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

Abstract

BackgroundTransmembrane 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.ResultsAs 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 GatewayR donor vector pDONR/ZeoR 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 GatewayR 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.ConclusionsMost 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.

RGF1 INSENSITIVE 1 to 5, a group of LRR receptor-like kinases, are essential for the perception of root meristem growth factor 1 in Arabidopsis thaliana

“…We therefore hypothesized that BAK1 may pair with another ligand-interacting LRR-RLK to regulate root development [34]. To identify this putative LRR-RLK which can physically interact with BAK1, we carried out yeast two-hybrid analysis to test the interaction of BAK1 with all other 161 LRR-RLKs, whose cDNAs were cloned previously [44]. Using BAK1 as bait, we totally identified 83 LRR-RLKs which can interact with BAK1 in an mbSUS yeast two-hybrid system [45].…”

CLAVATA Signaling Pathway Receptors of Arabidopsis Regulate Cell Proliferation in Fruit Organ Formation as well as in Meristems

“…This region through the start codon was cloned into pENTR/D-TOPO using the TOPO-TA cloning kit (Invitrogen), subcloned into pBIB: BASTA-GUS (Gou et al 2010), and transformed into Col plants via Agrobacterium-mediated transformation (Table S3). To visualize GUS expression, plants were incubated in X-Gluc staining solution (2 mM X-Gluc dissolved in 1 mL DMSO, 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 0.2% Triton X-100, 50 mM NaPO 4 , pH 7.2) for 1-16 hr, then cleared in 9:1 ethanol/acetic acid for 3-16 hr, followed by 1 hr in chloral hydrate solution (8:2:1 chloral hydrate/water/glycerol).…”

An Allelic Series of bak1 Mutations Differentially Alter bir1 Cell Death, Immune Response, Growth, and Root Development Phenotypes in Arabidopsis thaliana

“…For pBIR1:GUS, the region 1516 bp upstream of and including the start codon of BIR1 was amplified using PrimeSTAR polymerase (TaKaRa Bio, Inc.) and cloned into the pENTR/D-TOPO vector (Life Technologies), followed by Gateway LR recombination (Life Technologies) into the pBIB-GUS-BASTA vector (Gou et al 2010). Constructs were transformed into the Col accession or indicated mutant backgrounds using Agrobacterium GV3103 and the floral dip method (Clough and Bent 1998).…”

“…For pBIR1:BIR1:GFP, the region from 1516 bp upstream of the ATG up to, but not including, the stop codon of BIR1 was amplified from BAC clone MJE7 (Arabidopsis Biological Resource Center, Ohio State University) using TaKaRa Ex Taq polymerase (TaKaRa Bio, Inc.) and cloned into the pCR8/ GW-TOPO vector (Life Technologies), followed by Gateway LR recombination (Life Technologies) into the pBIBKAN-GWR-GFP vector (Gou et al 2010). For pBIR1:GUS, the region 1516 bp upstream of and including the start codon of BIR1 was amplified using PrimeSTAR polymerase (TaKaRa Bio, Inc.) and cloned into the pENTR/D-TOPO vector (Life Technologies), followed by Gateway LR recombination (Life Technologies) into the pBIB-GUS-BASTA vector (Gou et al 2010).…”