KO of ZmMTN1 but not ZmMTN2 by CRISPR/Cas9 causes interveinal chlorosis. A, Gene structure of ZmMTN1 and ZmMTN2, and target sites used for CRISPR/Cas9 editing. Two CRISPR/Cas9 constructs (Cas9-1 and Cas9-2) were generated, each targeted at one conserved site in ZmMTN1 and ZmMTN2 (indicated by arrows). The red and black arrows indicate edited and unedited sites, respectively. The premature stop-codons resulting from editing are indicated by red vertical bars in the gene structure. B and C, The target sites of CRISPR/Cas9 and edited sequences of ZmMTN1 and ZmMTN2. The protospacer-adjacent motif sequences are underlined and shown in blue color. The single T insertion in ZmMTN1 is indicated in red letters, while deletions are indicated with dashes. D, Interveinal chlorosis phenotype identified in homozygous Zmmtn1 KO plants harboring a heterozygous Zmmtn2 KO site, while no phenotype difference was observed from homozygous KO of ZmMTN2. Allelism test using seedlings from mic1-ref/+ × homozygous Zmmtn1-KO. The mic1-ref/Zmmtn1-KO seedlings showed the interveinal chlorosis phenotype, while ZmMIC1/Zmmtn1-KO seedlings exhibited the WT phenotype. The WT and KO images were digitally extracted for comparison. Bars = 1 cm.