Fig. 4

Functional analysis of CitHAG28 and its protein interaction with CitGATA7. A Transcript levels of CitHAG28 in CitHAG28-OE citrus callus. B Citric acid content in CitHAG28-OE citrus callus. C Transcript levels of CitHAG28 in CitHAG28-TRV citrus seedlings. D Citric acid content in CitHAG28-TRV citrus seedlings. E Expression pattern of CitHAG28 in ‘SY’ and ‘GZ’ fruit. Error bars represent the SE (n = 3). Asterisks denote significant differences using Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001). LSD values were calculated at p = 0.05. F Y2H assays showing the interaction between CitGATA7 and CitHAG28. Transformants were spotted on SD/-Trp/-Leu (DDO), SD /-Trp/-Leu/-His/-Ade (QDO), or QDO with 3-AT (1 mM) media. pOst1-NubI, positive control; pPR3-N, negative control. G LCI assays showing the interaction between CitGATA7 and CitHAG28 in Nicotiana benthamiana leaves. The color bar ranges from purple to red suggesting that the luciferase activity strength is from weak to strong. H BIFC assay showing the interaction between CitGATA7 and CitHAG28 in transgenic N. benthamiana leaves. The fluorescence of GFP represents protein–protein interactions. Bar = 20 μm. 3-AT, 3-amino-1,2,4-triazole; BIFC, bimolecular fluorescence complementation; GFP, green fluorescence protein; LCI, luciferase complementation imaging; SD, synthetic dropout; Y2H, yeast two-hybrid