AGAMOUS-LIKE 24 (AGL24) and AGAMOUS-LIKE 18 (AGL18) are two vital MIKC-type transcription factors to mediate flowering transition in plants. Previous studies indicated that AGL24 acted as an activator but AGL18 served as an inhibitor from vegetative to reproductive growth. However, it is still elusive whether and how AGL24 directly interacts with AGL18 protein. In this study, three homologues of AGL18 (AGL18-1, AGL18-2 and AGL18-3) and AGL24 were cloned in Brassica juncea. Three AGL18 homologues and AGL24 decoded MIKC-type proteins, which were most highly conserved in the M and C domains. AGL18-1, AGL18-2 and AGL18-3 were quite different in the tertiary protein structures. Yeast two-hybrid and pull-down assays showed that AGL18-1, -2 and -3 interacted with AGL24 directly and their K domains of AGL18s were sufficient for their protein interactions. Additionally, mutations in the K domains of AGL18s were costructed and protein interactions were further detected. The results indicated that AGL18-2(L113F), AGL18-2(E116H), AGL18-2(L118F), AGL18-2(K165T), AGL18-3(L114P), AGL18-3(E117G), AGL18-3(R118G) and AGL18-3(L119P) still interacted with AGL24. However, interestingly, AGL24 interacted with AGL18-1(L114V) and AGL18-1(E117V) but not AGL18-1(K190I). It suggested that the 190th amino acid residue of AGL18-1 played crucial roles in mediating the protein dimerization of AGL18-1/AGL24 in flowering time control. (C) 2019 Elsevier Inc. All rights reserved.