CRISPR/Cas9-mediated targeted mutagenesis of two homoeoalleles in tobacco confers resistance to powdery mildew

doi:10.21203/rs.3.rs-1892545/v1

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CRISPR/Cas9-mediated targeted mutagenesis of two homoeoalleles in tobacco confers resistance to powdery mildew

https://doi.org/10.21203/rs.3.rs-1892545/v1

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The CRISPR/Cas9 technology has proven to be an effective tool for crop improvement. In tobacco (Nicotiana tabacum L.), the Mildew Locus O (MLO) genes, NtMLO1 and NtMLO2, are responsible for powdery mildew susceptibility. However, modification of multiple homoeoalleles is complex in polyploidy crops. Therefore, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system containing a single-guide RNA (sgRNA) to enable targeted mutagenesis of both NtMLOs simultaneously and enhance the powdery mildew resistance of tobacco. The approach generated different mutants, of which two-thirds had both genes mutated. In the T₁ lines, three types of mutations were identified for NtMLO1 and two types for NtMLO2 at the expected position based on Sanger sequencing. Meanwhile, four mutation events were identified in the mutants with both genes edited in T₁ lines. These double mutants showed enhanced resistance to powdery mildew, whereas the mutants with only one edited gene showed no significant difference compared to wild-type plants. Our results suggest that CRISPR/Cas9-mediated targeted editing of NtMLO1 and NtMLO2 is a practical approach for improving the powdery mildew resistance of tobacco.

CRISPR/Cas9

Powdery mildew

NtMLO

Disease resistance

Tobacco

Powdery mildew, caused by the biotrophic fungal species of the Erysiphales order, is a common disease affecting a wide range of plant species worldwide (Quaglia et al. 2012). In tobacco (Nicotiana tabacum L.), powdery mildew caused by Golovinomyces cichoracearum (Gc) is one of the most devastating diseases (Cunnington et al. 2005). The infection changes the color of leaves from green to brown and eventually affects the yield and quality of tobacco (Darvishzadeh et al. 2010). Using cultivars containing disease resistance alleles is the most effective and eco-friendly strategy to manage powdery mildew incidence.

Wild tobacco species N. glutinosa and N. tomentosiformis harbor a dominant locus for powdery mildew resistance, which has been used in tobacco breeding (Davis and Nielsen 1999). Another strategy to achieve resistance to powdery mildew is using susceptible genes. Mildew Locus O (MLO) is a class of plant-specific proteins with seven transmembrane domains localized in the plasma membrane, which act as susceptibility factors towards powdery mildew (Devoto et al. 1999). The inactivation of MLOs in tomato induced papilla formation and callose deposition, preventing fungal penetration into the host cells (Li et al. 2007). Meanwhile, the loss-of-function alleles of MLO conferred broad-spectrum and durable resistance to various fungal species causing powdery mildew in barley (Büschges et al. 1997), Arabidopsis thaliana (Consonni et al. 2006), tomato (Bai et al. 2008), pea (Humphry et al. 2011; Pavan et al. 2011), pepper (Zheng et al. 2013), and bread wheat (Wang et al. 2014). In tobacco, naturally mutations in the intronic regions of the two NtMLO homoeologs triggered aberrant splicing of MLO transcripts and enhanced resistance to powdery mildew (Fujimura et al. 2016). These previous studies demonstrated the feasibility of using MLO mutagenesis in resistance breeding. However, in allotetraploid tobacco, the introgression of these two recessive homoeoalleles using traditional breeding methods is difficult and time-consuming. Therefore, technologies that can targeted mutate several homoeologous gene copies is necessary to improve the resistance.

Genome editing is an effective tool widely applied to improve the agricultural traits of various plants (Xu et al. 2019). The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system, which performs targeted genome editing under the guidance of an engineered sequence-specific single guide RNA (sgRNA), has revolutionized genome editing and crop improvement (Kato-Inui et al. 2018). CRISPR/Cas9 system-mediated targeted mutagenesis has been widely used to genetically modify disease resistance in rice (Wang et al. 2016; Kim et al. 2019), tomato (Nekrasov et al. 2017), citrus (Peng et al. 2017), cucumber (Chandrasekaran et al. 2016), and watermelon (Tian et al. 2018). The major advantage of this technology is its ability to edit multiple target sites simultaneously (Gao 2021). Therefore, it is ideal for genetically improving polyploid crops with traits controlled by multiple gene copies (Zaman et al. 2019; Gao 2021). Recent studies have demonstrated the success of this technique in the targeted mutagenesis of multiple genes. In bread wheat, targeted editing of three homoeoalleles of MLO simultaneously using this approach conferred powdery mildew resistance (Wang et al. 2014).

