A Collection of Mutants for CLE-Peptide-Encoding Genes in Arabidopsis Generated by CRISPR/Cas9-Mediated Gene Targeting

Abstract

The ligand-receptor-mediated intercellular communication system plays important roles in coordinating developmental and physiological events in multicellular organisms. In plants, CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides and their cognate receptors are thought to be involved in various aspects of the plant life cycle. Although the importance of this communication is broadly recognized, most CLE peptides are yet to be functionally characterized. A major problem in research on small signaling peptide-encoding genes is the limited number of loss-of-function mutants available due to their small gene size. CRISPR/Cas9-mediated gene targeting has the potential to overcome this problem, as it can be used to generate targeted mutations in essentially any gene, regardless of size. Here we generated a series of mutants of CLE-peptide-encoding genes. Newly generated clv3 and cle40 mutants reproduced the expected mutant phenotypes in the shoot apical meristem and root meristem, respectively. Our results show that CRISPR/Cas9-mediated gene targeting is a powerful tool for genetic analyses, even of small genes. We also report a novel mutant for CLE44 [which is thought to encode a tracheary elements differentiation inhibitory factor (TDIF)] and show that CLE44 contributes to vascular development. The bioresources presented here will be a powerful tool for further characterization of CLE peptides.

The signaling peptide-encoding genes CLE16, CLE17 and CLE27 are dispensable for Arabidopsis shoot apical meristem activity

“…The insertion site is located upstream of the CLE27 CLE domain, indicating that cle27-2 represents a loss-of-function allele. A second, independent CLE27 allele used was a CRISPR/Cas9-generated loss-of-function allele in the Col-0 accession designated cle27-cr1 [ 38 ]. This allele generates a frame shift that introduces a premature stop codon in the CLE27 coding sequence upstream of the CLE domain ( Table 1 ), indicating that it is a null allele [ 38 ].…”

Look Closely, the Beautiful May Be Small: Precursor-Derived Peptides in Plants

“…Subsequently, forward or reverse genetic approaches can be employed to identify genes encoding candidate receptors (or other signaling components) for the peptide of interest, either through genetic interaction studies or insensitivity of the receptor mutant to the (synthetic) peptide (4,18,19,27,108,120,133,153,184,209) (Figure 2a) ( Table 1). In the past, available T-DNA insertion lines and functional redundancy were limiting for genetic studies (19), but new approaches, such as CRISPR/Cas9-mediated mutagenesis, are now being employed to overcome this (215). In combination with synthetic or recombinant peptide variants, genetics is a powerful method to identify and characterize peptide-receptor pairs.…”

“…Nevertheless, a number of aspects have been explored only limitedly. For example, genome editing by CRISPR/Cas9 approaches can be used to create specific knockout lines that can partially solve the issue of genetic redundancy and may result in the identification of novel peptide-receptor pairs in the near future (144,215). Specificity in the expression patterns of proteins and the available signaling components in spatiotemporal contexts are largely responsible for regulating overall specificity.…”

Peptide signaling molecules CLE5 and CLE6 affect Arabidopsis leaf shape downstream of leaf patterning transcription factors and auxin

“…In addition CLE40 plays a role in root stem cell homeostasis, and CLE41 and CLE44 function in vascular development and lateral root formation (Matsubayashi, ; Wang, Zhang, & Wu, ). Although a comprehensive library of CLE loss‐of‐function alleles now exists (Yamaguchi et al., ), plants carrying most single cle null alleles show no obvious developmental or physiological phenotypes (Jun, Fiume, et al., ). One possible explanation is that due to a high degree of sequence homology, many CLE genes play largely redundant roles in plant biology (Strabala et al., ).…”