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. 2024 Jan 18;57(2):79–85. doi: 10.5483/BMBRep.2023-0210

Phenotypic characterization of pre-harvest sprouting resistance mutants generated by the CRISPR/Cas9-geminiviral replicon system in rice

Jong Hee Kim 1,2, Jihyeon Yu 3, Jin Young Kim 1, Yong Jin Park 4, Sangsu Bae 3, Kwon Kyoo Kang 1,2,*, Yu Jin Jung 1,2,*
PMCID: PMC10910094  PMID: 38303561

Abstract

Pre-harvest sprouting is a critical phenomenon involving germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. In this paper, we generated HDR mutant lines with one region SNP (C/T) and an insertion of 6 bp (GGT/GGTGGCGGC) in OsERF1 genes for pre-harvest sprouting (PHS) resistance using CRISPR/Cas9 and a geminiviral replicon system. The incidence of HDR was 2.6% in transformed calli. T1 seeds were harvested from 12 HDR-induced calli and named ERF1-hdr line. Molecular stability, key agronomic properties, physiological properties, and biochemical properties of target genes in the ERF1-hdr line were investigated for three years. The ERF1-hdr line showed significantly enhanced seed dormancy and pre-harvest sprouting resistance. qRT-PCR analysis suggested that enhanced ABA signaling resulted in a stronger phenotype of PHS resistance. These results indicate that efficient HDR can be achieved through SNP/InDel replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Keywords: CRISPR/Cas9, Geminiviral replicons, Gene replacement, Homology-directed repair (HDR), OsERF1

INTRODUCTION

Pre-harvest sprouting (PHS) is an important phenomenon associated with germination of parent plant seeds prior to harvest under relatively humid conditions and reduced dormancy, significantly reducing grain yields and grain quality (1-3). Therefore, crop breeders are putting a lot of effort into improving PHS resistance as a breeding goal (4). Understanding the physiology of seed dormancy and germination is important for controlling PHS in cereal crops. It has been reported that seed dormancy and PHS differ depending on the expression patterns of many genes in the ABA, ethylene, and GA signaling transduction pathways (5-9). In rice, the Sdr4 gene has been reported to be expressed by OsVP1 in the ABA signaling pathway, causing seed dormancy (10). Overexpression of the OsSdr4 gene results in resistance to PHS (4). It has also been reported that PHS can be caused by gene mutations involved in carotenoid biosynthesis in rice (11). QTL analysis revealed that qSD12 involved in seed dormancy contained candidate genes of PIL5 and bHLH (12). To date, several QTL-based fine mapping studies have explored genes associated with seed dormancy and germination (11, 13-17). However, the molecular mechanisms for seed dormancy and PHS are not yet clear. Therefore, the way to overcome PHS in response to climate change is to secure many alleles associated with seed dormancy and germination. Gene editing technology is widely used to generate mutations by deleting, inserting, or replacing the base of a target gene on the genome.

Among the gene editing tools known to date, the CRISPR/Cas9 system is the most widely used in animals, plants, and microorganisms (18). According to recent information, when gene editing is performed using CRISPR/Cas9, more base insertions and deletions occur than base substitutions, and the efficiency varies depending on the sgRNA of the gene (19, 20). Additionally, the CRISPR/Cas9 system has proven its feasibility by conducting experiments to induce gene targeting and gene replacement in plants (10, 19-24). HDR, such as GT or gene replacement, has a relatively low editing ratio and is not yet routinely used in plants. To increase HDR frequency, many researchers were interested in donor size and copy number (25-28). HDR and gene replacement in plants require not only induction of DSBs in the target DNA, but also adjustment of donor copy number within cells. To succeed in HDR in plants, stability must be ensured after DRT (Donor Repair Template) is delivered reliably within cells. However, HDR in plants is quite challenging for DSB induction and DRT delivery of target DNA. Base editing (BE), which is most suitable for changing single nucleotides in DNA sequences, has been developed, and because it chemically changes the target base, only one change can be made at a time (29-32). It has been recently reported that HDR efficiency can be improved using a CRISPR/Cas9-geminiviral replicon delivery system (33-36). In a previous experiment, we performed an HDR experiment using the CRISPR/Cas9-geminiviral replicon system in rice calli and found that the HDR frequency was 1.32% (37). Here, we used the CRISPR/Cas9-geminiviral replicon system to generate HDR mutant lines with a replacement of one region SNP (C/T) and a 6-bp insertion (GGT/GGTGGCGGC) in the OsERF1 gene for PHS resistance in rice. In addition, to enhance the frequency of HDR, heat shock treatment was performed for the callus for 12 hours after infection with Agrobacterium tumefacience (EHA105). Our results revealed that the ERF1-hdr line showed significantly enhanced seed dormancy and PHS resistance.