Therefore, the present study used the CRISPR/Cas9 system for mutating two MLO homoeologs simultaneously in the allotetraploid tobacco aiming at powdery mildew resistance. Results showed that the CRISPR/Cas9 system successfully edited the NtMLO1 and NtMLO2 simultaneously, and the double mutant lines displayed enhanced resistance to powdery mildew.

Plant materials

The N. tabacum cv. ZY300 susceptible to powdery mildew was obtained from the Tobacco Research Institute, Chinese Academy of Agricultural Sciences. The plants were maintained in a growth chamber at 22 ℃ under a 16 h light cycle with 75±15% relative humidity.

sgRNA designing and vector construction

The full-length coding sequences (CDS) of the NtMLO1 (Genebank accession number: LC089758.1) and NtMLO2 (Genebank accession number: LC089759.1) genes were retrieved from the National Center for Biotechnology Information database. Then, a single guide RNA (sgRNA) targeting a conserved region within exon 1 of NtMLO1 and NtMLO2 was designed using the online tool CRISPR MultiTargeter (Prykhozhij et al. 2015). The pORE-Cas vector (Gao et al. 2015) and the sgRNA expression cassette driven by the AtU6-26 promoter were ligated to generate the recombinant expression vector pORE-Cas-gRNA-MT. The pORE-Cas-gRNA-MT was used to transform the Agrobacterium tumefaciens strain EHA105 by the freeze-thaw method. Primers used for constructing the plasmid are listed in Supplementary Table 1.

Generation of transgenic tobacco plants

Tobacco plants were transformed following the leaf disc method (Li et al. 2020). Leaf discs from five-week-old tobacco plants were infected with the Agrobacterium strain EHA105 harboring the pORE-Cas-gRNA-MT and placed on a regeneration medium (MS medium containing 0.1 mg/L 1-naphthaleneacetic acid, 1.0 mg/L 6-benzylaminopurine, 250 mg/L carbenicillin sodium, and 50 mg/L kanamycin sulfate, pH 5.8–6.0). The regenerated shoots were then transferred to the root induction medium (MS medium containing 250 mg/L carbenicillin sodium and 50 mg/L kanamycin sulfate, pH 5.8–6.0). The drug-resistant seedlings were obtained and prepared for analysis.

PCR-based identification of the targeted mutants

Genomic DNA of kanamycin resistant transgenic plants and wild type plants was extracted using DNeasy Plant Mini Kit (Qiagen, CA) to analyze targeted mutagenesis using PCR amplification and Sanger sequencing. The DNA fragment flanking the sgRNA target regions was amplified by PCR using NtMLO1 and NtMLO2 gene-specific primers (Supplementary Table 1) and subjected to Sanger sequencing. The study used sequence alignment to detect the homozygous mutations within the target sites and the sequencing chromatograms to identify heterozygous mutations (double peaks at the targeted sites). These T₀ plants carrying the mutations were further selected and self-pollinated to produce the T₁ progeny. The T₁ generation of plants was then screened by PCR using NtMLO1 and NtMLO2 gene-specific primers and the amplicons were sequenced. The plants with homozygous mutations in NtMLO1 only, NtMLO2 only, and both NtMLO1 and NtMLO2 were selected and self-pollinated to obtain the T₂ progeny; these T₂ lines were used to assess powdery mildew resistance.

Off-target analysis

The potential off-target sites of sgRNA in tobacco were identified by searching against the tobacco genome (version: AYMY01) using the Cas-OFFinder (Bae et al. 2014; http://www.rgenome.net/cas-offinder/), allowing three or fewer mismatches between the target sgRNA and aligned sequences. The presence of these potential off-target sites was checked via PCR using specific primers (Supplementary Table 1), followed by Sanger sequencing.

