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

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 Ni during hypocotyl elongation and to Cd and Zn during root growth. Our finding supports the model of an organ specific network of positively and negatively acting CrRLK1Ls.