A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis

Abstract

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.

Figure 1.

A phylogenetic view of AtRLP protein domain configurations and the corresponding RLP gene structures as shown by exon/intron boundaries. Left, Phylogenetic tree of the AtRLP family that also includes CLV2 (AtRLP10) and TMM (AtRLP17). The tree was generated from the alignment of C3-F domains of all AtRLPs with 100 bootstrap replicates as indicated on the branch of the tree. The AGI code and AtRLP gene number are indicated on the left. Genes are organized according to the order along the chromosomes. Middle, Domain organizations as predicted by SMART/Pfam. Each colored box represents a domain as indicated. The arrowhead shows the putative N-terminal transmembrane domain. The open box indicates an amino acid fragment not showing any significant motif or domain. Right, RLP gene structure presented by gray boxes for exons and spaces for the introns.

Figure 2.

Characterization of the Atrlp17-1 mutant allele. A to E, Comparison of stomata distribution of wild-type (A and C) with the Atrlp17-1 mutant (B), tmm-1 mutant (D), and the Atrlp17-1 mutant after complementation with a wild-type TMM allele (E). The arrows indicate single stomata, while the circles indicate stomatal clusters. F to I, Comparison of ABA response of wild-type (top half of the plate) with Atrlp17-1 (F and G; bottom half of the plate) or tmm-1 (H and I; bottom half of the plate) in the absence (F and H) and presence (G and I) of ABA. J, Location of the T-DNA insertion in Atrlp17-1.

Figure 3.

Characterization of the Atrlp10-1 mutant allele. A, Wild-type inflorescence meristem. B, Atrlp10-1 inflorescence meristem. C, Atrlp10-1 inflorescence meristem upon complementation with a wild-type CLV2 allele. D and E, Cleared shoot meristem of wild type (D) and Atrlp10-1 (E). Arrowheads indicate meristem borders. F, Location of T-DNA insertion in Atrlp10 (CLV2). G, Comparison of inflorescence development of wild type (left) with Atrlp10-1 mutant (right). The zoom-in picture indicated no siliques were developed because of the temporary termination of inflorescence meristem of Atrlp10-1 mutant. H, The 8-d, wild-type seedling (left) showed a short root phenotype, while Atrlp10-1 (right) shows no effect with 10 μm CLV3p treatment. I, Comparison of 4-week-old plants of wild type (left) with Atrlp10-1 mutant (right). J and K, Comparison of 2-week-old plants of wild type (J) with Atrlp10-1 mutant (K). L to N, The mean rosette leaf number (L), height of the primary stem (M), and carpel number (N) of wild types, clv2-3, Atrlp10-1, and Atrlp10-1 upon complementation with a wild-type CLV2 allele. Asterisks indicate significant differences (P < 0.01) compared to the respective wild types.

Figure 4.

AtRLP30 is involved in bacterial resistance and localized at the plasma membrane. A to C, Symptom development in Arabidopsis leaves 4 d after inoculation with Psp. Areas in half leaves of Col-0 (A), RLP30-1 (B), and Col-0 fls2 (C) were syringe inoculated after wounding. Full details of symptom scores are recorded in Table II. D, Comparative analysis of the multiplication of Psp 1448A in Col-0 and Atrlp30 mutant plants. Infiltrated leaves were examined 3 d after inoculation; results are means from four replicates with ses. Statistical analysis using Student's t tests showed significantly higher numbers of bacteria in the mutants (P = 0.047, 0.014, and 0.088 for Atrlp30-1, -2, and -3, respectively). E to G, Localization of GFP-tagged AtRLP30 in leaf epidermis and petiole tissue as determined using confocal microscopy (E and F) and western blotting with an antibody directed against the HA tag (G). H, Locations of the T-DNA insertions in AtRLP30.

Figure 5.

Characterization of the Atrlp41 mutant alleles. A, Comparison of the leaf phenotype of wild type with mutants Atrlp41-1, Atrlp41-2, and Atrlp41-3 after exogenous application of ABA (right). B, The location of T-DNA in Atrlp41-1, Atrlp41-2, and Atrlp41-3.