A Method for Obtaining Low Luminescence Mutants of Col-LUC through Reverse Genetics in Arabidopsis

doi:10.21203/rs.3.rs-3131040/v1

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A Method for Obtaining Low Luminescence Mutants of Col-LUC through Reverse Genetics in Arabidopsis

https://doi.org/10.21203/rs.3.rs-3131040/v1

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Previous studies have shown that using transgenic reporter systems to screen mutants is one of the effective methods to study DNA demethylation. Many genes involved in the regulation of DNA methylation have been uncovered through forward genetic screens. However, forward genetic screens not only have a long period, high cost, but also a large workload and low efficiency. In order to address these problems, based on reverse genetics, this study used CRISPR technology to knockout selected co-expressed genes, so as to quickly obtain low LUC (luciferase) luminescence mutants of Col-LUC line which harbors a LUC transgene driven by a 2×35S promoter in Arabidopsis and uncover new genes involved in DNA demethylation pathway. In this study, we selected the ROS1 (REPRESSOR OF SILENCING 1) gene and RDM1 (RNA-DIRECTED DNA METHYLATION 1) gene as controls, with the co-expressed gene IDM3 (INCREASED DNA METHYLATION 3) of ROS1 as the target gene, and conducted gene knockout experiments in the Col-LUC line. The experiment results reveal that combining co-expressed gene list and CRISPR technology is feasible for obtaining low LUC luminescence mutants in the Col-LUC line. This study provides a new approach and solid basis for obtaining low luminescence mutants in the Col-LUC line through reverse genetics.

reverse genetics

Col-LUC genetic screening system

gene co-expression

CRISPR

It has been shown that screening for mutants by transgenic reporter systems is one of the effective ways for DNA demethylation study (Gong et al. 2002; Wang et al. 2013; Li et al. 2016). Researchers have identified several novel proteins involved in DNA demethylation process by forward genetic screens. For example, utilizing the 35S:SUC2 (SUC2, SUCROSE-PROTON SYMPORTER 2) transgene system, researchers have identified IBM1 (INCREASE IN BONSAI METHYLATION 1), EDM2 (ENHANCED DOWNY MILDEW 2), MBD7 (METHYL-CpG-BINDING DOMAIN 7), etc (Wang et al. 2013; Lei et al. 2014; Lang et al., 2015); ROS1 (REPRESSOR OF SILENCING 1), ROS3, and KTF1 (KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1) have been identified by the RD29A:LUC (LUC, luciferase) transgene system (Gong et al. 2002; Zheng et al. 2008; He et al. 2009); IDM2 (INCREASED DNA METHYLATION 2) and RPA2 (REPLICON PROTEIN A2) have been identified through 35S:NPTII transgene (Zhao et al. 2014; Kapoor et al. 2005); With reduced 2×35S:LUC transgene expression, MBD7, SUVH1 (SU(VAR)3–9 HOMOLOG 1) and other proteins have been identified (Li et al. 2017; Li et al. 2016; Hou et al. 2022). However, forward genetic screens are not only time-consuming and costly, but also inefficient with a large workload. To address these problems, this study aims to find a rapid method to obtain transgenic silencing mutants for DNA demethylation regulation study.

Owing to continuously screens, many key proteins of DNA demethylation pathway have been identified in the past 20 years since the isolation of ros1-1, including ROS1, IDM1 and EDM2 (Gong et al. 2002; Qian et al. 2012; Lei et al. 2014). Functional studies of these proteins have expanded the understanding of DNA demethylation pathway, which contributes to the study of the mechanism of newly identified anti-silencing factors (Nie et al. 2019; Zhao et al. 2019). The first problem of reverse genetic studies that needed to be solved is how to select target genes. A strategy utilizing co-expressed genes of known genes such as ROS1, DME (DEMETER) and DML2 (DEMETER-LIKE 2) was proposed to narrow down the selection range of target genes and the feasibility analysis was also performed. Although exceptions may exist, a large number of functionally related genes do have very similar expression profiles under the same condition, especially when their products comprising the same protein complex or involved in the same pathway (Wongpalee et al. 2019; Harris et al. 2018; Potok et al. 2019; Duan et al. 2017). In addition, acquiring corresponding mutants is another problem remaining to be solved. Since knocking out related genes in Arabidopsis by CRISPR is well studied (Xing et al. 2014), this study proposed to perform gene knockout experiments on lines harboring transgenic reporter systems utilizing CRISPR technology. However, the application of CRISPR technology may cause side effects on the transgenic reporter system, which may further lead to misunderstanding of the function of the target gene, resulting in the assessment requirement of the applicability of CRISPR technology to the Col-LUC line which harbors a LUC transgene driven by a 2×35S promoter (Miao et al. 2020).

