which reinforces the observation that high-level of functional redundancies among AtRLP genes.²⁰ Remarkably, a large number of AtRLP genes are transcriptionally regulated by multiple external stimuli and hormones,²⁰ suggesting that one single AtRLP gene may be involved in multiple biological processes and the existence of crosstalks among different signaling pathways. We found that the most homologous AtRLP genes resided at the same locus and AtRLP genes are underwent massive duplications.^11,12,20 The comparision of AtRLP phylogeny and their transcriptional profile indicates that there is no signifiant correlation between AtRLP phylogeny and the expression patterns, although culstered AtRLP genes with similar expression profiles are observed. This implies that the duplicated AtRLP homologs could acquire different functions through the diversification in their gene expression.

As noted in our studies, the lack of phenotypes in single loss-of-function mutants may be explained by huge functional redundancy among AtRLP genes.^12,19 In addition, it is nearly impossible for genetically crossing individual T-DNA insertion lines to obtain high-order knockout mutants because many AtRLP genes are clustered at the same locus.^11,12,20 Also it is difficult to elucidate the function of AtRLPs due to the high diversity and extremely low abundance of their putative ligands. Particularly, no phenotypic changes were found for a number of RNAi lines that were generated to knockdown the expression of several sets of AtRLP genes.¹ Nevertheless, the generation of gain-of-function mutants has produced invaluable information on the function of AtRLP genes such as TMM, Ve1, AtRLP23 and AtRLP44.^6,15,17,21 We thus performed a genome-wide functional analysis of AtRLP genes using overexpression analysis to gain additional insights into their potential roles in various aspects such as Arabidopsis growth and development, and salt tolerance.²⁰ Despite the absence of any phenotypic alterations under normal growth condition, the overexpression of AtRLP3 and AtRLP11 could rescue the clv2-1 mutant.²⁰ In addition, AtRLP28 was found to play a role in salt stress tolerance.²⁰ Most AtRLP genes were proposed to be involved in disease resistance based on the hypothesis that disease resistance genes are likely to be duplicated and underwent diversifying selection.^11,12,20 The notion is thus reinforced by our expression study showing that the majority of AtRLP genes exhibited altered responses to a broad series of abiotic and biotic stress treatments.²⁰ With the availability of the collection of AtRLP overexpressors generated in our study, more AtRLP genes are expected to be assigned biological functions in future. Therefore, the obtained transgenic lines overexpressing AtRLP genes provide a valuable tool for further investigations into the biological roles of AtRLP genes.

Previous studies have indicated that RLPs need to interact with additional components to activate cellular response upon ligand perception as they lack intracellular signaling domains.¹ For instance, it has been shown that CLV2 was functionally associated with CRN in a receptor complex to perceive the CLV3 signal.²² Likewise, TMM, forming a receptor complex with ERECTA family members, was found to regulate the proper stomatal distribution and differentiation.²³ Recently, SUPPRESSOR OF BIR1-1 (SOBIR1) was found to associate specifically with various RLPs that either function in plant development or in plant immunity. It has been demonstrated that SOBIR1 physically interacted with Cf-4, Ve1, LepR3, Rlm2, AtRLP1, AtRLP23, AtRLP30 and AtRLP42, and was required for the proper function of these RLPs.^{9,10,12,13,15,16,24-26} However, it is unclear about the biological relevance of the physical interaction between SOBIR1 and two developmental RLPs CLV2 and TMM, respectively. It could postulate that SOBIR1 may involve in the CLV2- and/or TMM-mediated developmental processes. Altogether, it seems that the heterodimerization of RLPs with SOBIR-type RLKs to form functional receptor complexes appears to be a crucial step for the proper function of RLPs. To bear this in mind, it may worthwhile, in the study of interested RLPs, to investigate whether SOBIR1 is required for the RLP-mediated biological process.

Disclosure of potential confllicts of interest.

No potential conflicts of interest were disclosed.

Funding

Work in our group is supported by the National Natural Science Foundation of China (31271575; 31200902), the Fundamental Research Funds for the Central Universities (GK201103005), the Specialized Research Fund for the Doctoral Program of Higher Education from the Ministry of Education of China (20120202120009), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Natural Science Basic Research Plan in Shaanxi Province of China (2014JM3064).