Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts

Abstract

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

Fluorescence complementation enables quantitative imaging of cell penetrating peptide-mediated protein delivery in plants including WUSCHEL transcription factor

“…Furthermore, technical limitations render microscopic analysis of uptake only suggestive of uptake rather than confirmative, and preclude quantification of relative CPP delivery efficiencies. Previously, final confirmation of delivery in plants has required lengthy functional analysis such as identifying delivery mediated gene editing (Martin-Ortigosa, Peterson et al 2013), protein expression (Demirer, Zhang et al 2018, Thagun, Horii et al 2022, or silencing (Zhang, Goh et al 2022). We therefore sought to develop a technique by which delivery designs could be rapidly and accurately assessed, shown here for CCP-mediated peptide and protein delivery, but could be generically extended to testing of other carriers.…”

“…CPPs, are small cationic or amphipathic peptides that when conjugated to cargoes, enable cytosolic delivery (Langel 2011). We utilized CPPs to test DCIP due to their synthetic accessibility, previous deployment in plant-tailored delivery schemes (Numata, Horii et al 2018, Thagun, Horii et al 2022), and because much of their underlying cell penetrating mechanisms in plants remain unstudied. In doing so, we show that DCIP can confirm the delivery of as little as 10µM of GFP11 peptide via confocal imaging and that nearly comprehensive (>78%) delivery to leaf cells is achieved with 300 µM of CPP delivered GFP11.…”

“…CPPs, are small cationic or amphipathic peptides that when conjugated to cargoes, enable cytosolic delivery (Langel 2011). We utilized CPPs to test DCIP due to their synthetic accessibility, previous deployment in plant-tailored delivery schemes (Numata, Horii et al 2018, Thagun, Horii et al 2022, and because much of their underlying cell penetrating mechanisms in plants remain unstudied.…”

Temperature-Responsive Bottlebrush Polymers Deliver a Stress-Regulating Agent In Vivo for Prolonged Plant Heat Stress Mitigation

“…There are a few potential ways to improve plant resilience to heat stress. Plant bioengineering may generate transgenic plants with heat stress tolerance. , However, the challenges in understanding the physiological effects of the transgenes at the whole plant level, the high upstream production costs from plant regeneration and elite line propagation, and public concerns regarding biosafety of transgene products limit their application . Foliar application of active agents is another potential solution. − For example, anionic cerium oxide nanoparticles (NPs) infiltrated into Arabidopsis thaliana leaves scavenged reactive oxygen species (ROS) in plants and alleviated heat stress .…”

“…Plant bioengineering may generate transgenic plants with heat stress tolerance. 21 , 22 However, the challenges in understanding the physiological effects of the transgenes at the whole plant level, 23 the high upstream production costs from plant regeneration and elite line propagation, and public concerns regarding biosafety of transgene products limit their application. 22 Foliar application of active agents is another potential solution.…”