Characterisation of a gain-of-function mutant of CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) in Arabidopsis thaliana
Piovesana, M
Date: 6 December 2021
Publisher
University of Exeter
Degree Title
PhD in Biological Sciences
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 ...
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.
Doctoral Theses
Doctoral College
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