Pathogen inoculation and disease resistance evaluation

The resistance of T₂ mutants to powdery mildew was analyzed. The conidiospores obtained from the infected tobacco leaves were suspended in distilled water and adjusted to a concentration of about 3.0 × 10⁵ spores mL^-1to prepare the inoculum. Six-week-old plants from the homozygous T₂ lines with different alleles (lines C2-8 and C8-10 for only NtMLO1, lines C3-1 and C1-15 for only NtMLO2, and lines C1-7 and C13-21 for both NtMLO1 and NtMLO2) were inoculated by spraying the conidiospore suspension. A transgenic line (C4) obtained from the same CRISPR transformation experiment with the wild-type alleles was used as a control. The disease severity was assessed 20 days post-inoculation (dpi) using a scale of 0, 1, and 3 and expressed as disease index (DI) (Bai et al. 2008). Meanwhile, to measure the relative fungal biomass, the fungal and plant genomic DNAs were extracted from the infected leaves (20 dpi) using the MiniBEST universal genomic DNA extraction kit (Takara, Dalian, China). Then, a real-time PCR was carried out using LightCycler®96 system (Roche, Switzerland). Relative fungal biomass was determined using 15 ng of the total DNA as a template and primers designed for the Gc-specific internal transcribed spacer sequence (ITS; GenBank accession number AF011292) (Zheng et al. 2016). The tobacco Ef1α gene was used as a reference to normalize the amount of fungal DNA in different samples. Finally, the fold change of the ratio of fungal genomic DNA to tobacco genomic DNA was calculated using the ΔΔC_T method (Livak et al. 2001). Primers used for real-time PCR are listed in Supplementary Table 1.

Vector construction for the CRISPR/Cas9 system

Initially, we aligned the full-length CDS of NtMLO1 and NtMLO2 to identify a site to design sgRNA targeting both genes simultaneously. The alignment showed 95.62% similarity between the NtMLO1 and NtMLO2 sequences (Supplementary Fig. 1). To increase the chances of obtaining complete loss-of-function mutations, a site targeting a conserved region, including a 20 bp nucleotide with the protospacer adjacent motif (PAM) at the 3’ region, was chosen within exon 1 of these genes (Fig. 1A). The sgRNA was cloned into the pORE-Cas vector to generate the pORE-Cas-gRNA-MT construct used to transform the susceptible tobacco cultivar ZY300 (Fig. 1B).

CRISPR/Cas9-mediated mutagenesis of NtMLO

In total, 20 transgenic plants (T₀) were obtained from the leaf discs transformed with the Agrobacterium (Supplementary Fig. 2). The genomic DNA of these T₀ individuals was amplified by PCR using NtMLO1-specific (MJC2F/MJC2R) and NtMLO2-specific (MJC8F/MJC8R) primers and sequenced to characterize the mutations. Sequence analysis showed that 13 out of the 20 T₀ transgenic plants had heterozygous mutations in NtMLO1, 12 had heterozygous mutations in NtMLO2 (Table 1, Supplementary Fig. 3). Among these mutants, 10 plants had heterozygous mutations in both genes. The approach achieved a mutation frequency of 65.0% in NtMLO1 and 60.0% in NtMLO2. Approximately 66.7% of the mutants had both genes mutated (Table 1).

We further characterized the T₁ progenies derived from the T₀ plants to obtain the homozygous mutants. Plants homozygous for the mutant alleles were selected from the T₁ segregating lines by sequencing the specific PCR fragments. Three homozygous mutation types were identified for NtMLO1, including a 1 bp deletion (-T), a 2 bp deletion (-GA), and a 1 bp insertion (+A) (Fig. 2A). For NtMLO2, two homozygous mutation types, including a 1 bp insertion (+A) and an 11 bp deletion (-ACGGTCGATGG), were identified (Fig. 2B). We identified individual plants with simultaneous homozygous mutations in both genes from the progenies of the 10 T₀ plants (C1, C5, C7, C10, C11, C12, C13, C18, C19, C20). Four different mutation events were identified in these simultaneous homozygous mutants (Fig. 2C). Event 1 resulted in a 1 bp deletion in NtMLO1 and a 1 bp insertion in NtMLO2 in the T₁ plants obtained from the T₀ lines C5, C10, C11, C12, and C20. Event 2 resulted in a 2 bp deletion in NtMLO1 and a 1 bp insertion in NtMLO2 in the T₁ plants from the T₀ lines C1, C7, and C18. Event 3 generated a 1 bp insertion in NtMLO1 and an 11 bp deletion in NtMLO2 in the T₁ plants obtained from the T₀ line C13. Event 4 created 1 bp insertions in NtMLO1 and NtMLO2 in the T₁ plants obtained from the T₀ line C19. We then obtained the self-pollinated seeds from the homozygous T₁ mutants for the further analysis.