RESULTS

Efficient allelic replacement by the geminiviral replicon

A previous rice genome-wide association study (GWAS) has shown that PHS resistance is caused by a total of two SNPs and one InDel in the open reading frame (ORF) region of the OsERF1 gene (38) (Supplementary Fig. 1). The SNP at position 4, which changed C to T in the ERF1 gene, caused an amino acid change from threonine to methionine. The increase of two glycine units by inserting a 6 bp insertion at position 96 resulted in resistance to PHS. Therefore, we improved our previously reported method to precisely replace alleles present in the genome with the elite alleles. First, we mutated the PAM site in the DRT in the CRISPR/Cas9 vector to prevent Cas9/gRNA from cleaving the DRT. And to select mutants in which HDR occurred, a SnaBI recognition site was created in DRT as a marker. We synthesized DRF for HDR by changing C to T at position 4 of the ERF1 gene and inserting two glycine units at position 96 followed by constructing it in the Cas9-gemini replicon vector (Supplementary Figs. 2-4, and Supplementary Table 1). After transformation, callus was subcultured three times using 2N6 medium containing 6 mg/L phosphinothricin. To confirm transgenic calli, PCR analysis was performed using the NOS-barF/R primer set, and the expected band was amplified in 95% of those tested (Supplementary Figure 5). In order to increase HDR efficiency, heat treatment was performed after infection with Agrobacterium tumefaciens during the transformation experiment. As a result of selecting HDR events by decomposing a total of 463 calli DNA with SnaBI or performing deep sequencing analysis, the highest HDR efficiency was shown under a 12-hour treatment condition of 42°C. In addition, to select variants in which HDR occurred, DNA from a total of 463 calli was digested with SnaBI or deep sequencing analysis was performed (Supplementary Fig. 5). The results showed the exact HDR events intended by 12 calli, regeneration plants (T0) were obtained from these calli, and T1 seeds were harvested from each plant (Fig. 1A, B, and Supplementary Table 2). Additionally, most mutations showed the NHEJ mutation type with a frequency of 58.1% (Fig. 1C and Supplementary Table 2). We selected T1 generation individuals with HDR events for further research and analysis and named them ERF1-hdr.

Fig. 1.

Fig. 1

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Detection of HDR mutant lines. (A) Position of key SNP and insertion replacement of the ERF1 targeted site (SnaBI restriction enzyme site was created inside the donor). (B) Screening by using PCR and restriction enzyme analysis. The second PCR products were digested with the SnaBI restriction enzyme. M: 1 kb DNA ladder; N: negative control; P: pGemBos::ERF1 plasmid vector; WT: wild type. (C) Mutation patterns of the target sequence region through deep sequencing. The target DNA sequence of ERF1 is shown in the WT with blue text at the top of the aligned sequences. The PAM sequences are underlined. Deletions are indicated as dashes; insertions are lowercase; and substitutes are in yellow highlighter. Indel sizes are shown on the right (i: insertion; d: deletion).

