ERD7-mCherry-Expressing and CRISPR/Cas9 erd7 Knockout Arabidopsis Lines

To generate Arabidopsis Col-0 plants stably-expressing ERD7-mCherry, selected hygromycin-resistant T₁ seedlings obtained after Agrobacterium-mediated plant transformation with pMDC32/ERD7-mCherry binary vector were advanced and resulting T₂ progeny were analyzed for ERD7-mCherry fluorescence using CLSM. Unfortunately, for reasons unknown, we were unable to generate homozygous T₃ lines stably-expressing ERD7-mCherry and, thus, experiments in this study were limited to assessing the subcellular localization of ERD7-mCherry in T₂ lines.

Arabidopsis erd7-2 mutant plants were generated using CRISPR/Cas9 genome editing of Col-0 based on the procedures described by Wang et al. (2015). Briefly, a pBEE401E-based vector containing the egg-cell-expressed CAS9 open reading frame (ORF) and a pair of single-guide RNAs (sgRNAs) corresponding to sequences near the 5' and 3' ends of the ERD7 ORF was introduced into Arabidopsis using the floral dip method (Clough and Bent, 1998); refer to Molecular Cloning and Plasmid Construction section for additional details on the construction of the pBEE401E-based vector used for CRISPR/Cas9 genome editing of ERD7 and Supplementary Table 1 for sequences of sgRNAs and all other oligonucleotide primers used in this study. Genomic DNA (gDNA) of BASTA [i.e., phosphinothricin (Gold Biotechnology)]-resistant T₁ plants was screened for the loss of the ERD7 ORF using the PCR and gene-specific primers (Supplementary Table 1) and selected erd7 mutant plants were advanced. gDNA from T₂ progeny plants was then screened by PCR [and with the appropriate primers (Supplementary Table 1)] for the CAS9-containing T-DNA insert in order to identify plant lines that had lost the T-DNA through genetic segregation. Thereafter, edited erd7 mutant and CAS9-deficient T₃ plants were analyzed by PCR genotyping and deletion of a sequence encoding 398 amino acids of the 452-amino-acid-long ERD7 polypeptide was confirmed by sequencing of the ERD7 PCR product. Refer to Supplementary Figure 1 for details on the relative locations of the sgRNAs and the region of the ERD7 ORF that was removed by CRISPR/Cas9 genome editing in the erd7-2 mutant line and the position of the T-DNA insertion in the erd7-1 mutant line, as well as the genotyping and reverse transcriptase (RT)-PCR results for both mutant lines.

All DNA sequencing carried out in this study, including the sequencing of newly-constructed plasmids (see Molecular Cloning and Plasmid Construction section), was performed at the University of Guelph Genomics Facility or by Retrogen Inc.