Figure 1.
OsPHO2 interacts with OsTrxh1 and OsTrxh4. A, Phylogeny of h-type thioredoxin (Trxh) proteins from Arabidopsis (At), rice (Os), Hordeum vulgare (Hv), and Triticum aestivum (Ta). Phylogenetic analyses were conducted in MEGA6, and the tree was generated using the neighbor-joining method. Trxh proteins were divided into three subgroups as indicated. B, Identification of the interaction between OsPHO2 and rice Trxh family members by Y2H assays. For bait, OsTrxh1/h2/h3/h6 (subgroup I), OsTrxh4/h5 (II), and OsTrxh7/h8/h9/h10 (III) were used. For prey, full-length OsPHO2, OsPHO2N1–633 (N-terminal amino acids 1 to 633) and OsPHO2C589–876 (C-terminal amino acids 589 to 876) were used. Yeast transformants were cultured on selection medium QDO (SD/-Leu-Trp-His-Ade). Positive control (+), pGAD-SV40 mated with pGBK-53; negative control (−), pGAD-SV40 mated with pGBK-Lam; empty, pGBKT7 or pGADT7-Rec without any insert. C, Y2H assays to probe for interaction between PHO2 and Trxh proteins of rice and Arabidopsis. OsTrxh1, OsTrxh4, and their homologs in Arabidopsis were used as bait. For prey, full-length AtPHO2, full-length OsPHO2 or OsPHO2N1–633, OsPHO2C589–876, and chimeric PHO2 proteins, AtPHO2 N-terminal fused with OsPHO2 C-terminal (AtN+OsC), OsPHO2 N-terminal fused with AtPHO2 C-terminal (OsN+AtC), and the OsPHO2 homologous protein OsUBC23 were used. Yeast cells were cultured on selection medium QDO and using X-α-gal as substrate for colorimetric detection of α-galactosidase activity. Blue box indicates the UBC domain. D, BiFC visualization of the interaction between OsPHO2 and OsTrxh1/h4 in rice protoplasts. Images were captured 12 to 15 h after polyethylene glycol-mediated transformation under a ZEISS LSM710nlo confocal laser scanning microscope. The ER marker was AtWAK2 (Nelson et al., 2007). The combinations of nYFP-OsPHO2 with empty vector cYFP, AtCNX1-nYFP with OsTrxh1-cYFP, nYFP-OsPHO2 with OsBiP3-cYFP, and AtCNX1-nYFP with OsTrxh4-cYFP, respectively, served as negative controls. Bars = 10 μm.