Protein model according to 1 bp SNP and 6 bp insertion of OsERF1 gene

The ERF1 protein is an ethylene-responsive transcription factor containing a DNA binding domain. The ERF1-hdr line has a 1 bp SNP and 6 bp insertion mutation in OsERF1 gene. Therefore, in order to determine whether changes in bases in the ERF1-hdr line cause any changes in the ERF1 protein structure, we examined the ERF1 structure and key functional residues by protein homology modeling (Fig. 2). In the ERF1-hdr line, T was changed to M in the second amino acid sequence of the ERF1 gene, which plays an important role in ERF1 protein stabilization. Accordingly, the subsequent peptide chains were connected with Gly29, Gly30, Gly31, Gly32 in wild-type ERF1, whereas in ERF1 of the ERF1-hdr line there were connected to Gly29, Gly30, Gly31, Gly32, Gly34, and Gly35. Also, the ERF1 structure from the ERF1-hdr lines converted the structure of the C-terminal end by creating a new residue from 298. In the ERF1-hdr line, the C terminus of the ERF1 protein was linked by a peptide chain of Ala298, Pro305, Val306, Glu307, Leu318, Val319, and Ile320. The ERF1-hdr line showed significant differences in ERF1 protein structure due to the effect of replacing one SNP in the ERF1 motif domain and adding two Gly (Fig. 2). Therefore, this suggests that the newly produced ERF1 protein in the ERF1-hdr line may act differently from the initially existing ethylene-responsive transcription factor.

Fig. 2.

Fig. 2

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Structural comparison of the wild-type OsERF1 and the ERF1-hdr-mutated version OsERF1. (A) Wild-type OsERF1 showing the position of Thr2. The black box shows all of the key amino acids (Pro286, Pro287, Val294, Gln295, Val296). (B) Mutant OsERF1 showing the position of Met2 and Gly34,35. The black box shows all of the key amino acids (Ala298, Pro305, Val306, Glu307, Leu318, Val319, Ile320).

Evaluation of PHS resistance of the ERF1-hdr line

To evaluate PHS resistance of the ERF1-hdr line, we harvested mature seeds at 35 days post anthesis (DPA) and performed germination experiments using Dongjin as a control. Dongjin seeds began to germinate at 4 days after imbibition (DAI), with a GR of ∼30% at 11 DAI, whereas seeds from ERF1‐hdr line germinated more slowly, with a GR of < 10% at 11 DAI (Fig. 3A, B, and Supplementary Fig. 6). Furthermore, we examined GR over time after treatment with ABA and H2O2 at 35 DPA in the ERF1-hdr line and Dongjin, respectively. As a result, compared to Dongjin, the ERF1-hdr line germinated more slowly with GR less than 10% at 15 DAI following ABA treatment, while the GR of the ERF1-hdr line was improved following H2O2 treatment (Fig. 3C-F and Supplementary Fig. 6). Additionally, after artificially breaking dormancy by heat treatment, GRs of Dongjin and ERF1-hdr line were almost similar at 97% (Supplementary Fig. 7A, B). Also, we compared seed viabilities of Dongjin and the ERF1-hdr line’s naturally aged seeds after 6 months. As a result, the GR of Dongjin exceeded 86%, whereas that of the ERF1-hdr line was below 73% (Supplementary Fig. 7C, D). To evaluate ABA signaling response, we measured ABA in seeds of the ERF1-hdr line and Dongjin. As a result, the ERF1-hdr line had increased endogenous ABA levels compared to Dongjin (Supplementary Fig. 8).

Fig. 3.

Fig. 3

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Improvement of pre-harvest sprouting resistance in rice using CRISPR/Cas9-Geminiviral replicon system to target OsERF1 gene. (A, C, E) Gemination phenotypes of seeds in freshly collected panicles at 35 days after pollination (DAP) from ERF1-hdr line and wild type after 4 days imbibition in water (A), 10 μM ABA (C) and 20 mM H2O2 (E). (B, D, F) Time-course germination percentages for (A), (C) and (E). Each analysis was repeated with four biological replicates. Error bars: ± SEM.

