Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

Abstract

Background: Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated.

Results: As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the Gateway donor vector pDONR/Zeo and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into Gateway destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community.

Conclusions: Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

Figure 1

Gene transformation constructs generated in this study. Four Gateway^R-compatible cloning vectors developed specifically in this study. All the four vectors were derived from pBIB vectors [60] by inserting the Gateway^R module and BASTA resistance gene. Gateway^R-mediated addition of GFP and FLAG epitope tags to the C-terminal ends of target sequences in vectors pB35GWG and pB35GWF. The attB sites are from the recombination between attL and attR sites. The target LRR-RLK sequence without stop codon is inserted between the attB1 and attB2 sites. To make the sequence in-frame with the epitope tags, one extra G is attached to the end of the C-terminus of the target sequence. Amino acids are indicated with a single-letter code. Additional amino acids from attB sites and linking sequences in destination vectors are added to the final protein.

Figure 2

Functional examination of the generated Gateway^Rdestination vectors. Full-length cDNA sequence of BAK1 was PCR amplified from Arabidopsis Col-0 plants and introduced into two destination vectors, pB35GWG and pB35GWF, by in vitro DNA recombination mediated by BP clonase and LR clonase. Expression constructs harboring BAK1 cDNA were transformed into the bri1-5 mutant, a weak allele of bri1. Overexpression of BAK1 using the vectors can suppress the bri1-5 mutant phenotype, indicating that the vectors are functional. Expression of BAK1 was confirmed by Western hybridization in transgenic plants. Anti-FLAG and anti-GFP antibodies were mixed to detect the signals on one membrane.

Figure 3

Experimentally derived LRR-RLK cDNAs displaying different coding sequences and containing one continuous ORF. Black boxes indicate exons, and lines between exons represent introns. Vertical dotted lines indicate the differences between predicted and isolated sequences. Size of produced amino acid sequence is indicated under each molecule. The accession numbers for each sequence can be found in Additional file 1: Table S7.

Figure 4

Experimentally derived LRR-RLK cDNAs showing different coding sequences but not containing one continuous ORF. Black boxes indicate exons, and lines between exons represent introns. Vertical dotted lines indicate the differences between predicted and isolated sequences. Size of produced amino acid sequence is indicated under each molecule. The accession numbers for each sequence can be found in Additional file 1: Table S8.

Figure 5

Two LRR-RLKs producing alternatively spliced transcripts. At4g31250, the isolated sequence in this report is the same as the TAIR8 prediction with an ORF of 675 aa. A previously reported sequence, AK176245, shows an unpredicted intron, producing a shorter protein of 588 aa with delayed start codon and early stop codon. The GenBank accession number for the isolated sequence is FJ708761. At5g01950, the first two exons in TAIR5 prediction are shown as three exons in both experimental sequences. AK229912 displays a different intron/exon boundary with a shorter exon 7, resulting in a shorter protein of 631 aa. The GenBank accession number for the isolated sequence is FJ708768.

Figure 6

Confirmation of alternative splicing of LRR-RLKs by RT-PCR. Total RNA was extracted from inflorescence and leaf of Arabidopsis, respectively. The reverse transcribed single-stranded cDNA was used as template for nested PCR with variant-specific primers to confirm potentially alternatively spliced variants emerged from the predicted mRNA sequences and previous reports. The nested PCR products were separated on a 1.5% (w/v) agarose gel. The AGI numbers for examined genes are shown on the top of the lanes. For each gene, the left lane shows the result from inflorescence and the right lane shows the result from leaf. a, Previously reported variant [GenBank:BT011697] of At1g56140; b, Predicted sequence [GenBank:NM_104492] of At1g56140; c, Predicted sequence [GenBank:NM_125922] of At5g65240; d, Previously reported variant [GenBank:AY059844] of At5g65240. M, molecular weight markers (kb).

Figure 7

Detection of epitope-tagged recombinant LRR-RLK proteins in Arabidopsis with Western hybridization. Full-length cDNAs of SERK1, SERK2, SERK3, SERK4, SERK5 and BRI1 were recombined into pB35GWF and pK35GWG. FLAG-tagged fusion proteins, prepared by immunoprecipitation of total membrane protein, and total proteins from leaves of transgenic plants expressing GFP-tagged fusion proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membrane and epitope-tagged proteins were detected using either (A) anti-FLAG or (B) anti-GFP antibody (diluted 1:3,000) followed by anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) secondary antibody (diluted 1:10,000).