Plasmid constructs and plant transformation

To make CRISPR-Cas9 knockout constructs, 20-bp gene-specific guide RNA sequences targeting MKP1and MYB4 in Arabidopsis were fused to pCAMBIA1300-pYAO:Cas9 and sgRNAs targeting exons of OsMKP1, OsMPK3, OsMPK6, OsMYB102 and OsMYB108 in rice were fused to pYLCRISPR/Cas9-MT. For overexpression constructs, the coding sequence (CDS) sequences were inserted into pCambia1300-MYC or pCambia1300-GFP (green fluorescent protein) vectors for Arabidopsis transformation or cloned into PUN1301-pUBI-Flag vector driven by the maize UBIQUTIN1 promoter for rice transformation. The constructs were introduced into Agrobacterium tumefaciens strain GV3101 (for Arabidopsis) and EHA105 (for rice) and then transformed into different genetic backgrounds to produce more than 15 independent transgenic lines for each construct. Further selection and validation were based on PCR-based sequencing or Western blotting.