Targeted Mutagenesis of NtCPS2 by CRISPR/Cas9 in Tobacco
In this study, Cas9 gene was optimised to edit the tobacco genome. To generate Cas9-induced mutations in NtCPS2, a vector was designed that harboured chimeric guide RNA (gRNA) to guide Cas9 to target sequences where it bound and cleaved genomic DNA to generate double-strand breaks [22]. Two target sites of CPS2 were selected (Supplementary Fig. 1). The gRNA for each target site, which was generated by overlap-extension PCR. Cas9 were subcloned into a single expression vector [23]. The Cas9 and gRNA expression cassette was located in one expression vector (pRGEB32-Cas9-NPT II-CPS2-gRNA). Through the Agrobacterium tumefaciens-mediated method, 36 transformed regenerated plants in the T0 generation were obtained. After amplification with target-specific primers, all positive samples were sequenced to assess the mutation efficiency. Of 36 plants, eight were transgenic lines. Most of the transgenic lines had a single-base insertion of A, C, or T at Target 2. Thus, as the peptide chain was being formed, the stop codon was encountered early in the process, and the translated amino-acid chain was greatly shortened. To test the heritability of the mutations, homozygous transgenic plants in the T0, T1, and T2 generations were analyzed. Detailed information about the homozygous T2 plants (M1–M9) is shown in Fig. 1A, and these plants were used for the following experiments.
NtCPS2 Knockout Affects cis-Abienol Content
To verify whether the gene mutations caused changes in gene expression, quantitative real-time PCR (qRT-PCR) was used to detect expression levels of NtCPS2 in the leaves of mutant and wild-type (8306) plants. The results showed that NtCPS2 expression decreased significantly in transgenic plants compared to wild-type plants (Fig. 1B). To detect changes in cis-abienol content in the leaves, exudates were collected from the mutant plants and analyzed using GC-MS. Contents of cis-abienol also decreased significantly in mutant plants compared to wild-type plants (Fig. 1C). The results indicate that NtCPS2 is one of the key genes regulating the cis-abienol biosynthesis pathway, and NtCPS2 knockout results in low levels of cis-abienol biosynthesis and accumulation. NtABS is another key gene involved in cis-abienol biosynthesis [16]. A previous study reported that cis-abienol was detected in plants expressing both NtCPS2 and NtABS but not in plants expressing just one of the two genes [16]. NtABS expression was weak in the mutant plants compared to the wild-type plants, implying that NtCPS2 knockout negatively influenced NtABS expression. This is possibly because NtCPS2 is located upstream of NtABS in the cis-abienol biosynthesis pathway.
NtCPS2 Has a Minor Effect on the Development of Glandular Trichomes in Tobacco
Agronomic characteristics were analyzed to assess the mutant phenotypes (Fig. 2 and Supplementary Fig. 2). Differences in plant height, internode length, number of leaves, and stem girth between mutant and wild-type plants did not exhibit the same trend. T2-2 mutants had longer internodes and wider stems than other mutants and wild-type plants, whereas all mutants except for T2-1 had shorter plant heights than the wild-type plants (Supplementary Fig. 2). These results indicate that NtCPS2 expression does not strongly affect tobacco plant morphology. As NtCPS2 is specifically expressed in glandular cells [16], the morphology of the glandular trichomes on the largest leaf of each plant was examined. Both the length and width of the largest leaf were significantly shorter in mutant plants compared to wild-type plants. The average diameter of glandular trichomes was smaller in mutant plants, especially T2-1, whereas both longer and shorter glandular trichomes were observed in mutant plants compared to wild-type plants (Fig. 2). Other trichome characteristics, such as numbers of long and short trichomes, did not differ significantly between mutant and wild-type plants (data not shown). Thus, in the absence of NtCPS2 expression in tobacco plants, the diameter of glandular cells and the area of the largest leaf decrease, but not the length of glandular trichomes. The T2-1 line was selected and used to profile transcriptomic changes after NtCPS2 knockout in tobacco 8306.
Overview of Transcriptome Sequencing
To profile gene expression after NtCPS2 knockout, RNA-seq libraries were constructed for the mutant and wild-type plants. Six samples of each line were sequenced, and 41.64 G of clean data was obtained. In total, 6.70–7.02 G of effective data was collected from each sample, with a Q30 distribution of 94.29–94.91%, and an average GC content of 43.41%. More than 95.58% of the clean reads had quality scores that met the Q30 criterium (probability of base-calling error = 0.1%) [24]. Furthermore, the GC content ranged from 43.15–43.66%. The sequencing data are summarised in Table 1.