Evaluation of transcription levels of genes associated with ABA, ethylene, and GA signaling pathways

Transcriptional reprogramming plays a critical role in seed dormancy and PHS resistance. Therefore, in this study, qRT-PCR analysis was performed for 16 genes present in ABA, ethylene, and GA signaling pathways reported so far in rice. First, in the case of ABA signaling-related genes, expression levels of PYL, SnRK2, ABI3, ABI5, and Sdr4 were higher in the ERF1-hdr line than in Dongjin (Fig. 4 and Supplementary Fig. 9). Additionally, in the case of genes related to ethylene and GA signaling, expression levels of EIN3, ERF1, GID1, and GYMYB except for the EIN2 gene were much lower in the ERF1-hdr mutant than in Dongjin (Fig. 4 and Supplementary Fig. 9).

Fig. 4.

Fig. 4

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Heatmap diagram of genes related to signaling of ABA, ethylene and GA in rice. Immature seeds were collected in the panicles at 14 days after pollination (DAP) from ERF1-hdr line and Dongjin. The color gradient shows the green to red gradient bar represent the log2 fold change in expression.

Agronomic traits of ERF1-hdr line

Key agronomic traits, such as plant height, number of tillers, panicle length and stem length of the ERF1-hdr line in the paddy field were examined during 3 years (Supplementary Fig. 10 and Supplementary Table 3). Compared to Dongjin, the ERF1-hdr line was similar in all characteristics investigated except for panicle length.

DISCUSSION

Pre-harvest sprouting (PHS) is a phenomenon in which grains germinate from mature spikes during prolonged wet weather just before harvest within mordern varieties. Therefore, PHS not only causes a decrease in grain yield, but also affects quality of the grain, resulting in significant economic losses (39, 40). So far, many researchers have reported that more than 140 quantitative trait loci (QTLs) or genes are related to seed dormancy and PHS in rice. Although several genes including Sdr4, qSD7-1/Rc, qSD1-2/SD1, PHS8, PHS9, and VP1 have been cloned to reveal functional validity, knowledge about the exact molecular mechanism for PHS resistance is still very limited (10, 41-47). Recently, rice GWAS results have shown PHS resistance by one SNP and one InDel in the coding area of the OsERF1 gene (Supplementary Fig. 1) (38). Base editing, a recently known evolution of CRISPR-Cas-based techniques, can introduce point mutations directly into cellular DNA without inducing double-stranded DNA breakage (DSB). They have two known DNA base editors: the cytosine base editor (CBE) and the adenine base editor (ABE). In addition, priming editing (PE) can cause transition mutations as well as small insertion or deletion mutations in CRISPR-based editing toolkits. Therefore, DNA base editing and prime editing tools have the advantage of being able to accurately substitute nucleotides in a programmable manner without the need for donor templates. Unlike these technologies, HDR, which DNA replacement, has the advantage of being able to use relatively large base substitution, insertion, and deletion fragments in donor templates (48). In this study, HDR experiments were performed in rice calli using the CRISPR/Cas9-Geminiviral Replicon System (Fig. 1 and Supplementary Table 2). Our HDR strategy enables PHS resistance by replacing SNPs at the 4th position with insertion of 6 bp fragments at the 96th position on the ORF of the ERF1 gene. As described in our previous report, we adjusted bases to donor DNA so that when HDR occurred, it could be distinguished by restriction enzymes SnaBI (Supplementary Fig. 2). The geminiviral replicon used in the HDR experiment in this experiment is a plant virus that can infect both monocots and dicots, and is a single-stranded circular DNA of 2.5-3.0 kb in size. It is known that infection with these viruses results in rolling circle replication, generating numerous copies, which can serve as repair templates for HDR (49, 50). In addition, we increased HDR efficiency by heat treatment at 42°C for 12 hr after infecting rice calli with Agrobacterium tumefaciens during transformation experiments (Supplementary Table 2). In this experiment, the frequency of HDR was 2.6%, which was twice as high as in our previous report (37) and much higher than the frequency of 0.66% in tomato (51). The difference of the present study from the previous experiment was that heat stress was performed for 12 hours after Agrobacterium infection. Therefore, the increase in HDR frequency can be explained as a heat stress effect. However, it has been reported that although heat stress can increase inheritable mutations in some CRISPR/Cas9 experiments, the frequency of HDR is not significantly increased (52). Therefore, in order to use the HDR system for plant breeding, research to increase the frequency of HDR must be continued. In this respect, our study differs from previously reported studies in that there are no selectable markers for HDR. This experiment also demonstrated the possibility of increasing the success rate of HDR in plants. Therefore, we believe that if factors such as the number of copies of donors, the duration of HDR in cells, and the time of cell replication are fully investigated in plant HDR experiments, it will be possible to increase the HDR frequency even further. Here, the ERF1-hdr line newly generated by HDR exhibited pre-harvest sprouting resistance (Fig. 3). In the ORF of the ERF1 gene, it was found that the 1 bp SNP at position 4 and the 6 bp insertion at position 96 positively regulated rice seed dormancy status. Meanwhile, the ERF1-hdr line had a significantly higher ABA content than Dongjin (Supplementary Fig. 8), suggesting that this allele was associated with seed dormancy and PHS resistance. We also showed a large structural difference between the ERF1 protein produced by HDR and the ERF1 protein of WT. Therefore, it was believed that PHS resistance was due to changes in the structure of ERF1 protein caused by HDR (Fig. 2).

