The expression methods of foreign gene include stable expression and transient expression in the plants (Walmsley et al., 2000), the transient expression system has the advantages of simplicity, rapidity and high efficiency (Shivprasad et al., 2000). Viral vector-mediated leaf-infection is a common method for transient expression of foreign gene in the plants. People have transiently expressed a variety of foreign proteins in vitro tobacco leaves by using this inoculation (Kathuria et al., 2002; Wang et al., 2002). Some researchers used viral vector-mediated leaf-infection to expressed high levels green fluorescent protein (GFP) in tobacco (N. tabacum) (Jia et al., 2003). Liu et al successfully expressed human acidic fibroblast growth factor (ha FGF) with viral vector-mediated leaf-infection in tobacco (N. tabacum) (Liu et al., 2007). In 2008, Yang et al transiently expressed GFP in tobacco (N. tabacum) by using viral vector 30B that is derived from Tobacco mosaic virus (TMV) (Yang et al., 2008). In 2013, we used viral vector-mediated method to transiently express haFGF in pea (Pisum sativum) plants (Yang et al., 2013). In 2016, we established and optimized the viral vector-mediated root vacuum inoculation in tobacco (N. tabacum), and successfully expressed the foreign gene efficiently with this method (Yang et al., 2016). The studies have shown that these plant transient expression systems and the study of inoculation methods provide a research basis for plant bioreactors application, and also provide an effective platform for the rapid production of medicinal proteins and vaccines.

In this study, we explored an Agrobacterium-mediated inoculation for rapid expression of foreign gene and gene function study in the plants. Agrobacterium-mediated leaf-infection was used to transiently express the reporter gene GUS in Arabidopsis thaliana plants. The results of gene expression detection analysis and GUS staining showed that we successfully expressed the GUS gene in Arabidopsis by using Agrobacterium-mediated leaf-infection. Further research results had shown that the viral suppressor HC-Pro was expressed by this method in the plant, which provides a new expression system for the study of its function. Agrobacterium-mediated leaf-infection has the advantages of simplicity, rapidity and high expression efficiency, which provides a research basis for the fast expression and the functional identification of foreign gene in the plants.

1 Results and Analysis

1.1 The expression of GUS gene by Agrobacterium-mediated method

First, we transformed the expression vector pCAMBIA2301-GUS into Agrobacterium EHA105, then we used the Agrobacterium-mediated leaf-infection to transiently express the reporter gene GUS in Arabidopsis thaliana, and also observed and photographed for 14 days continuously. GUS staining was used to detect the expression of GUS gene in Arabidopsis. The results showed that 7 days after agro-inoculation, GUS began to expressed in the wound site of infected leaves (Figure 1A); 10 days after agro-inoculation, Agrobacterium was passed around through vascular bundles, and it can be observed that GUS expressed spread out from the wound site of infected leaves (Figure 1B; Figure 1C); 14 days after leaf-infection, GUS expressed heavily in uninfected stems (Figure 1D) and new leaves (Figure 1E).

Figure 1 Detection of GUS gene expression by GUS staining method Note: A: The expression of GUS appeared in the infection leaves at 7 days after agro-inoculation; B-C：The expression of GUS appeared in the infection leaves at 10 days after agro-inoculation; D-E：The expression of GUS increased in new stems and leaves at 14 days after agro-inoculation

1.2 The rapid expression of GUS gene in Arabidopsis thaliana

The plant materials from 7-10 days after inoculation were extracted for gene expression detection. The results showed that the reporter gene GUS was successfully expressed in Arabidopsis thaliana, indicating the effectiveness of Agrobacterium-mediated leaf-infection for fast expression of foreign gene (Figure 2A). In order to further determine whether the foreign gene transiently expressed in the new leaves and new stems, we detected the reporter gene GUS by RT-semi-quantitative PCR. The total RNA was extracted form the infected leaves (15 days after inoculation) and the new leaves to reverse transcription, and obtained cDNA was used as template to detect the expression of reporter gene. The result showed that GUS was expressed in both leaves on 15 days after Agrobacterium inoculation and new leaves (Figure 2B), that indicated the Agrobacterium-mediated leaf-infection can realize the expression of foreign gene in Arabidopsis.

Figure 2 The detection of GUS gene expression by RT- sqPCR Note: A: The expression of GUS was detected in the plants at 10 days after agro-inoculation; B: The expression of GUS was detected in the new and local leaves at 15 days after agro-inoculation

1.3 The expression of HCPro by Agrobacterium-mediated method in Arabidopsis thaliana

We have successfully constructed the expression vector PBI121-HCPro. In order to further study the function of HCPro, we used Agrobacterium-mediated method to express HCPro in Arabidopsis thaliana. Arabidopsis with stretched leaves were selected as plant materials for leaf-infection inoculation. When Arabidopsis infected with Agrobacterium carrying empty vector was used as control (Figure 3A), the phenotype of Arabidopsis expressing HCPro were obviously changed. 7 days after inoculation, the infected leaves showed mild curled and deformed (Figure 3B); 10 days after inoculation, some infected leaves were obvious curled and deformed, and the new leaves were serrated (Figure 3C), which was very similar to the phenotype of HCPro transgenic Arabidopsis obtained previously (Yang et al., 2016), indicating that HCPro began to express in the new leaves. 14-15 days after inoculation, the leaves were more serrated, some leaves were severely curled and deformed, and appeared yellow and wilting symptoms (Figure 3D). The phenotype change of Arabidopsis after inoculation suggested that HCPro expression changed the expression of host endogenous gene and the regulation mechanism of gene expression. The total RNA were extracted form infected plants to semi-quantitative RT-PCR detection, and the result confirmed that HCPro expressed in Arabidopsis (Figure 3E).