Table 1
Summary of RNA-sequencing outcomes.
Sample | Raw reads | Clean reads | Raw bases | Clean bases | Q30 (%) | GC (%) |
Con1 | 49.05 M | 47.90 M | 7.36 G | 6.89 G | 94.60 | 43.46 |
Con2 | 49.76 M | 48.74 M | 7.46 G | 7.02 G | 94.88 | 43.25 |
Con3 | 47.66 M | 46.51 M | 7.15 G | 6.70 G | 94.29 | 43.30 |
L1 | 49.71 M | 48.57 M | 7.46 G | 6.97 G | 94.67 | 43.66 |
L2 | 49.77 M | 48.72 M | 7.47 G | 6.99 G | 94.88 | 43.41 |
L3 | 49.70 M | 48.62 M | 7.45 G | 6.97 G | 94.85 | 43.47 |
Not: Con, wild type; L, mutant. |
Analysis of Differentially Expressed Genes (DEGs) and Their Functions
Volcano plots were used to assess the variation in gene expression between mutant and wild-type plants (Fig. 3A). In total, 9,514 DEGs were detected. Among them, 4,279 were upregulated and 5,235 were downregulated in the transgenic tobacco plant compared to 8103 using the thresholds p < 0.05 and |log2(fold change [FC])| > 1 (Fig. 3B).
Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analyses of the differentially expressed mRNAs were performed to determine the functions of the DEGs. The 20 most significantly enriched pathways (lowest q values) according to KEGG metabolic pathway annotation were examined in detail (Fig. 4A). Based on GO analysis, the DEGs were most likely to be associated with biological processes (Fig. 4B) and cellular components (Fig. 4C). A large percentage of the DEGs was assigned to the categories metabolic process, cellular process, catalytic activity, binding, and single-organism process, with only a few genes assigned to channel regulator activity, cell killing, and protein tag. The DEGs involved in the pathways for diterpenoid biosynthesis, plant hormone signal transduction, and plant-pathogen interactions were analyzed in detail.
Validation of Selected DEGs Using qRT-PCR
To validate the RNA-seq data, 12 DEGs, including genes involved in cis-abienol and gibberellin (GA) biosynthesis as well as genes related to plant-pathogen interactions and other hormone signalling pathways, were selected randomly for qRT-PCR analysis. The gene-expression patterns as determined using qRT-PCR were consistent with those determined via transcriptome sequencing (Fig. 5). FC values differed between qRT-PCR and RNA-seq, possibly due to differences in the sensitivity of each method or because different samples were used for qRT-PCR and RNA-seq.
Expression Levels of Genes Related to cis-Abienol Biosynthesis Decreased Significantly in Mutant Plants
NtCPS2 (Nitab4.5_0001630g0010) was identified as a DEG via RNA-seq, and its expression level was 9.27-fold lower in the mutant compared to the wild type. The expression level of another key gene related to cis-abienol biosynthesis, NtABS (Nitab4.5_0015240g0010), also decreased 2.43-fold in the mutant. NtCPS2 and NtABS operate in succession to synthesise cis-abienol [16]. When both NtCPS2 and NtABS are expressed, cis-abienol is synthesised and can be detected in plants. However, cis-abienol synthesis does not occur in plants that express only one of these genes [16]. NtCPS2 encodes 8-hydroxy-copalyl diphosphate synthase, which synthesises 8-hydroxy-copalyl diphosphate, and NtABS encodes a kaurene synthase-like (KSL) protein, abienol synthase, which uses 8-hydroxy-copalyl diphosphate to produce cis-abienol. Our results indicate that weak expression of NtCPS2 directly or indirectly results in a decrease in the expression level of NtABS and consequently, low cis-abienol contents. Another putative cis-abienol synthase (Nitab4.5_0008024g0010) was also found to be downregulated in the mutant, indicating that this enzyme may have the same substrate as NtABS and thus be involved in the cis-abienol biosynthesis pathway. By contrast, other putative cis-abienol synthases, including Nitab4.5_0004164g0070 and Nitab4.5_0004164g0010, were found to be upregulated in the mutant. These two enzymes may have other functions in tobacco. Other DEGs involved in the cis-abienol biosynthesis pathway were also identified (based on KEGG analysis) and had lower expression levels in the mutant (Fig. 4). This included KSL4 (Nitab4.5_0000029g0200, FC = 2.43) and genes predicted to encode ent-kaur-16-ene synthase (Nitab4.5_0002280g0060, FC = 5.61; and Nitab4.5_0002862g0030, FC = 1.57). As with KSL4, NtABS is a KSL gene (Table 2). Hence, KSL4 and genes that putatively encode ent-kaur-16-ene synthase may be involved in cis-abienol biosynthesis. This needs to be verified in future work.