In summary, a new scheme for obtaining low LUC luminescence mutants of the Col-LUC line in Arabidopsis by reverse genetics is proposed, which is a combination of co-expressed genes of know genes and CRISPR technology. In addition, the applicability is also evaluated where mutants are created by CRISPR technology, with ROS1 and RDM1 (RNA-DIRECTED DNA METHYLATION 1) as controls and IDM3, one of co-expressed genes of ROS1, as target gene. As a result, the combination of co-expressed genes of known genes and CRISPR technology has been verified as a practical method for the creation of low LUC luminescence mutants of the Col-LUC line. This study provides a new idea for the acquisition of low LUC luminescence mutants in Col-LUC background by reverse genetics and finding new proteins in DNA demethylation pathway.

Materials

All Arabidopsis plants used in this study, including mutants and transgenic plants, were in the Columbia-0 (Col-0) genetic background. Col-LUC: Columbia (Col-0) background, bearing a 2×35S:LUC transgene and a homozygous rdr6 mutation, with stable high LUC luminescence, was used as parent material in the experiment (Miao et al. 2020). ros1-7: A line with stable low LUC luminescence phenotype that was isolated from Col-LUC line, which was used as a negative control in the experiment.

Cultivation of Arabidopsis thaliana

Seedlings were grown on half-strength Murashige and Skoog (MS) media containing 2.0% sucrose with 0.74% (for horizontal plate) or 0.85% (for vertical plate) agar. After stratifcation at 4°C for 2 d plants were grown under a long-day photoperiod (16-h light/8-h dark) at 22°C in a growth room. After 12 d of cultivation, the seedlings were transferred to the culture soil composed of vermiculite and nutrient soil whose proportion is 3:1, and continued to be cultured in the light incubator until the bud stage for the subsequent gene knockout experiments.

The selection of target genes

The co-expressed gene lists of ROS1, DME and DML2 were downloaded from ATTED-II (https://atted.jp), an online plant gene co-expression database ( Obayashi et al. 2022). The selection range of target genes for subsequent knockout experiments were narrowed down by overlapping first 2000/1000/500 co-expressed genes using jvenn (Bardou et al. 2014).

Selection and primer design of targets gene gRNA

From online database CRISPR-PLANT (http://www.genome.arizona.edu/crispr), gRNA of target gene was screened (Minkenberg et al. 2019). gRNA should be located as close to the 5' end of the gene as possible in the exon. As listed in Supplementary Table 1, the sgRNA sequence of the target gene is highlighted in red according to the designed primers.

PCR amplification

Using pCBC-DT1T2 (Xing et al. 2014) diluted 25 times as a template for four primer PCR amplification to obtain a PCR fragment (626 bp). The primer concentration of DT1-BsF/DT1-BsR is 10 µmol/L, and DT1-F0/DT1-R0 is diluted to 1 µmol/L. Amplification system (20 µL) is as follows: 2 × PCR Mix 10 µL; Forward and reverse primers (10 mmol/L) 1 µL; DNA Template 2 µL; ddH₂O 6 µL. Amplification program: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, repeated cycling 35 times, holding at 72 ℃ for 5 min, and storage at 4 ℃. Primers are listed in Supplementary Table 1.

Construction of CRISPR vector

The PCR product (626 bp) was combined with the PHEC401 plasmid to establish the enzyme digestion linkage system (Table 1). The reaction was incubated in a thermos-cycler (Bio-Rad) for 5 h at 37°C to construct a CRISPR vector.

