Knockout (KO) mutants of PASPRO1 and PASPRO2 were generated using the CRISPR/Cas9 genome editing system. We chose single-guide RNA (gRNA) using the CRISPRdirect (36 (link)) tool (http://crispr.dbcls.jp/ ), and the annealed oligomers were ligated into the BsaI-digested pRGEB32 binary vector (Addgene no. 63142). For expression validation of the two MIF-Is using a reporter system, we amplified approximately 2kb of the promoters and ligated into the linearized RUBY vector (37 (link)) using the In-Fusion HD PCR Cloning Kit (#639648; Takara, Japan). The constructed plasmid was transformed into Agrobacterium (strain LBA4404) using the freeze-thaw method. Stable transformation was achieved using the Agrobacterium-mediated cocultivation method as described previously (38 ). For the subcellular localization assay, the coding regions of PASPRO1 and PASPRO2 were amplified and ligated into the HindIII-digested pGreen vector (35S promoter-driven GFP vector) using the In-Fusion HD PCR Cloning Kit (#639648; Takara, Japan). The information of all gRNAs and primers is presented in Supplementary Table S5 .
Prgeb32 binary vector
Manufactured by Addgene
The PRGEB32 binary vector is a plasmid used for gene expression in plant cells. It contains a multiple cloning site for inserting genes of interest and a selection marker for identifying transformed cells.
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2 protocols using prgeb32 binary vector
1
CRISPR-Mediated Knockout of PASPRO1 and PASPRO2
2
Subcellular Localization and Knockout of OFF
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To investigate subcellular localization, the coding sequences of OSAT.v7_FAR1.Chr10.2 (hereafter called OFF) and OsRH36 were amplified and fused with GFP and mCherry, respectively, in the HindIII-digested pGreen vector using the In-Fusion HD PCR Cloning Kit (Takara). The plasmids were transformed into Agrobacterium tumefaciens GV3101 individually and used for tobacco infiltration assays as described below. To generate a knockout mutant of OFF using CRISPR/Cas9 genome editing, we designed a single-guide RNA through CRISPRdirect (http://crispr.dbcls.jp/ ) (31 (link)). Designed oligomers were synthesized, and annealed oligomer was ligated into the BsaI-digested pRGEB32 binary vector (Addgene plasmid ID: 63142). After the plasmid was transformed into A. tumefaciens LBA4404, stable transformation of rice was performed using cv. Dongjin through the Agrobacterium-mediated co-cultivation method as described in Lee et al. (32 ). The primers for vector construction are listed in Supplementary Table S9 .
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