Typically, transcriptional reprogramming of the ERF1 gene is critical for seed dormancy and PHS resistance. ABA is a plant hormone that can promote seed dormancy, while GA can break dormancy and promote seed germination (22, 29). Until now, studies on ABA, ethylene, and GA signaling mutations have revealed their antagonistic roles in dormancy and germination. Therefore, the endogenous levels of these hormones and the differences in expression of genes on each signaling transduction pathway are closely correlated with maintaining dormancy and promoting (23, 53). Therefore, the present study performed qRT-PCR analysis of 16 genes present in ABA, ethylene, and GA signaling pathways reported so far in rice. First, in the case of ABA signaling-related genes, expression levels of PYL, SnRK2, ABI3, ABI5, and Sdr4 were higher in the ERF1-hdr line than in Dongjin (Fig. 4). When seeds germinate, the ABI5 gene, which is involved in ABA signal transduction, acts as a transcription factor. It has been known that the ABI5 gene is regulated by post-translational protein modifications such as phosphorylation, ubiquitination, sumoylation, and S-nitrosylation (54). Also, in the case of genes related to ethylene and GA signaling, expression levels of EIN3, ERF1, GID1, and GYMYB except for the EIN2 gene were much lower in the ERF1-hdr line than in Dongjin (Fig. 4). These results suggest that the expression level of genes in the ERF1-hdr line plays an important role in determining PHS resistance. Further research on biological functions and agricultural properties of the ERF1-hdr line is needed in the future.

MATERIALS AND METHODS

Vectors construction, Agrobacterium-mediated transformation of rice and heat treatment, Homology modeling and protein structure analysis, Mutation detection by PCR-restriction enzyme pattern and sequencing, Phenotype assessment for seed dormancy and pre-harvest sprouting, Quantitative RT‐PCR analysis, ABA concentration and Statistical analysis are described in the supplementary information.

Funding Statement

ACKNOWLEDGEMENTS This work was supported by a grant from the New Breeding Technologies Development Program (Project No. RS-2022-RD010342). Rural Development Administration and basic science research program through the National Research Foundation of Korea (NRF) funded by the ministry education (2022R1A2C1092904) Republic of Korea.

Footnotes

bmb-57-2-79-supple.pdf (973.1KB, pdf)

CONFLICTS OF INTEREST

The authors have no conflicting interests.

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