Table 1

CRISPR Enzyme-Linking System
Component	Concentration	Volume (µL)	Reaction conditions
PCR fragment (626 bp)	300–400 ng/µL	2	5 h at 37°C
pHEC401	300–400 ng/µL	2
10 × NEB T4 Buffer	/	2
10 × BSA	/	2
BsaI (NEB)	2000000 U/mL	1
T4 Ligase (NEB)	350 U/µL	1
ddH₂O	/	10
Total	/	20

Identification and sequencing

1–2 µL enzyme-ligand products were transformed into the receptive state of Escherichia coli, then kanamycin was used to screen positive clones. The selected clones were identified and sequenced using the following primers: U626-F and U629-R for PCR identification and U626-F and U629-F for liquid sequencing (General Bio.) to ensure the gRNA vectors containing the correct sequence of ROS1, RDM3 and IDM3, separately. Primers are listed in Supplementary Table 1.

Positive clones cultivation and Plasmid extraction

The above identified positive clones were inoculated into 500 µL LB liquid medium and mixed, then resumed culture in 180 r/min shaker at 37℃ for 40–60 min. After resuming culture, receptive cell fluids were inoculated into 5 mL LB liquid medium containing kanamycin and cultured in a shaking bed at 37℃ and 180 r/min for 14–16 h. Then, expanded bacterial solution was extracted using a plasmid microextraction kit (Aidlab Bio.).

Agrobacterium transformation

1–2 µL plasmid was added into Agrobacterium Competent Cell and placed on ice for 30 min. Then the Agrobacterium Competent Cell was frozen in liquid nitrogen for 3 min and quickly transferred to a thermostat water bath at 37℃ for 5min. Place the Agrobacterium Competent Cell on ice for 2 min, add 500 mL liquid medium with YEP, and resume culture for 3 h in a shaker at 28℃, 180 r/min. After the culture is completed, pour it with YEP solid medium containing kanamycin antibiotics to select positive clones and then identified by PCR using U626-F and U629-R.

Transgenic seedling screening

Using pollen-tube pathway, Col-LUC was transfected. The harvested T₁ seeds were planted on MS medium containing 50 mg/L hygromycin. Cultured in a growth room for about 14 d, transfer the growing seedlings to the nutrient soil and harvest T₂ generation seeds individually. The T₂ seeds were seeded on MS medium containing 50 mg/L hygromycin, and the strains without phenotypic isolation were selected as positive transgenic seedlings. Finally, the mutation location of transgenic plants was determined by PCR product sequencing. Primers are listed in Supplementary Table 1.

Imaging of LUC luminescence

For imaging LUC luminescence, seedlings were grown on plates with half-strength MS media supplemented with 0.74% agar and 2.0% sucrose. Ten- to twelve-day-old seedlings were sprayed with 1 mM d-luciferin which was dissolved in 0.01% Triton X-100. After 5-min incubation in the dark, the seedlings were placed in a Princeton Dark Box equipped with a Roper VersArray 1300B camera controlled by the WinView32 software, and then imaged for 30 s.

Analysis of gene expression

RNA was extracted from 12-day-old seedlings by Tripure method followed by DNase I (Turbo) treatment. For gene expression analysis, 1 µg of total RNA was used for reverse transcription by oligo dT with Trelief™ SoSoo RT kit (TSINGKE). Real-time PCR was carried out using iQ SYBR Green Supermix (Bio-Rad) on a CFX96 real-time PCR detection system (Bio-Rad). A housekeeping gene TUBULIN 8 or ACTIN2 served as an internal control for all reactions. RT-PCR (qPCR) reactions were performed as follows: 1 (0.5) min at 94°C; 18–40 cycles [94°C for 30 (5) s, 55°C (58°C) for 30 s, and 72°C for 30 (0) s] followed by 72°C for 5 min. The qPCR results were from two biological replicates with three technical replicates each. Fold changes of gene expression values were calculated according to a method reported previously (Li et al. 2016). All the primers used for RT-PCR (qPCR) reactions are listed in the Supplementary Table 1.

