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. 2020 Jul 6;30(13):2645–2648. doi: 10.1016/j.cub.2020.06.015

Figure 1.

Figure 1

CRISPR/Cas9 Strategy for Generating Ppclv1 Mutants

(A) Strategy for mutagenizing PpCLV1a to generate Ppclv1a single mutants and Ppclv1a1b double mutants. Two guide RNAs were designed against the LRR domain of PpCLV1a and cloned into pU3::Ppclv1a sgRNA5 and pU3::Ppclv1a sgRNA7 expression vectors (original paper). Plants were co-transformed with pU3::Ppclv1a sgRNA5, pU3::Ppclv1a sgRNA7, pNRF, and pACT::Cas9 (original paper), or with pU3::Ppclv1a sgRNA7, pNRF, and pACT::Cas9 for the Correction. Sanger sequencing of full-length PpCLV1a and PpCLV1b loci in all lines verified the mutations illustrated. Sequences in bold represent sgRNA target sites on WT loci, gray boxes highlight mutations in each line, and schematics to the right illustrate the predicted effect of mutations on the protein. Lines with labels in blue were reported in the original paper.

(B) Strategy for mutagenizing PpCLV1b to generate Ppclv1b single mutants and Ppclv1a1b double mutants. An sgRNA was designed against the LRR domain of PpCLV1b and expressed under the U6 promoter. Plants were co-transformed with pU6::Ppclv1b sgRNA, pNRF, and pACT::Cas9 (original paper). Sanger sequencing of full-length PpCLV1a and PpCLV1b loci in all lines verified mutations shown in three independently generated lines. Sequences in bold represent sgRNA target sites on WT loci, gray boxes highlight mutations arising, and schematics to the right illustrate the predicted effect of the mutation on the protein. Lines with labels in blue were reported in the original paper.

(C) Summary of PpCLV1 mutations at each locus in nine mutant lines currently in use. Lines with labels in blue were reported in the original paper.

(D) Previously described phenotypes of Ppclv1a, Ppclv1b, and Ppclv1a1b mutants are shared by three independently generated lines. Whereas WT plants, Ppclv1a, and Ppclv1b mutants have well-developed gametophores, Ppclv1a1b mutants have a defective 2D-3D growth transition. Ppclv1b and Ppclv1a1b, but not Ppclv1a mutants or WT plants, have tissue outgrowths at the base of gametophores. Lines with labels in bold were included in Figure 4 of the original paper.