CORRECTION

Construction of the Triple Mutants

The triple mutants were generated by either knocking down PLIP1 using artificial microRNA or knocking out PLIP1 using the CRISPR-Cas9 system in the background of the plip2-2/3-1 double mutant.

The amiRNA constructs were produced using the protocol provided by WMD3-Web MicroRNA Designer (http://wmd3.weigelworld.org/downloads/Cloning_of_artificial_microRNAs.pdf). The amiRNA containing precursor sequence was amplified by overlapping PCR with the primers listed in Supplemental Table 1, before finally inserting it into the pEarley100 plasmid. The resulting construct was introduced into the plip2-2/3-1 double mutant. The progeny lines were first selected for Basta resistance and then confirmed by qPCR analysis. The two lines used for this study were selected based on their strong repression of PLIP1.

For generation of the CRISPR lines, the one-plasmid CRISPR-Cas9 cloning system (Feng et al., 2014) was used to mutate PLIP1 in the plip2-2/3-1 double mutant background. The oligo duplex generated from the PLIP1 single guide (sg)RNA primers (Supplemental Table 1) was first ligated into the Bbs I-digested px260 plasmid before the fragment containing both PLIP1-sgRNA and Cas9 were inserted into the pCAMBIA1300 plasmid. The final construct was then introduced into Agrobacterium tumefaciens for plant transformation.

For genotyping of PLIP1 mutated lines, total genomic DNA was extracted from individual lines and the regions containing the CRISPR target sites were amplified by PCR using the primers listed in the Supplemental Table 1. The PCR products were digested with Dpn II and plant lines showing a partially or completely uncut band were chosen. The homozygous T₂ lines were identified based on PCR products that could not be cut by Dpn II. The PCR products were further confirmed by sequencing. The lines showing frame-shift mutations were identified as homozygous lines.

Germination Assay

Freshly harvested seeds from plants grown in the same chamber were dried in a 37°C chamber for 4 days before use. Seeds were first sterilized in 30% bleach for 15 min with gentle rotation, then rinsed with autoclaved deionized water for 5 times in a sterile bench. Rinsed seeds were kept in water at 4°C in the dark for 2 days for stratification. The seeds were sown on 0.6% agar-solidified plates containing specific concentration of ABA (Sigma; Catalog No. A4906). A dissecting microscope was used to assess seed germination, which was defined by radicle emergence exceeding half of the seed length. Observation of 100 seeds in one plate was treated as one biological repeat. Germination rates were recorded on the indicated day past sowing.