Off-target analysis

Furthermore, we predicted the putative off-target mutations induced by the sgRNA in the whole genome using Cas-OFFinder (Bae et al. 2014), allowing a mismatch of three or fewer nucleotides between the sgRNA and the potential off-target regions to assess the specificity of this CRISPR/Cas9 system. Four potential off-targets with a PAM sequence were identified in the whole genome (Supplementary Table 2), of which one off-target, located on chromosome AYMY01S000835.1, had two mismatches, and the other three off-targets, located on chromosomes AYMY01S001145.1, AYMY01S001369.1, and AYMY01S024885.1, had three mismatches. We then checked for these potential off-target sites in three plants randomly selected from the homozygous T₂ lines C1-7 and C13-21 by PCR using different primer combinations (Supplementary Table 1). Sequencing showed no differences between the wild type and the mutant on these four potential sites (Supplementary Fig. 4), suggesting that no off-target mutation occurred in the potential sites.

Resistance of mutants to powdery mildew

Finally, to assess the impact of CRISPR/Cas9-induced mutations on powdery mildew resistance in tobacco, the T₂ lines with homozygous mutations in only NtMLO1 (C2-8 and C8-10), only NtMLO2 (C3-1 and C1-15), and both NtMLO1 and NtMLO2 (C1-7 and C13-21) (Supplementary Fig. 5) were inoculated with Gc. No apparent disease symptom was observed on the leaves of the C1-7 and C13-21 lines with homozygous mutations in both NtMLO1 and NtMLO2 (Fig. 3A, Supplementary Fig. 6). Meanwhile, fungal growth was obvious in the wild-type plants and the mutants with only one edited gene (Fig. 3A). Consistent with these findings, the DI and relative Gc biomass on the leaves of the C1-7 and C13-21 lines were significantly lower than the wild-type plants. However, no significant differences in the disease index or the relative Gc biomass were observed among the wild-type plants and single mutants (Fig. 3B, C). These results indicated the role of both NtMLO1 and NtMLO2 genes in responding to powdery mildew infection, and mutating both genes could confer resistance to powdery mildew in tobacco.

The CRISPR/Cas9 system has significantly boosted the application of targeted genome editing in crop breeding (Hua et al. 2019). Genome editing helps eliminate the susceptible genes or negative elements and enhance disease resistance in cultivars (Chen et al. 2019). MLO is one of the best-studied susceptibility genes of plants; a loss-of-function mutation in MLO confers broad-spectrum and durable resistance against powdery mildew (Santillán Martínez et al. 2020). In common tobacco, two MLO homoeologs control the resistance to powdery mildew, and these two homoeologs are functionally redundant (Appiano et al. 2015; Fujimura et al. 2016). However, the introgression of these two homoeologs into elite cultivars by traditional backcross is time-consuming. Recently, the CRISPR-Cas9-based approach edited three homoeoalleles of MLO simultaneously and created a bread wheat cultivar with powdery mildew resistance; this study suggested the suitability of the CRISPR-Cas9 system for polyploid crop improvement (Wang et al. 2014; Li et al. 2022). Similarly, in the present study, we used the CRISPR/Cas9 genome-editing system in allotetraploid tobacco and edited NtMLO1 and NtMLO2 simultaneously. Our results showed that the sgRNA, targeting a conserved region in exon 1, efficiently induced mutation. Among the 15 T₀ mutants, 10 had both genes mutated simultaneously (Table 1). Three types of homozygous mutants were obtained in T₁ generation, including NtMLO1 edited only, NtMLO2 edited only, and both genes edited simultaneously. In the double mutants, four different mutation events were identified (Fig. 2). Six T₁ plants (C2-8 and C8-10 NtMLO1 edited only, C3-1 and C1-15 NtMLO2 edited only, C1-7 and C13-21 both genes edited) were selected to further analyzed the transmission of homozygous mutations to their T₂ offspring. The homozygous mutations from six T₁ plants were all transmitted to T₂ offspring (Supplementary Fig. 5). These results suggested that CRISPR/Cas9-mediated mutations in NtMLOs can be stably transmitted to subsequent generations.

