Fig. 3: Genetic confirmation of the GTRC locus.
From: A minus-end directed kinesin motor directs gravitropism in Physcomitrella patens
a–d Targeted mutagenesis of GTRC in wild type. a The native sequence encoding Q598 was mutated to reproduce the stop codon (X) mutation identified in the gtrC-5 mutant. WT P. patens was transformed with a linear 1537-bp fragment of DNA comprising the four exons surrounding the Q598 codon in exon 14 using a transient selection strategy. The WT and mutant sequences are shown above and below. b Gravitropism in WT, gtrC-5 and gene-targeted WT P. patens. Caulonemal growth in darkness for 3 weeks on vertical BCD agar medium. The untransformed WT and gtrC-5 mutant lines are shown in the upper panel together with a transgenic line A85 that did not alter the Q598 residue. The lower panel shows three transgenic lines (B59, D25, and G85) with a Q598X mutation. c Sequence analysis of the mutant locus in the gene-targeted lines, compared with WT and the original gtrC-5 mutant. Lines B59, D25, and G85 contain the targeted mutation (CAGAGT > TAATCA), resulting in Q598X (CAG > TAA). Line A85 contains the targeted mutation of Ser599 (AGT > TCA) but unmutated Q598 (CAG > CAA). The nucleotides coding these two amino acids were labeled in the black box. d Southern blot analysis of the gene-targeted mutant lines. The genomic DNA was digested by Eco RI and then detected by labeled ssDNA. Only one band of the expected size (ca. 4.64 kb) was detected in each line, confirming that no off-target integration occurred. The experiment was carried out once, and the result is unambiguous. See methods for details, and source data are provided as a Source Data file. e Gravitropic phenotypes of knockout lines of GTRC. GTRC-HRKO was generated via homologous recombination by substituting GTRC genomic region with the NptII cassette. GTRC-Cas9KO was generated by editing the GTRC locus via CRISPR-Cas9 mutagenesis. f Gravitropic phenotypes of complementation lines of GTRC. HARes/gtrC-16 was generated by substituting the mutated nucleotide of gtrC-16 with wild-type genomic nucleotide. ProGTRC::GTRC/gtrC-16 was generated by substituting the mutated genomic DNA of GTRC in gtrC-16 with wild-type GTRC coding sequence. The details of the constructs and genotypes for the P. patens lines in e–f are shown in Supplementary Fig. 1. g Gravitropic phenotypes of knockout lines of KCH family genes. All lines except kchb (gtrC-16) were generated by editing the corresponding KCH loci via CRISPR/Cas9 mutagenesis. Genotypes for these lines are shown in Supplementary Fig. 3. In (b, e, f, g), arrows labeled with “g” indicate the directions of gravity vectors, and the scale bars are 3 mm.
