Polymeric Nanocarriers Autonomously Cross the Plant Cell Wall and Enable Protein Delivery for Stress Sensing

Abstract

Delivery of proteins in plant cells can facilitate the design of desired functions by modulation of biological processes and plant traits but is currently limited by narrow host range, tissue damage, and poor scalability. Physical barriers in plants, including cell walls and membranes, limit protein delivery to desired plant tissues. Herein, a cationic high aspect ratio polymeric nanocarriers (PNCs) platform is developed to enable efficient protein delivery to plants. The cationic nature of PNCs binds proteins through electrostatic. The ability to precisely design PNCs' size and aspect ratio allowed us to find a cutoff of ≈14 nm in the cell wall, below which cationic PNCs can autonomously overcome the barrier and carry their cargo into plant cells. To exploit these findings, a reduction-oxidation sensitive green fluorescent protein (roGFP) is deployed as a stress sensor protein cargo in a model plant Nicotiana benthamiana and common crop plants, including tomato and maize. In vivo imaging of PNC-roGFP enabled optical monitoring of plant response to wounding, biotic, and heat stressors. These results show that PNCs can be precisely designed below the size exclusion limit of cell walls to overcome current limitations in protein delivery to plants and facilitate species-independent plant engineering.

Keywords: nanocarrier; plant engineering; polymers; protein delivery; stress sensing.

Figure 1.. Synthesis and characterization of cationic high aspect ratio polymer nanocarrier (PNC) for protein delivery in plants and their complexation with protein.

(a) Synthesis procedure of high aspect ratio bottlebrush polymer nanocarriers with permanent positive charge. (b) Protein grafting onto cationic PNCs. (c) Binding energy between GFP and polymer arm in PNC investigated by molecular dynamic simulation. Total interaction energy and binding energy of polymer-GFP interactions are reported in Table S2. (d) Height profile of unloaded PNC and PNC complexed with green fluorescence protein at PNC to GFP mass ratios of 0.5, 1.5 and 3.0. (e) AFM height of PNC before and after loading with GFP at different mass ratios. Scale bars, 100 nm. (f) AFM width of PNC before and after loading with GFP at different mass ratios. (g) GFP complete complexation with PNC confirmed by PAGE-gel electrophoresis. (h) Apparent zeta potential of unloaded and GFP loaded PNC in PBS buffer at pH 7.4. Apparent zeta potential was calculated from electrophoretic mobility using the Smoluchowski approximation. (i) Degradation of free GFP and PNC-GFP complex by major plant proteases bromelain, ficin and papain. Error bars represent standard deviation (n=4). ANOVA testing followed by Fisher’s LSD testing was used for multiple comparisons, P≤ 0.05.

Figure 2.. Protein delivery into Nb plant cells by PNC complexed with different PNC to GFP mass ratios post foliar application.

(a) Schematic illustration of PNC enables protein uptake into plant cell (b) PNC-GFP complex internalization into plant cells post infiltration shown by imaging GFP with confocal microscopy for PNC to GFP mass ratio 3.0, 1.5, 0.5 and free GFP. (c) High magnitude image of PNC-GFP after taken up by plant leaf cells. The cell wall was stained by calcofluor white (CFW). (d) Quantitative fluorescence intensity analysis of Nb confocal images for free GFP and PNC-GFP complexes. Error bars represent standard deviation (n=6). ANOVA testing followed by Fisher’s LSD testing was used for multiple comparisons, P≤ 0.05. (e) PNC-GFP complex entered Nb leaf protoplast 6 h after incubation in W5 solution. (f) Z-stack analysis of the fluorescence profile of the PNC-GFP treated Nb leaf epidermis and mesophyll close to the infiltration area. Green: GFP, blue: DAPI, magenta: chlorophyll. Scale bars, 50 μm.

Figure 3.. Protein delivery into Nb plant cells by PNC composed of 10, 20 or 50 degree of polymerization (DP) arms with different height and widths.

GFP delivery by (a) PNC with 10 DP polymer arms. (b) Wider PNC20 composed of 20 DP polymer arms. (c) GFP delivery by widest PNC50 composed of 50 DP polymer arms. (d) GFP delivery by widest PNC50. Plant leaves were treated by cell wall degrading enzymes before PNC-GFP infiltration. Scale bars, 50 μm. (e) Quantitative fluorescence intensity analysis of Nb confocal images for GFP delivered by PNC, PNC20, PNC50 and PNC50 with cell wall degrading enzymes. Error bars represent standard deviation (n=4-6). ANOVA testing followed by Fisher’s LSD testing was used for multiple comparisons, P≤ 0.05. (f) Schematic illustration of cell wall inhibiting uptake of PNC50 into plant cells, while the thinner PNC with 10 or PNC20 with 20 DP arms can be taken up by plant cells autonomously, suggesting a rigid size cutoff at ~14-24 nm in cell wall.

Figure 4.. Reduction-oxidation sensitive green fluorescent protein (roGFP) and PNC-roGFP complex response to ROS and reducing agent DTT in vitro.

(a) Excitation spectra, and (b) fluorescence emission spectra at 405 nm, and (c) Emission spectra at 488 nm for free roGFP in response to 0.1-5 mM H₂O₂ for 20 min. (d) Excitation spectra, (e) fluorescence emission spectra at 405 nm, and (f) Emission spectra at 488 nm for PNC-roGFP complex in response to 0.1-5 mM H₂O₂ concentrations. Time course for the 405 and 488 nm emission ratio (R_405/488) values during successive 0.1-5 mM H₂O₂ oxidation and 5 mM DTT reduction for (g) free roGFP and (h) PNC-roGFP complex. (i) PNC-roGFP sensor did not show significant change in R_405/488 after exposed to stress-associated plant ions, sugars, and hormones. Error bars represent standard deviation (n=3).

Figure 5.. In-vivo plant stress monitoring by PNC-roGFP complex sensor in Nicotiana benthamiana, tomato and maize plants.

(a) Schematic depicting PNC-roGFP delivered into Nb plant leaf cells detect plant stress by responding to ROS and shift their fluorescence emission profiles. (b) roGFP fluorescence at 405 and 488 nm excitation in Nb plants before stress conditions, after immersed in 10 mM H₂O₂, wounding, infiltrated with flg22 peptide, immersed in 10 mM DTT. (c) 405 and 488 nm emission ratio (R_405/488) values in Nb plants calculated from CLSM images. roGFP fluorescence at 405 and 488 nm excitation in dicot tomato plants (d) before stress conditions, treated with 10 mM H₂O₂ and heat stress. (e) roGFP fluorescence at 405 and 488 nm excitation in monocot maize plants before stress conditions, treated with 10 mM H₂O₂ and heat stress. (f) 405 and 488 nm emission ratio (R_405/488) values in tomato and maize plants before and after stressors calculated from CLSM images. Scale bars, 50 μm. Error bars represent standard deviation (n=6). ANOVA testing followed by Fisher’s LSD testing was used for multiple comparisons, P≤ 0.05.