Determining the Roles of CLS Genes in the Cytokinin Signaling Pathway in Arabidopsis
Creators: name: Sackett, Scott
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2018

description: Introduction
Cytokinins are plant hormones involved in numerous growth and development processes, such as cell division, leaf senescence, shoot initiation and growth, cell development, and biotic and abiotic stress responses5. The cytokinin signaling pathway (Figure 1) in plants is similar to the two-component phosphorelay systems commonly found in prokaryotes. In plants, this pathway is initiated when cytokinin binds to the CHASE domain of an ARABIDOPSIS HISTIDINE KINASE (AHK), which causes autophosphorylation of a histidine residue contained within its kinase domain. This phosphate is subsequently transferred to an aspartic acid contained within the receptor’s receiver domain. The phosphate group is then transferred to an ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN (AHP), which takes the phosphate group into the nucleus to phosphorylate the aspartic acid of ARABIDOPSIS RESPONSE REGULATORS (ARR). There are two types of ARRs: type-A ARRs, which lack DNA binding domains and act as negative regulators of this signaling pathway, and type-B ARRs, which control the expression of cytokinin-regulated genes, including the type-A ARRs5.
Recent evidence has suggested that cytokinin can activate type-B ARR’s by a mechanism independent of a phosphorylated aspartic acid. A type-B ARR double mutant line, arr1,12, is hyposensitive to cytokinin. Introduction of wild-type (WT) ARR12 rescued type-B ARR function in the mutant plants. However, introduction of ARR12:D69N, in which the aspartic acid was replaced by the non-phosphorylatable asparagine, also rescued function in the plants, though to a lesser extent than the ARR12 (unpublished data). An alternative possible mechanism is phosphorylation through
serine/threonine (Ser/Thr) residues in addition to the canonical Asp residue. To explore this possibility, analysis of ARR12 and ARR12:D69E on Phos-tag gels exhibited several retarded bands, which is consistent with several sites of phosphorylation. Upon inspection, it was noted that there were several Ser/Thr residues within the receiver domain of ARR12. After creating phospho-mimics of these sites, it was found that these mutants can activate the cytokinin signaling pathway in the absence of cytokinin. Altogether, this data supports the notion that phosphorylation of serine and threonine may act as an alternative mechanism to aspartic acid phosphorylation in the cytokinin signaling pathway (unpublished data).
To identify the Ser/Thr kinase that may act in cytokinin signaling, we took inspiration from the ethylene signaling pathway (Figure 2), which involves a His kinase coupled to a Ser/Thr kinase. In the absence of ethylene, the ethylene receptors (ETR1, ERS1, ETR2, EIN4, and ERS2), which are His kinases, act as negative regulators by interacting with and activating a Ser/Thr kinase called CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1). CTR1 phosphorylates ETHYLENE INSENSITIVE 2 (EIN2), which is then degraded. In the presence of ethylene, ethylene receptors undergo a conformational change to inactivate CTR1, and CTR1 no longer functions to repress EIN2. Thus, EIN2 is free to signal to the next part of the pathway and activate transcription factors that regulate various primary and secondary ethylene response genes4. Interestingly, the CTR1 protein kinase has several paralogs, which we have termed CTR1-LIKE SEQUENCES (CLS) (Figure 3). Other than CTR1, the only CLS gene characterized to date is ENHANCED DISEASE RESISTANCE 1 (EDR1). EDR1
functions as a negative regulator in disease resistance, stress response signaling, and cell death regulation3. The other CLS genes currently have unknown functions.
Since CTR1 is a Ser/Thr kinase that is regulated by the ethylene receptors, which are two-component histidine kinases similar to the cytokinin receptors, we hypothesize that the CLS genes, including EDR1, may encode Ser/Thr kinases that can phosphorylate type-B ARRs in the cytokinin signaling pathway. In this report, I made use of two reverse genetics approaches, CRISPR-Cas9 and T-DNA insertional mutagenesis, to knockout the CLS genes to determine if they function in the hypothesized Ser/Thr phosphorylation. Clustered Regularly Interspaced Short Palindromic Sequences (CRISPR)-Cas9 is genome editing tool that has recently been developed in plants. It utilizes a synthetic guide RNA that recruits the Cas9 enzyme to induce site-specific DNA cleavage and generate insertion/deletion (in/del) mutations through the cell's error prone DNA repair program8. As an alternative approach to CRISPR-Cas9 and to isolate additional mutant alleles for each target gene, I am also screening the T-DNA insertional mutant libraries. The T-DNA libraries consist of large numbers of random insertion events of the Agrobacterium T-DNA into the Arabidopsis genome. These insertion events have been mapped onto the genome, and the libraries are publicly available1. To date, I have isolated 1 heterozygous and 6 homozygous cls mutant lines using the T-DNA approach as well as obtained 4 homozygous cls mutations via the CRISPR-Cas9 method.