A vector-free gene interference system using delaminated Mg-Al-lactate layered double hydroxide nanosheets as molecular carriers to intact plant cells

He Zhang; Xinyu Li; Dong Yu; Junqi Guan; Hao Ding; Hongyang Wu; Qiang Wang; Yinglang Wan

doi:10.1186/s13007-023-01021-1

A vector-free gene interference system using delaminated Mg-Al-lactate layered double hydroxide nanosheets as molecular carriers to intact plant cells

Plant Methods. 2023 May 8;19(1):44. doi: 10.1186/s13007-023-01021-1.

Authors

He Zhang^#^{1

2}, Xinyu Li^#¹, Dong Yu¹, Junqi Guan¹, Hao Ding¹, Hongyang Wu³, Qiang Wang⁴, Yinglang Wan⁵

Affiliations

¹ Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Tropical Crops, Hainan University, Haikou, 570228, China.
² Key Laboratory of Integrated Pest Management On Tropical Crops, Ministry of Agriculture and Rural Affairs, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China.
³ Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
⁴ College of Environmental Science and Engineering, Beijing Forestry University, 35 Qinghua East Road, Haidian District, Beijing, 100083, China.
⁵ Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Tropical Crops, Hainan University, Haikou, 570228, China. ylwan@hainanu.edu.cn.

^# Contributed equally.

Abstract

Background: The Mg-Al-lactate layered double hydroxide nanosheet (LDH-NS) has shown great potential as an optimal nanocarrier for extensive use in plants. However, previous studies in plant sciences have not provided a clear description of the application for the LDH-NSs-based double-stranded RNA (dsRNA) delivery (LDH-dsRNA) system in different tissues of both model and non-model species.

Results: LDH-NSs were synthesized by using the co-precipitation method, while the dsRNAs targeting genes of interest were prepared in vitro using T7 RNA polymerase. The LDH-dsRNA bioconjugates with a neutral charge were produced by incubating with the mass ratio of LDH-NSs to dsRNA at 3:1, which were then introduced into intact plant cells using three different approaches, including injection, spray, and soak. The LDH-dsRNA delivery method was optimized by inhibiting the expression of the Arabidopsis thaliana ACTIN2 gene. As a result, soaking A. thaliana seedlings in a medium containing LDH-dsRNA for 30 min led to the silencing of 80% of the target genes. The stability and effectiveness of the LDH-dsRNA system were further confirmed by the high-efficiency knockdown of plant tissue-specific genes, including that encoding phytoene desaturase (PDS), WUSCHEL (WUS), WUSCHEL-related homeobox 5 (WOX5), and ROOT HAIR DEFECTIVE 6 (RHD6). In addition, the LDH-dsRNA system was employed in cassava, where it was found that the expression of the gene encoding nucleotide-binding site and leucine-rich repeat (NBS-LRR) was significantly reduced. As a result, the resistance of cassava leaves to pathogens was weakened. Noteworthy, the injection of LDH-dsRNA into leaves resulted in a significant downregulation of target genes in both stems and flowers, indicating the successful transport of LDH-dsRNA from leaves to other parts of plants.

Conclusions: LDH-NSs have proven to be a highly effective molecular tool for delivering dsRNA into intact plant cells, enabling accurate control of target gene expression.

Keywords: Bioconjugates; RNA delivery; RNAi; Target gene expression; dsRNA.

Abstract

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