Quantification of cell penetrating peptide mediated delivery of proteins in plant leaves

Background:

The delivery of molecules into plant cells is so far mainly based on genetic transformation by introduction of DNA into the genome. But not only is this a time-consuming, tedious process, the use of transgenic plants is also highly debated and tightly regulated by laws. Direct delivery of proteins presents a tool for DNA-free genome editing, however, this direct delivery is challenging in plant cells because of the presence of a cell wall, which serves as barrier for pathogens and macromolecules like DNA, RNA and proteins. To date, mostly small interfering RNAs are delivered into the cells via microcarriers like carbon dots and gold nanoparticles (Vega-Vásquez et al., 2020), while less tools are available for the delivery of peptides. Biolistic delivery of peptides to cells via nanoparticles requires protein dehydration which is accompanied by the potential loss of their function. A great limitation of exploring new delivery tools is the lack of a control system for the successful delivery of peptides into plant cells and its quantitative validation. Most quantification tools rely on the use of fluorescent markers, either by delivering a macromolecule encoding for a fluorescent protein or vice versa, silencing the expression of a fluorescent protein by siRNA (Watanabe et al., 2021). However, plant tissues are heterogeneous, light scattering and highly autofluorescent, making it difficult to distinguish signal from background noise. Furthermore, the vacuole in plant cells is very spacious, while the other cell organelles are pushed towards the periphery of the cells. This makes it challenging to distinguish between signal from successful delivery into the cytosol and signal from the cell wall directly adjacent, and to distinguish successful delivery from cargo accumulated in the cell wall. Therefore, the traditional approach of relying on confocal microscopy to quantify the presence of a fluorescent marker in the cell does not provide consistent and quantifiable results.

Key findings:

Wang et al. here introduce a feasible method for the quantification of successful delivery of peptides and bigger proteins to plant cells, by utilizing a fluorescence complementation-based method (Wang et al., 2022). In their system, Delivered Complementation in planta (DCIP), GFP is split between a larger non-fluorescent fragment (GFP1-10) and a smaller peptide strand of 16 amino acids (GFP11). Fluorescence is only reconstituted if both fragments are present in the same cellular compartment.  This requires the peptide shuttled together with GFP11 to stay intact, that no sequestration to any other than the target organelle occurs and that the cargo is not trapped in the apoplast. To test the system, the authors used GFP1-10 fused to mCherry with an N-terminal SV40 nuclear localisation signal and transiently expressed the construct in tobacco leaves (Figure 1). 3 days after the infiltration, the leaves were additionally infiltrated with an aqueous solution containing cargo that contains the GFP11 tag. After confocal imaging 4-5 h later and analysis with Cell Profiler for nuclear GFP and mCherry fluorescence, they quantified the green/red ratio in mCherry expressing nuclei. Cells transformed with the GFP-mCherry construct and successfully delivered cargo show red fluorescence of mCherry in the nucleus and green fluorescence of the reconstituted GFP.

For a proof of concept, Wang et al. used different cell penetrating peptides (CPPs) as carriers for the cargo. CPPs are small cationic or amphipathic peptides that consist of 5-30 amino acids and that enable cytosolic delivery when conjugated to cargoes. They tested different CCPs as carriers, such as nona-arginine (R9), in various concentrations, and discovered that concentrations above 50 μM were required for successful delivery and trespassing the background fluorescence.

They found that delivery in plants was not as efficient as in mammalian cells, but >40 % positive delivery could be achieved when leaves were infiltrated with 100 μM of R9-GFP11 or more, with a maximum positive delivery of 75 % at 300 μM. Then they tested different CPPs, whose ability for delivery into plant cells was previously proved by other methods. Two of them were successfully delivered, the third one only showed a successful delivery in rare instances, indicating that conjugation to different cargoes can alter the uptake rate. Temperature-treatment experiments and co-infiltration with endocytosis inhibitors showed that the CPP penetration was not dependent on endocytosis, and suggest that the cargo enters cells through direct membrane permeation. Analysing the delivery over a time course showed that the delivery could be detected 4 h after infiltration with the cargo, but was undetectable after 24 h. Together with immuno blotting, this indicates that the peptide is unstable in the leaf tissues, potentially affected by apoplastic proteases and intracellular degradation.

To show that DCIP also enables the detection of bigger recombinant proteins, the authors tagged mCherry N-terminally with GFP11 and C-terminally with a CPP and used it as cargo, with a final weight of 34.5 kDa. They detected GFP complementation also with this cargo, however, the delivery was less efficient than the one of smaller peptides. To test if the delivered proteins are able to mediate protein-protein interaction or mislocalisation of proteins, the authors delivered an actin binding peptide, tagged with GFP11 and a CPP into cytosolic DCIP (cytoDCIP) expressing tobacco leaves. They detected GFP fluorescence along the actin filaments, indicating that the GFP11 fused to the actin binding peptide was able to tether the cytoDCIP to actin.

Importance and future directions:

The described quantification method for the delivery of proteins to plant cells can be used to rapidly screen the effectivity of delivery peptides, and also account for the durability of the uptake and stability of the delivered peptides in the tissue. A similar system could be used for other delivery systems that transport RNA or DNA.

Direct delivery of macromolecules to cells enables DNA-free genome editing and DNA-free interference into physiological processes, avoiding tedious plant transformation and legal regulations of genetically modified organisms. Given that mCherry was able to penetrate the cells fused to CPPs, the delivery of Cas9 for DNA-free genome editing might ultimately become possible. Direct delivery of macromolecules might play an important role in future agriculture: it can drive the development of more precise pesticides forward, because bioactive molecules can be directly delivered into plant cells and thereby their effectivity is increased, while applied concentrations can be decreased. Furthermore, controlling the uptake and turnover rate of the active compound enables a time-controlled release to the target site (Vega-Vásquez et al., 2020).

 

References:

Vega-Vásquez, P., Mosier, N. S., & Irudayaraj, J. (2020). Nanoscale Drug Delivery Systems: From Medicine to Agriculture. Frontiers in Bioengineering and Biotechnology, 8, 1–16. https://doi.org/10.3389/fbioe.2020.00079

Wang, J. W., Goh, N., Lien, E., González-grandío, E., & Landry, M. P. (2022). Quantification of cell penetrating peptide mediated delivery of proteins in plant leaves. BioRxiv, 1–33. https://doi.org/https://doi.org/10.1101/2022.05.03.490515

Watanabe, K., Odahara, M., Miyamoto, T., & Numata, K. (2021). Fusion Peptide-Based Biomacromolecule Delivery System for Plant Cells. ACS Biomaterials Science & Engineering, 7(6), 2246–2254. https://doi.org/10.1021/acsbiomaterials.1c00227

Tags: fluorescence complementation, peptide delivery

doi: https://doi.org/10.1242/prelights.32247

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