Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions

Abstract

Resolving functions of closely linked genes is challenging or nearly impossible with classical genetic tools. Four members of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) family are clustered on Arabidopsis chromosome five. To resolve the potentially redundant functions of this subclass of CrRLK1Ls named MEDOS1 to 4 (MDS1 to 4), we generated a single CRISPR/Cas9 transformation vector using a Golden Gate based cloning system to target all four genes simultaneously. We introduce single mutations within and deletions between MDS genes as well as knock-outs of the whole 11 kb gene cluster. The large MDS cluster deletion was inherited in up to 25% of plants lacking the CRISPR/Cas9 construct in the T2 generation. In contrast to described phenotypes of already characterized CrRLK1L mutants, quadruple mds knock-outs were fully fertile, developed normal root hairs and trichomes and responded to pharmacological inhibition of cellulose biosynthesis similar to wildtype. Recently, we demonstrated the role of four CrRLK1L in growth adaptation to metal ion stress. Here we show the involvement of MDS genes in response to Ni2+ during hypocotyl elongation and to Cd2+ and Zn2+ during root growth. Our finding supports the model of an organ specific network of positively and negatively acting CrRLK1Ls.

Comparative genomics of six Juglans species reveals disease‐associated gene family contractions

“…These fungal peptides target various members of transmembrane receptor‐like kinases encoded by the Catharanthus roseus receptor‐like kinase (CrRLK1L) gene family. There are 17 reported CrRLK1L protein orthologs in Arabidopsis that have been implicated in a variety of processes, including immunity signaling, abiotic stress response and cell wall dynamics (Kessler et al , ; Richter et al , , ; Guo et al , ). Several Basidiomycete genomes have been reported to encode RALF homologs, making it plausible that Armillaria is among the fungi that utilize RALF‐homolog effectors in infection.…”

Meiotic DNA Repair in the Nucleolus Employs a Nonhomologous End-Joining Mechanism

“…To specifically delete the rDNA portion of the R4 transgene (Wanzenböck et al, 1997) but retain the resistance marker and the left border and the right border of the vector after integration in the plant genome, a CRISPR/Cas9 vector was constructed as described before (Richter et al, 2018). Two guide RNAs (gRNAs) were designed that bind between the left border of the R4 binary vector and the start of the rDNA sequence and between the 39ETS and the promoter of the hygromycin-resistant gene, respectively.…”

Meiotic DNA repair in the nucleolus employs a non-homologous end joining mechanism

“…In order to specifically delete the rDNA portion of the R4 transgene (Wanzenbock et al 1997) but retain the resistance marker and the Left Border (LB) and the Right Border (RB) of the vector after integration in the plant genome, a CRISPR Cas9 vector was constructed as described before (Richter et al 2018). Two gRNAs were designed that bind between the LB of the R4 binary vector and the start of the rDNA sequence and between the 3'ETS and the promoter of the hygromycin resistant gene, respectively.…”