However, off-target activity-induced mutation at sites other than the target site is undesirable and a major concern of the CRISPR/Cas9 system, which has less happened in plant genome editing than in animals (Zhang et al. 2021). In rice and cotton, CRISPR/Cas systems did not induce sgRNA-independent off-target effects (Li et al. 2019; Tang et al. 2018). This study examined the sgRNA-dependent off-target effect of the CRISPR/Cas9 construct targeting the MLO genes in double mutants. We searched the tobacco genome for potential off-target sites allowing three or fewer mismatched nucleotides between sgRNA and the off-target regions. Four potential off-targets with a PAM sequence were found in the genome. Further verification of mutants by PCR showed no off-target mutations (Supplementary Table 2; Supplementary Fig. 4), suggesting that the target sequence was specific.

In common tobacco, natural mutations in the intronic regions of the two NtMLO genes, which lead to aberrant splicing of MLO transcripts, are responsible for the occurring broad-spectrum powdery mildew resistance (Fujimura et al. 2016). In the double mutants of the present study, both NtMLO1 and NtMLO2 genes had frameshift mutation, which caused abnormal protein synthesis. Finally, disease resistance evaluation showed lower powdery mildew incidence and relative Gc biomass in the double mutant than the control and the mutants with a single edited gene (Fig. 3). These results confirmed that both NtMLO1 and NtMLO2 contribute to the susceptibility of tobacco to Gc infection, and editing one single gene could not reduce the incidence of powdery mildew, which is consistent with the previous reports (Wang et al. 2014; Fujimura et al. 2016).

However, susceptible genes are implicated in various other biological functions, and therefore, recessive gene-mediated disease resistance is often associated with growth defects and yield losses (Li et al. 2022). In hexaploidy bread wheat, Tamlo-aabbdd mutants with three TaMLO homoeologs simultaneously edited exhibited reduced plant height and grain yield compared to wild-type plants, although they were highly resistant to powdery mildew (Li et al. 2022). To solve this problem, an alternative breeding strategy was the use of the weak mlo alleles to attain a balance between disease resistance and plant growth (Acevedo-Garcia et al. 2014). Meanwhile, Li and his colleagues demonstrated a gain-of-function mutation that overcame the growth defects caused by mlo recessive resistance alleles in wheat postulating a promising strategy to take full advantage of the susceptible genes in crop breeding (Li et al. 2022). These observations suggest that the double mutant of tobacco generated in this study may influence growth and yield; therefore, the pleiotropic effects of NtMLO mutations on the yield and quality of tobacco should be further tested.

The present study identified a highly efficient and specific CRISPR/Cas9 system for editing two NtMLO homoelogs simultaneously in the allotetraploid tobacco for powdery mildew resistance. We characterized four different mutation events in mutant lines with two homoelogs mutated simultaneously and confirmed enhanced powdery mildew resistance of these lines. Our study presents an excellent approach based on the CRISPR/Cas9 system in breeding resistance against pathogens using the susceptibility genes of polyploid crops.

Acknowledgements

We would like to thank Topedit for English language editing of this manuscript.

Author contribution

LD, FXK, and YAG conceived and designed the experiment. WXB, LDD performed vector construction and transformation. TXL and CCC performed mutant identification. LDD, ZXY, and SZ performed Gc inoculation. WXB, LD, and FXK wrote the manuscript. All authors revised the manuscript.

Funding

This work was supported by the Agricultural Science and Technology Innovation Program of CAAS (ASTIP-TRIC01), Tobacco Genome Project of China National Tobacco Corporation [110202001022(JY-05), 110202001019(JY-02), 110201801025(JY-02)], the key Science and Technology Program of Hubei Tobacco Corporation (027Y2020-014), and the China Tobacco Hunan Industrial Co., Ltd. Research Project (KY2019YC0003).

Data availability

The data sets supporting the results of this article are included within the article and its additional files.

Ethics approval and consent to participate

No applicable.

Consent for publication

No applicable.

Conflict of interest

The authors declare no competing interests.

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Table 1

Frequencies of CRISPR/Cas9-induced mutation in the T₀ transgenic tobacco plants
Variety	Total number of plants^a tested	Number of NtMLO1 mutated plants	Number of NtMLO2 mutated plants	Number of NtMLO1 and NtMLO2 mutated plants	Number of plants without mutation
ZY300	20	13 (65.0%)^b	12 (60.0%)	10 (50.0%)	5 (25.0%)
^a Referred to transgene-positive plants
^b Frequencies based on the number of mutated plants over the total number of transgene plants tested.

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CRISPR/Cas9-mediated targeted mutagenesis of two homoeoalleles in tobacco confers resistance to powdery mildew

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