Fig 1.
Schematic representation of CPP-FP/Citrine complexes formation.
The negatively charged Citrine was ionically combined with molecular binding sequence of the CPP-FP to form the CPP-FP/Citrine complexes.
Fig 2.
Physicochemical properties of CPP-FP/Citrine complexes prepared at different molar ratios.
(A) Hydrodynamic diameters and zeta potentials of BP100(KH)9/Citrine prepared at molar ratio from 1 to 30. (B) Hydrodynamic diameters and zeta potentials of BP100CH7/Citrine prepared at molar ratios from 1 to 100. All data represent means ± standard deviations (n = 3).
Fig 3.
Plant regeneration test from callus in different size.
Rice seeds were grown on N6D medium for (A) 5 days or (B) 21 days at 30°C with continuous light. Mature embryo-derived rice callus (C, D) were cut into small pieces of different sizes, then placed on regeneration medium (E, F) and cultured under the same conditions for one week. The smallest callus show plant regeneration is marked in red boxes.
Fig 4.
Single-cell quantification of Citrine fluorescence.
The single cell area was randomly selected, and their fluorescence intensity were calculated by CLSM. The results of Citrine delivery by (A) BP100(KH)9, (B) BP100CH7, and (C) Milli-Q water into the 5-day callus cells and of (D) BP100(KH)9 and (E) Milli-Q water into 21-day callus cells are shown. (F) The averaged intensity of the fluorescence from single cell was calculated by CLSM. All data represent means ± standard deviations (n = 10). The scale bar is 10 μm.
Fig 5.
Observation of intracellular distribution of BP100(KH)9/Citrine and BP100CH7/Citrine complexes by CLSM.
The red color comes from FM4-64 dye, which was used for cell membrane staining. The yellow arrows represent the Citrine, the red arrows represent cell membrane debris stained with FM 4–64, the orange arrows represent a complex of Citrine and cell membrane debris. The white arrows indicate symbiotic bacteria inside the cell. The negative control and Citrine represent samples infiltrated with Milli-Q water or Citrine only, respectively. The scale bar is 10 μm.
Fig 6.
Observation of intracellular Citrine delivery using BP100(KH)9, BP100CH7 and Citrine only.
The yellow spots represent Citrine positions. The red spots represent the cell membrane stained by FM4-64. The orange spots represent of FP-CPP/Citrine and cell membrane debris complexes. The 3D structures were constructed by Imaris, and the pictures used are all from the CLSM Z-stack results.
Fig 7.
Western blot analysis of Citrine extracted from 5-day rice callus after 72 hours post-infiltration.
Western blot bands from the cell debris. (A) The reversible ponceau staining is performed to check equal loading of the gel. (B) Quantitative analysis of those bands from western blot by ImageJ. All data represent means ± standard deviations (n = 5).