Table 2
Genes related to diterpenoid biosynthesis that are differentially expressed between NtCPS2-knockout and 8306 plants.
Gene name | Gene ID | log2FC | Protein properties |
CPS2 | Nitab4.5_0001630g0010 | -3.21 | PREDICTED: copal-8-ol diphosphate hydratase, chloroplastic |
KSL4 | Nitab4.5_0000029g0200 | -1.21 | PREDICTED: ent-kaur-16-ene synthase, chloroplastic isoform X3 |
DLO2 | Nitab4.5_0000129g0310 | -2.53 | PREDICTED: gibberellin 2-beta-dioxygenase 8-like |
GA2OX2 | Nitab4.5_0000222g0140 | -5.09 | PREDICTED: gibberellin 2-beta-dioxygenase 2 |
GA2OX1 | Nitab4.5_0000923g0050 | -3.82 | PREDICTED: gibberellin 2-beta-dioxygenase 1-like |
GA2OX2 | Nitab4.5_0001013g0080 | -2.88 | PREDICTED: gibberellin 2-beta-dioxygenase 2-like |
KAO2 | Nitab4.5_0001476g0100 | 4.06 | PREDICTED: ent-kaurenoic acid oxidase 1-like isoform X2 |
GA20OX2 | Nitab4.5_0001573g0060 | 2.08 | gibberellin 20 oxidase 1-like |
GA2OX1 | Nitab4.5_0002209g0240 | -1.46 | gibberellin 2-beta-dioxygenase 1-like |
KO | Nitab4.5_0002280g0060 | -2.49 | PREDICTED: ent-kaurene oxidase, chloroplastic |
GA2 | Nitab4.5_0002862g0030 | -1.19 | PREDICTED: ent-kaur-16-ene synthase, chloroplastic-like isoform X1 |
KS1 | Nitab4.5_0004164g0010 | 1.46 | PREDICTED: cis-abienol synthase, chloroplastic-like |
TPS1 | Nitab4.5_0004164g0070 | 3.00 | PREDICTED: cis-abienol synthase, chloroplastic-like |
GA2 | Nitab4.5_0008024g0010 | -1.20 | PREDICTED: cis-abienol synthase, chloroplastic-like |
CPS1 | Nitab4.5_0010312g0010 | 3.60 | PREDICTED: ent-copalyl diphosphate synthase, chloroplastic-like isoform X1 |
ABS | Nitab4.5_0015240g0010 | -1.28 | cis-abienol synthase, chloroplastic |
Note: FC, fold change. |
GA Biosynthesis Increased Significantly in Mutant Plants
According to diterpenoid biosynthesis pathways, the same substrate, geranylgeranyl pyrophosphate (GGPP), is used for cis-abienol and GA synthesis. In this study, most of the DEGs involved in GA biosynthesis were strongly upregulated in the mutant, including KAO2 (Nitab4.5_0001476g0100, FC = 16.67), KS1 (Nitab4.5_0004164g0010, FC = 2.76), and CPS1 (Nitab4.5_0010312g0010, FC = 12.14) (Table 2). From these genes, ent-copalyl diphosphate synthase 1 (encoded by CPS1) and ent-kaurene synthase (encoded by KS1) were found to separately catalyse the synthesis of ent-kaurene from GGPP. However, KO (encodes ent-kaurene oxidase, which converts ent-kaurene to kaur-16-en-18-oate) expression was downregulated in the mutant. DEGs participating in the latter stages of the pathway, such as KAO2 and GA20OX2, were upregulated compared to the wild type. KO and KAO belong to the CYP701A, P450, and CYP88A clade. Accordingly, KAO is localised in the endoplasmic reticulum, whereas KO is localised in both the endoplasmic reticulum and plastid envelope [25]. The differential expression of KO1 and KAO2 in response to NtCPS2 knockdown was explored further. GA contents in mutant plants were also analyzed via GC-MS. The results showed that the GA contents in transgenic plants were significantly lower than those in wild-type plants (Fig. 6). GA12 is considered the precursor of all GAs in plants [26], and other GA forms are produced through oxidative steps catalysed by GA12. Genes involved in the production of these GA forms were up- and downregulated in the mutant.