Figure 3 The phenotype of Arabidopsis thaliana expressed HCPro and the detection of gene expression Note：A：mock-infected Arabidopsis thaliana served as controls; B：The leaves appeared curled and deformed after agro-inoculation; C-D：The infected Arabidopsis thaliana exhibited obvious deformed leaves, new leaves exhibited obvious serration; E：The detection of HCPro gene expression in infected plants, Actin served as controls

2 Discussion

Viral-derived expression vectors are often used for the transient expression of foreign genes in plants. The main inoculation methods of the plant transient expression system mainly include viral vector-mediated leaf-infection and vacuum infiltration (Kapila et al., 1997). The researchers used viral-mediated method to transiently express acidic fibroblast growth factor (aFGF) in tobacco (Liu et al., 2007). This method has the advantages of fast and high expression efficiency and is widely used by people. In this study, we transformed plant expression vectors pCAMBIA2301-GUS and pBI121-HCPro into Agrobacterium EHA105, and studied the transient expression of foreign genes in Arabidopsis plants by Agrobacterium-mediated leaf infection. In this method, the expression vectors and foreign genes carried by Agrobacterium are rapidly expressed mainly through the transmission and spread of Agrobacterium (Figure 1). Our results indicated that the reporter gene GUS (Figure 2) and functional protein HCPro (Figure 3) were successfully expressed with this inoculation method.

In this Agrobacterium-mediated leaf-infection, the expression vector carried by Agrobacterium may be a plant binary expression vector, Arabidopsis or other crops can be selected as the infection material. However, the host is limited by viral-derived expression vectors in viral-mediated transient expression systems. For example, expression vectors derived from Tobacco mosaic virus (TMV) are limited to tobacco and other host materials (Liu et al., 2007; Yang et al., 2008). Therefore, this Agrobacterium-mediated inoculation method further expands the host range and is suitable for the rapid expression of genes and the study of gene function in plants, providing a new system for plant transient expression systems.

3 Materials and Methods

3.1 Materials and sources

The Arabidopsis thaliana seeds and strains are kept by our laboratory; plant expression vector pCambia-GUS is kept by our laboratory.

3.2 Preparation of Agrobacterium tumefaciens resuspension

The EHA105-pBI121-HC-Pro Agrobacterium liquid was activated and cultured in 50 mL LB liquid medium, and 20 μmol AS and 100 μmol MES were added. Centrifuge at 4 000 r/min for 10 min to collect Agrobacterium. Formulated solution (100 mL of ddH₂O was added with 1 mmol of MgCl₂, 1 mmol of MES and 100 μmol of AS), Agrobacterium was resuspended at a concentration of about OD₆₀₀=1.0, and the Agrobacterium tumefaciens resuspension was placed at room temperature for 3 hours for leaf infection.

3.3 Agrobacterium-mediated Arabidopsis inoculation

The stretched leaves of Arabidopsis thaliana were selected, and the syringe with the needle removed was used to infect the Agrobacterium tumefaciens resuspension into the leaf tissue, and each plant was infected with 2-3 leaves. The plants were cultivated under dark conditions for 24 hours and then transferred to normal conditions. The cultivation temperature of Arabidopsis thaliana was 22℃~25℃.

3.4 Semi-quantitative RT-PCR to detect gene expression

We used TRIzol method to extract the total RNA of the plant, semi-quantitative RT-PCR detection, pre-denaturation: take 2.5 μg of RNA, adjusted to 10 μL system with ddH₂O, pre-denatured the RNA first, the pre-denaturation conditions: treatment at 70℃ for 5 min; 4℃, 5 min. The pre-denatured RNA was reverse-transcribed, and the post-transcriptional cDNA was used as the template for PCR detection.

3.5 GUS staining analysis

The GUS staining solution and the GUS staining buffer were configured as the GUS staining working solution according to the volume ratio of 1:50, and placed in darkness at -80℃ for standby use. Put the Arabidopsis plant material in a 50mL beaker, added the appropriate amount of GUS staining working solution to completely cover the plant material. Wrapped the beaker with foil paper and used the vacuum infiltration for staining. The vacuum pressure was 0.08 MPa and the time was 5 minutes. We observed that the plant material was immersed in the bottom of the GUS staining solution, and the experiment was repeated twice. The whole beaker was incubated at 37℃ for 24 hours. With the extension of incubation time, GUS expression parts or sites showed blue or blue spots (The whole process is protected from light). Plant materials were fixed with 30 mL Carnoy's fluid at room temperature for 1-3 hours. Then the decolorization process was carried out. The volume fractions of ethanol were set as 25%, 50%, 70%, 85% and anhydrous ethanol, and the decolorization process was conducted at each concentration for 1-3 hours. We observed and took pictures under the microscope. Samples can be stored in 75% ethanol at 4℃ for 1-3 months.

Authors’ contributions

Taicheng Jin was the executor of this experimental study, completed the data analysis and paper writing. Liping Yang directed the experimental design and paper modification; Chenjing Lang, Yue Wang, Dawei Meng and Yanju Wu participated in the completion of relevant experiments. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (Grant Number 31301043) and the Department of Finance of Jilin Province (Grant Number JJKH20191013KJ).

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