Characterisation of a gain-of-function mutant of CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) in Arabidopsis thaliana

Abstract

Receptor-like kinases (RLKs) comprise a large superfamily of proteins in plant genomes, and play essential roles in plant growth, development and response to biotic and abiotic stresses. The CYSTEINE-RICH RECEPTOR-LIKE KINASES (CRKs) comprise one of the largest subfamilies of RLKs with over 40 members in Arabidopsis thaliana, and although a few members of the family have been initially characterised, their precise biological functions remain largely unknown. This thesis reports the characterisation of a novel gain-of-function allele of CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) in A. thaliana which was isolated from a chemical mutagenesis screen. This mutation causes the substitution of alanine 397 with a threonine residue in subdomain III / αC-helix of the kinase domain of CRK10, and this novel allele has been accordingly registered as crk10-A397T with the Arabidopsis community database. The crk10- A397T mutant is a dwarf, and anatomical characterisation unveiled severely collapsed xylem vessels in the root and hypocotyl of the plant. Reporter lines suggested CRK10 is expressed in close association to vascular tissues, and a translational fusion with the fluorescent protein mCherry indicates that CRK10 is a plasma membrane-bound protein. Analysis of the recombinant WT and crk10- A397T versions of the cytoplasmic kinase domain of CRK10 demonstrated their auto-phosphorylation activity, and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis concluded that Thr397 acts as an additional auto-phosphorylation site in situ. Furthermore, an RNA-seq experiment revealed the constitutive induction of defence-related genes in the transcriptome of crk10- A397T mutant hypocotyls, including genes involved in the signalling pathways of the stress hormones salicylic acid (SA) and abscisic acid (ABA). Analysis of the composition of cell walls in the crk10-A397T mutant hypocotyls revealed extensive differences compared to the WT, an indication of cell wall remodelling mechanisms that are likely associated with the collapse of xylem vessels in this organ. Bioassays with the soil-borne vascular pathogen Fusarium oxysporum revealed that crk10-A397T mutant has a greater probability of survival to infection compared to WT plants. Analysis of genetic crosses demonstrated that key components of SA signalling pathways are required for the disease resistance phenotype of the crk10-A397T mutant.