Changes in Abscisic Acid (ABA) Biosynthesis and Signal Transduction in Mutant Plants
In carotenoid biosynthesis pathways, GGPP is also a substrate for ABA synthesis. RNA-seq analysis showed that four PSY genes (encoding phytoene synthases) were upregulated at the first step, which involves GGPP, in the mutant compared to the wild type. PSY is a transferase enzyme that is involved in the biosynthesis of carotenoids. It catalyses the conversion of GGPP to phytoene. Two genes encoding LCYs (lycopene epsilon cyclases) were also upregulated in the mutant at the next step. These results indicate that NtCPS2 knockout positively regulates ABA synthesis, likely because substrate competition decreases. In addition, two ABA 8'-hydroxylases, which are involved in ABA degradation, were downregulated in the mutant. In the ABA signal transduction pathway, five of six ABA receptors (PYLs), which inhibit the expression of protein phosphatase 2C, were upregulated in the mutant. At the next step, serine/threonine-protein kinase expression was upregulated in the mutant. This might have been related to stress responses and stomatal opening and closure in tobacco leaves.
Transcriptomic Analysis of Genes Involved in Plant-pathogen Interactions
In plants, cis-abienol may participate in insect resistance and disease resistance [27]. Plant resistance to pathogen attack can induce the accumulation of pathogenesis-related proteins (PRs) that contribute to systematically acquired resistance. In this study, PRs were identified through RNA-sEq. Of 17 PRs, 14 (82.35%) were significantly upregulated in the mutant than in the wild type, including genes that encode PR proteins 1A, B, and C (Table 3). Among the 17 families of PRs, PR 1–5, 9–11 and 17, were related to the acquisition of defence against the pathogen infections. In addition, calcium is involved in regulating diverse physiological processes as a second messenger [28]. Results of transcriptomic analysis revealed that, 15 of 19 CDPKs and most CAM/CML were significantly downregulated upon NtCPS2 knockout and low content of cis-abienol, which disturbed the balance among active oxygen species, including rubidium hydroxide, reactive oxygen species, and nitric oxide synthase.
Table 3
Properties of DEGs encoding pathogenesis-related proteins.
Gene name | Gene ID | log2FC | Protein properties |
PRB1 | Nitab4.5_0003771g0010 | 4.52 | Pathogenesis-related protein 1A |
OSM34 | Nitab4.5_0004097g0050 | 3.76 | PREDICTED: pathogenesis-related protein R minor form |
PRB1 | Nitab4.5_0014031g0010 | 3.51 | PREDICTED: pathogenesis-related protein 1B-like |
- | Nitab4.5_0006088g0020 | 3.46 | PREDICTED: pathogenesis-related protein PR-4B |
- | Nitab4.5_0018960g0010 | 3.35 | PREDICTED: pathogenesis-related protein PR-4B |
- | Nitab4.5_0008835g0020 | 3.27 | PREDICTED: pathogenesis-related protein STH-2-like |
- | Nitab4.5_0004861g0030 | 3.24 | PREDICTED: pathogenesis-related protein 1C-like |
PRB1 | Nitab4.5_0004861g0040 | 3.16 | PREDICTED: pathogenesis-related protein 1C |
- | Nitab4.5_0008375g0050 | 3.06 | PREDICTED: pathogenesis-related protein STH-2-like |
HEL | Nitab4.5_0009495g0020 | 2.73 | PREDICTED: pathogenesis-related protein PR-4A |
TL1 | Nitab4.5_0008011g0010 | 2.29 | PREDICTED: pathogenesis-related protein 5-like isoform X1 |
- | Nitab4.5_0000194g0120 | 2.17 | PREDICTED: pathogenesis-related protein STH-2-like |
CRF2 | Nitab4.5_0000105g0290 | 2.06 | PREDICTED: pathogenesis-related genes transcriptional activator PTI6-like |
CRF2 | Nitab4.5_0002902g0060 | 1.11 | PREDICTED: pathogenesis-related genes transcriptional activator PTI6-like |
MOS11 | Nitab4.5_0002073g0060 | -1.47 | PREDICTED: pathogenesis-related protein PRMS-like |
CRF2 | Nitab4.5_0000586g0010 | -2.67 | PREDICTED: pathogenesis-related genes transcriptional activator PTI6-like |
CRF2 | Nitab4.5_0007730g0010 | -2.72 | PREDICTED: pathogenesis-related genes transcriptional activator PTI6-like |