A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Yongpeng Li,Wei Wang,Yaojie Zhang,Tiantian Chen,Xin Yan,Xueqing Fu,Kexuan Tang,Bowen Peng,Chen Wang,Hang Liu,Sadaf-Ilyas Kayani,Wei Qin,Lihui Xie,Kuanyu Wu-Zhang,Xinghao Yao,Ling Li

doi:10.1186/s13007-021-00807-5

Yongpeng Li, Wei Wang + Show 14 more

Open Access

https://doi.org/10.1186/s13007-021-00807-5

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Journal: Plant Methods	Publication Date: Oct 16, 2021
Citations: 17	License type: open-access

Affiliation: Tsinghua University, Shanghai Jiao Tong University

Abstract

BackgroundThe Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging.ResultsThe first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua.ConclusionsA simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

Highlights

The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency
Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phe‐ notypes resulting from gene expression in a heterologous system
The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection Normally, the leaves of A. annua are odd-pinnately compound with deeply indented margins [38]

Summary

Introduction

The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. While the stable genetic transformation endows transformed plants with hereditable new characteristics, transient plant transformation mediated by A. tumefaciens is widely applied for high-throughput gene function studies due to its simplicity, rapidity and high efficiency [2,3,4]. Given the advantages of high efficiency transformation of tobacco, the leaf agro-infiltration method was firstly established in Nicotiana sylvestris and adapted for fast gene functional characterization studies [8, 9]. Agro-infiltration has proved effective in diverse plant species including Arabidopsis thaliana [7, 13, 14], Vitis vinifera [15], Theobroma cacao [16], Gossypium hirsutum [17], Malus domestica [18] and Caragana intermedia [19]

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