Analysis of DNA methylation levels

DNA methylation-sensitive PCR (also known as Chop-PCR) was performed according to a method reported previously (Wang et al. 2013). First, genomic DNA was extracted from 12-day-old seedlings using cetyl trimethylammonium bromide (CTAB) method and was quantified with NanoDrop2000c UV–Vis Spectrophotometer (Thermo SCIENTIFIC). Second, 600 ng of DNA was separately digested with 40 U of DNA methylation-sensitive restriction enzyme HpaII for 4 h. Following the digestion, 10 µg of digested DNA was used as template for PCR. Undigested genomic DNA served as a control template. All Chop-PCR amplifications were performed as follows: 1 min at 94°C; 27–33 cycles (94°C for 30 s, 55–58°C for 30 s, and 72°C for 30 s) followed by 72°C for 5 min. The Chop-PCR primers are listed in the Supplementary Table 1.

The feasibility analysis of narrowing down target genes using co-expressed genes

Co-expressed genes of known genes have been used to narrow down the selection range of target genes (Lu et al. 2020). Although exceptions existing, functionally related genes indeed have similar expression patterns under the same condition when their products consisting of the same protein complex or participating in the same pathway (Wongpalee et al. 2019; Harris et al. 2018; Potok et al. 2019; Duan et al. 2017). ROS1, DME and DML2 are the main DNA demethylases in Arabidopsis (Penterman et al. 2007). Many co-expressed genes of ROS1 are members of DNA demethylation pathway, including DME, IDM1, DML2, IDM3 and MBD7. DNA demethylation pathway related genes, including ROS1, PKL (PICKLE) and DML2, are also in co-expressed gene list of DME while DME, ROS1, IDM3 and ROS3 are in co-expressed gene list of DML2. This indicates that co-expressed genes are feasible as candidate target genes of reverse genetics. In addition, we downloaded the co-expressed gene lists of ROS1, DME and DML2 and found the overlapped genes of the first 2000/1000/500 genes (Fig. 1). As shown in Table 2, most of the identified genes involved in DNA methylation and demethylation pathways belong to the overlapped genes of the first 1000 co-expressed genes, which further narrowed down the candidate target genes.

CRISPR technology is suitable for Col-LUC screening line

Although mutants can be created by CRISPR, it’s still unknown whether knocking out the target gene will result in side effects on 2×35S:LUC transgene and mislead the assessment of target gene function. Previous studies have shown that ROS1 can antagonize LUC transgene silencing and ros1 in Col-LUC background exhibits low LUC luminescence while rdm1 doesn’t play a role in direct antagonism of LUC transgene silencing (Gong et al. 2002; Miao et al. 2020; Gao et al. 2010). Therefore, ROS1 and RDM1 are selected as target genes for subsequent knockout experiments. As shown in Fig. 2A & 3A, ros1 and rdm1 mutants of the T₁ plants both have heterozygous and homozygous genotypes. After self-crossing, the result of T₂ genotyping revealed that ros1 and rdm1 are both homozygous (Fig. 2A & 3A). In order to further confirm the effects of knocking out ROS1 and RDM1, we conducted qRT-PCR and the expressions of ROS1 and RDM1 were both significantly reduced after mutation (Fig. 2B & 3B). The sequencing results showed that there is a 143 bp deletion in ROS1 and a 33 bp deletion in RDM1 as well (Fig. 2E & 3E). As expected, ros1 exhibited a significantly reduced luminescence signal while rdm1 not (Fig. 2D & 3D), indicating that CRISPR did not bring side effects to the Col-LUC line and LUC luminescence change of mutants can reflect the function of corresponding genes. These results demonstrated that CRISPR is suitable for the Col-LUC line.

idm3, a lowLUCluminescence mutant of Col-LUC line, was created by CRISPR

IDM3 and ROS1 are co-expressed through co-expression analysis. However, idm3 mutant of Col-LUC line has not been isolated by forward genetic screens so far. A knockout experiment was performed to create idm3 mutant of Col-LUC line. As shown in Fig. 4A, the phenotyping of T₁ plants showed that the idm3 mutants have both homozygous and heterozygous genotypes while T₂ plants were homozygous (Fig. 4A). To further confirm the results, qRT-PCR was conducted, which showed a significant reduction of IDM3 expression (Fig. 4B). There is 1 bp deletion mutation in IDM3 gene (Fig. 4E), which results in a low-luminescence phenotype because of a stop codon induced by frameshift mutation (Fig. 4C). In addition, Chop-PCR experiments were performed and the results showed that the methylation levels of certain loci were elevated after IDM3 mutation (Fig. 5), which is consistent with IDM3 function (Lang et al. 2015). The above results indicate that based on reverse genetics, idm3, a low-luminescence mutant of Col-LUC line, was successfully created, which confirms the feasibility of the scheme proposed in this study to obtain low LUC luminescence mutants in Col-LUC line in Arabidopsis.

