A Vector-Free Gene Interference System Using Delaminated Mg–Al-lactate Layered Double Hydroxide Nanosheets as Molecular Carriers to Intact Plant Cells

Abstract

Background: Mg–Al-lactate Layered Double Hydroxide Nanosheets is an optimal nanocarrier for widely application in plant, However, a standardize vector-free LDH-dsRNA system doesn’t clear describe in past research in different tissues in model and non-model species in plant sciences. Results: Here, we introduce a vector-free system to interfere with gene expression in intact plant cells. This method uses delaminated Mg–Al-lactate layered double hydroxide nanosheets (LDH-NSs), which can easily be synthesized via co-precipitation method. Briefly, double-stranded RNA (dsRNA) targeting genes of interest are synthesized in vitrousing T7 RNA polymerase, adsorbed onto the LDH-NSs, and transported by the LDH-NSs into intact plant cells. The LDH-dsRNA system was tested against housekeeping gene, ACTIN2, finding that only 30 minutes of soaking in medium containing LDH-dsRNA led to 80% gene silencing in Arabidopsis thaliana seedlings. The LDH-dsRNA system also efficiently knocked-down tissue-specific genes in Arabidopsis and tobacco (Nicotiana benthamiana), such as phytoene desaturase (PDS), WUSCHEL(WUS), WUSCHEL related homeobox 5 (WOX5), and ROOT HAIR DEFECTIVE 6 (AtRHD6). Finally, we used the LDH-dsRNA system to target nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes in cassava (Manihot esculenta), an economically important plant. The LDH-dsRNA system successfully downregulated these genes, decreasing the bacterial resistance of the cassava leaves. Conclusions: Therefore, we believe that the vector-free LDH-dsRNA system described, optimized, and validated herein has wide application prospects as an efficient, broad-spectrum RNA interference method for the plant and agricultural sciences.

Mg–Al LDH nanosheets as a nanotechnological tool in agriculture: an exploratory toxicity evaluation study

“…This indicates that the delivery protocol is a crucial component to consider in evaluating the NP effects. 29 Considering this and compared to previously published works on the use of Mg-Al LDH as a molecule carrier, 24,34,36 this is the first study carried out in whole plant leaves with a technique that allows better control of the nanoparticle concentration to which leaves have been exposed since spray experiments may have more significant variability in the final nanoparticle quantity that encounters plant cells. 44,68 Scanning electronic microscopy was performed to unravel any possible effect of the Mg-Al LDH on the leaf's surface (Fig.…”

“…, where M 2+ is a divalent metal cation (e.g., Mg 2+ , Ni 2+ , or Zn 2+ ), M 3+ is a trivalent metal cation (e.g., Al 3+ , Fe 3+ , or Cr 3+ ), A n− represents the interlayer inorganic or organic anions that compensate the charge (e.g., ), and x is a calculated ratio between M(II)/ M(III) + M(II), with values ranging from 0 to 0.4. 28 Among their relevant characteristics, those related to their adsorption/ desorption and thermal stability properties have captivated researchers' attention because they can be used in drug delivery, 21,29 adsorbents, 30 catalysts, 31 and fire retardants. 25 Although Mg-Al LDH has been proposed and probed as a delivery system for pathogen control compounds and plant fertilizers in different organisms, 24,29,32,33 these studies do not focus directly on the toxicity of the nanostructure itself on target organisms; instead, they evaluate the general effect that involves other components, making its possible toxicological effect hard to establish.…”

“…28 Among their relevant characteristics, those related to their adsorption/ desorption and thermal stability properties have captivated researchers' attention because they can be used in drug delivery, 21,29 adsorbents, 30 catalysts, 31 and fire retardants. 25 Although Mg-Al LDH has been proposed and probed as a delivery system for pathogen control compounds and plant fertilizers in different organisms, 24,29,32,33 these studies do not focus directly on the toxicity of the nanostructure itself on target organisms; instead, they evaluate the general effect that involves other components, making its possible toxicological effect hard to establish. Despite this, a previous study has established that Mg-Al LDH did not induce cellular damage in A. thaliana plants at different concentrations.…”

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes

“…LDHs belong to a family of inorganic layered materials comprising positively charged hydroxide layers that are intercalated with anions. 24 Furthermore, it has the advantages of high reliability, good biological compatibility, and minor side effects, with the average particle size being 50− 300 nm. Studies showed that LDH nanosheets can be loaded with large dsRNA to form dsRNA−LDH complexes, resulting in successful dsRNA uptake into plant cells and RNAi induction, thus providing sustained plant disease protection.…”