Fig. 3

Interactions of CAT with OsNCA1a and / or OsNCA1b. (A) BiFC assay. NCA1 interacts with CATC in the cytosol. Plasmids were introduced into rice protoplasts and fluorescence was detected by confocal microscopy. Top row, NYFP-NCA1a and CYFP-CATC were cotransformed. Second row, NYFP-NCA1b and CYFP-CATC were cotransformed. Third row, NYFP-NCA1b and CYFP-NCA1a were cotransformed. Bottom row, NYFP vectors and CYFP-CATC were cotransformed. In each row, the first panel, bright-field image (BL); the second panel, chlorophyll autofluorescence (chlorophyll) in red; the third panel, YFP signal in yellow; and the fourth panel, merged image (Merge). Scale bars, 5 μm. (B) Yeast three-hybrid assay 4. Prey (pGADT7) and bait (pBridge) constructs were transformed into Y2Hgold yeast strain solely or together to examine the interactions. pBridge contains two distinct multiple cloning sites (MCS) allow expression of the BD (DNA binding domain) fusion as well as a third protein. “pBridge BD and pBridge MCSII” represent the first and the second MCS link gene in pBridge vector, and expressed separately in yeast. “-” represent pBridge BD or pBridge-MCSII without any gene fusion was used as a control. The pGADT7-NCA1a and pGADT7-NCA1b indicated that the pGADT7 AD (DNA Activation Domain) vector MCS were connected to the NCA1a, NCA1b respectively and expressed in yeast Y2Hgold cells. The picture shows yeast growth on selective dropout media lacking Methionine/Tryptophan/Leucine (SD-MWL) or Methionine/Tryptophan/Leucine/Histidine/Alanine (SD-MWLHA) to check for interactions between the three partners. (C) The activity of CATC from pBridge MCSII site. NCA1a: the yeast transformed with pBridge BD-NCA1a + MCSII-NCA1a and pGADT7-NCA1a grow on SD-MWL; NCA1a + CATC: the yeast transformed with pBridge BD-NCA1a + MCSII-CATCa and pGADT7-NCA1a grow on SD-MWL. The CAT activities of the total protein extracted from yeast cells were measured. Data represent means ± SD of at least three replicate experiments. Different letters on the top of columns indicate significant difference at p < 0.05 according to Duncan’s multiple range test