Currently, many laboratories are still screening mutants through forward genetic screens using transgenic reporter systems (Hou et al. 2022; Pei et al. 2022; Zhou et al. 2023; Wang et al. 2022). However, this method is not only time-consuming, costly, but also inefficient with a large workload. This study proposed a novel scheme to obtain low LUC luminescence mutants in Col-LUC background by reverse genetics, which combines co-expression theory and CRISPR technology.

There are two main problems remaining to be solved for reverse genetics, which are how to narrow down the selection range of target genes and whether there is a suitable method to obtain mutants of transgenic reporter systems. This study applied the co-expression theory and found that most of the identified genes involved in DNA demethylation pathway were in the overlap list of co-expressed genes of ROS1, DME and DML2.

Many ways can be applied to obtain mutants, such as ordering T-DNA insertion lines of target genes. However, this method can only obtain plants in Col-0 background and can not confirm the effect of target gene mutation on transgenic reporter systems. This study applied CRISPR technology to create knockout mutants of Col-LUC line, but whether the mutants obtained by CRISPR can properly reflect the function of corresponding genes still needs to be verified. In this study, ROS1, which is the main demethylase in DNA demethylation pathway, was chosen as the positive control while RDM1, an important protein in DNA methylation pathway, was chosen as the negative control (Gong et al. 2002; Gao et al. 2010). After knocked out, the change of LUC luminescence is consisted with previously reported gene functions, indicating the suitability of CRISPR application to Col-LUC line (Gong et al. 2002; Gao et al. 2010).

After years of forward genetic screens, idm3 mutants have not been isolated in Col-LUC background. IDM3 is one of the co-expressed genes of ROS1 and functions in ROS1-mediated DNA demethylation pathway. Following the proposed scheme, this study obtained the idm3 mutant in Col-LUC background for the first time (Fig. 4C).

As the development of CRISPR technology, it has become possible to identify all effective genes of a given transgenic reporter system (e.g., Col-LUC line) by a large-scale gene knockout project using co-expressed gene lists of identified genes and build related databases. With such a database, researchers can directly explore the gene function and working model of a given effective gene, which will greatly accelerate the progress of related fields. Therefore, the method proposed in this study is of great practical significance.

Acknowledgements

This work was supported by Jiangsu Agri-animal Husbandry Vocational College.

Subject area

regulation of gene expression

Competing interests The authors declare no competing interests.

Statement of author contributions

Wei Miao and Jie Dai prepared a draft of the manuscript. Li Zhang and Aqin Zhang reviewed and completed the text of the paper. Wei Miao and Jie Dai prepared the final version which was approved by all authors.

Accession Numbers

The Arabidopsis Genome Initiative identifiers for the genes described in this work are: ROS1, AT2G36490; RDM1, AT3G22680; IDM3, AT1G20870.

Funding

This work was supported by Jiangsu Agri-animal Husbandry Vocational College research project of China (NSF0222CB07). Wei Miao has received research support from Jiangsu Agri-animal Husbandry Vocational College.

Data Availability

There are no new data associated with this article.

Human and animal rights statement

This article does not contain any studies with human participants performed by any of the authors.

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A Method for Obtaining Low Luminescence Mutants of Col-LUC through Reverse Genetics in Arabidopsis

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Version 1

Abstract

Figures

Introduction

Materials and methods

Materials

Cultivation of Arabidopsis thaliana

The selection of target genes

Selection and primer design of targets gene gRNA

PCR amplification

Construction of CRISPR vector

Identification and sequencing

Positive clones cultivation and Plasmid extraction

Agrobacterium transformation

Transgenic seedling screening

Analysis of gene expression

Analysis of DNA methylation levels

Results

The feasibility analysis of narrowing down target genes using co-expressed genes

CRISPR technology is suitable for Col-LUC screening line

Discussion

Declarations

References

Supplementary Files

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