Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system

Abstract

A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe. With this clone, various MADS box genes that encode for protein-to-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method. On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family. One of the interaction partners, OsMADS14, was selected for further study. Both genes began expression at early stages of flower development, and their expression was extended into the later stages. In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/lemmas, stamens, and carpels. Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14. Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction. Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement.

Figure 1

Deduced amino acid sequence comparisons of MADS box proteins. A, Alignment of the amino acid sequences of OsMADS6, ZAG3, ZAG5, and AGL6. The two conserved motifs of the C region are indicated in bold. B, Alignment of the amino acid sequences of OsMADS14 and ZAP1, an AP1 homolog of maize. The MADS box regions are underlined and the K domains are double underlined. Asterisks indicate identical amino acid residues. Dashes indicate gaps introduced to maximize alignments.

Figure 2

The nucleotide sequence and the deduced amino acid sequence of OsMADS14. The MADS box region is underlined, and the K domain is double underlined. The 10 repeats of the GGA sequence in the 5′-UTR are indicated in bold type. The primer sequence used in isolation of the 5′ region of the gene is underlined. Arrowheads and numbers below the amino acid sequence indicate positions of the first amino acid of the fusion proteins selected by the yeast two-hybrid screening and the number of selected clones with the same first amino acid, respectively. The XhoI site used for generation of the gene-specific probe (accession no. AF058697) is indicated in bold type.

Figure 3

Genomic DNA blot analysis and RNA blot analysis of OsMADS6 and OsMADS14. A, Southern blot analysis of OsMADS6 and OsMADS14. The rice genomic DNA was digested with EcoRI (E), HindIII (H), or PstI (P). The numbers indicate the size (in kb) of the DNA markers. B, RNA blot analysis of OsMADS6 and OsMADS14. Ethidium bromide staining of 25S and 17S rRNAs demonstrated equal amounts of RNA loading (data not shown). Lane L, Leaves; lane R, roots; lane Sl, sterile lemmas; lane P, paleas/lemmas; lane Lo, lodicules; lane S, stamens; lane C, carpels; lane 1, young flowers with a panicle size of 1 to 5 cm; lane 2, flowers at the early vacuolated pollen stage; lane 3, flowers at the late vacuolated pollen stage.

Figure 4

Amino acid sequence alignment of the region containing the K domain of OsMADS6 with those of OsMADS1, OsMADS5, OsMADS7, OsMADS8, OsMADS14, OsMADS15, OsMADS17, and OsMADS18. The region of OsMADS6 was divided into three regions: the KI region (amino acids 86–110), the KII region (amino acids 109–137), and the KIII region (amino acids 138–170). The entire K domain of OsMADS6 is underlined. The replaced amino acids are indicated below each Leu with arrows. The conserved hydrophobic residues, such as Leu, Ile, Val, and Met, are shaded. The numbers indicate the positions of mutagenized Leu residues and the first and last amino acids of the K region elucidated in this study.

Figure 5

Phylogenetic tree showing the relationship among AP1/AGL9 family proteins. Rice MADS box proteins are indicated in bold type. The horizontal branches are proportional to the number of base substitutions. 1, 7, 8, 18, 23, 24, and 25, Arabidopsis; 9, 19, 20, and 26, snapdragon; 10, 14, and 15, maize; 11, 12, 13, 16, 17, 27, 28, 29, and 30, rice; 21, petunia; 3, potato; 4 and 22, tomato; 5, Silene latifolia; 2 and 6, Sinapis alba.