Manipulation of CBTS1 Expression Alters Tobacco Resistance to Spodoptera frugiperda and Phytophthora nicotianae

Abstract

Cembranoids produced by tobacco glandular trichomes have bioactivities in resistance to insect pests and pathogens. Cembratrien-ol synthase (CBTS) plays a key role in the biosynthesis of cembranoids and directly determines the cembranoid content in tobacco. This study examined the effect of changing CBTS1 expression on tobacco resistance to the insect pest Spodoptera frugiperda and oomycete pathogen Phytophthora nicotianae. The CDS sequence of CBTS1 was cloned into gene overexpression and silencing vectors and introduced into tobacco (Nicotiana tabacum L. cv. TN90) to obtain CBTS1-overexpression plants (CBTS1-OE) and CBTS1-silenced plants (CBTS1-RI). Compared with control plants, the content of cembratrien-ol (CBT-ol) was increased 4.48 times in the CBTS1-OE plants but decreased by 68% in the CBTS1-RI plants, while that of cembratrien-diol (CBT-diol) was increased 3.17 times in the CBTS1-OE plants but decreased by 76% in the CBTS1-RI plants. The S. frugiperda resistance of transgenic tobacco plants was evaluated by in vitro toxicity test, and the results showed that the resistance of CBTS1-OE plants to S. frugiperda was significantly improved but that of CBTS1-RI plants was reduced. The P. nicotianae resistance of transgenic tobacco plants was assessed by the detached leaf assay, and the results showed that the resistance of CBTS1-OE plants to P. nicotianae was enhanced, while that of CBTS1-RI plants was attenuated. Further gene expression analysis showed that overexpression of CBTS1 increased the expression of the pathogen-related gene PR-1a, while silencing of CBTS1 decreased its expression. This study demonstrated that manipulating the expression of CBTS1 could change the cembranoid content in tobacco plants and alter their resistance to both insect pests and oomycete pathogens.

1. Introduction

The glandular secretory trichomes of tobacco (Nicotiana tabacum L.) accumulate a large number of metabolites, among which are the cembranoids [1,2]. Cembranoids are a class of macrocyclic skeleton compounds formed by four isoprene units connected end-to-end [3,4]. These kinds of compounds have good antimicrobial, insecticidal, cytotoxic, and anti-cancer bioactivities, and have potentials to be developed as plant fungicide [5,6,7], cytotoxic [8,9], and antitumor drugs [10]. Thus, cembranoids are attracting great attention from multiple research fields.

In nature, tobacco is the terrestrial plant with the most abundant amount of cembranoids, which are also a kind of aromatic compounds of tobacco [11,12]. Cembratrien-ols (CBT-ols) and cembratrien-diols (CBT-diols) are the major cembranoids in tobacco, which are biosynthesized and secreted by the glandular trichomes on tobacco leaves and flowers [13,14]. CBT-ols are biosynthesized from geranylgeranyl pyrophosphate (GGPP) under the catalyzation of cembratrien-ol synthase (CBTS), which are then catalyzed by CYP450 to form CBT-diols [15,16]. Silencing of CYP450 could inhibit the production of CBT-diols and increase the content of CBT-ols in tobacco plants, which resulted in the enhanced resistance to aphids [17]. These facts showed that CBT-ols have an anti-insect bioactivity. On the other hand, CBT-diols are antimicrobial compounds and can inhibit the growth of both fungal pathogens, such as Aspergillus niger, Alternaria alternata, and Candida albicans, and bacterial pathogens, such as Staphylococcus aureus, Bacillus subtilis, and Proteus vulgaris [8]. Studies have also suggested that β-CBT-diol has better antimicrobial activities than α-CBT-diol [8].

Tobacco plants are often attacked by herbivores and pathogens in nature. Insect pests and microbial pathogens always cause great yield loss to tobacco production, and insect pests also mediate the transmission of microbial pathogens [18]. The insect pest S. frugiperda, also known as fall armyworms, is a polyphagous, migratory and destructive lepidopteran pest originating from the Americas, and is invading the sub-Saharan countries, India, and China in recent years [19,20]. S. frugiperda can feed on the leaves, stems, and reproductive organs of tens of crop species [21]. The survival rate of S. frugiperda larvae on tobacco is relatively lower than that on other crops, but their offspring can establish themselves on tobacco and pose a potential threat to tobacco cultivation [22]. Tobacco black shank caused by P. parasitica was first discovered by van Breda de Haan in 1896 in Java, Indonesia [23]. As one of the major diseases of tobacco, black shank may occur in the seedbed and field, and often leads to the death of tobacco plants, showing severe effects on tobacco production [24]. The breeding of black shank resistant varieties always takes long periods of time, and thus comprehensive control measures are still the main approach for relieving black shank damage to tobacco [25]. An effective way for enhancing tobacco resistance to both the insect pest S. frugiperda and the oomycete pathogen P. parasitica is of great importance for tobacco cultivation practices. Tobacco cembranoids are a kind of terpenoid compounds with multiple bioactivities against both insect pests and microbial pathogens and has great potential for developing eco-friendly pesticides. Thus, manipulation of the CBTS genes may be an approach for tobacco resistance breeding against insect pests and microbial pathogens.

In this study, the function of the CBTS1 gene in tobacco was examined in transgenic plants with overexpression or silencing of the CBTS1 gene. The findings of this work showed that manipulation of CBTS1 could affect the biosynthesis of cembranoids in tobacco and alter tobacco resistance to both an insect pest and oomycete pathogen. This work provides insights into the function of the tobacco CBTS1 gene and evidenced its potential roles in tobacco resistance breeding against both insect pests and oomycete pathogens.

2. Results

2.1. Development of CBTS1 Gene-Overexpressing and Gene-Silencing Plants

The CBTS1 gene-overexpressing and gene-silencing plants were developed using the agrobacterium-mediated transformation method. Twenty CBTS1 overexpression lines (CBTS1-OE, expressing Flag-tagged CBTS1 protein) and 15 CBTS1 silencing lines (CBTS1-RI) were obtained. The genomic PCR results showed that an expected 1 kb fragment could be amplified from the CBTS1-OE lines, and that an expected 392 bp fragment could be amplified from the CBTS1-RI lines, whereas neither of these fragments could be amplified from control (empty-vector transformed) plants (Figure 1A,B). Further qRT-PCR analyses showed that the expression of the CBTS1 gene in CBTS1-OE plants was 5–13 times higher than that in control plants (Figure 1C). Line #15 of the CBTS1-OE plants had the highest abundance of transcripts of CBTS1 (Figure 1C). The expression of the CBTS1 gene was decreased by 80–92% in the lines #2, #4, and #5 of the CBTS1-RI plants compares with that in control plants. The expression of CBTS1 was the most suppressed in the line #2 of the CBTS1-RI plants (Figure 1D). The CBTS1-OE plants were further identified by Western blotting using an anti-Flag antibody. The 70 kDa CBTS1 protein could be detected in the CBTS1-OE line #2, #9, and #15, but could not be detected in control plants (Figure 1E). The CBTS1-OE lines with detectable CBTS1 protein and the CBTS1-RI lines with CBTS1 expression suppressed by over 80% were selected for further analyses.

2.2. Changing the Expression of CBTS1 Altered the Cembranoid Content of Tobacco

The contents of CBT-ols and CBT-diols in the CBTS1-OE plants and CBTS1-RI plants were quantified using UPLC as described previously [26]. The results showed that the content of CBT-ols was 0.41–0.95 mg/g in CBTS1-OE plants, whose average was over 4.48-fold higher than that of control plants (0.16 mg/g), and the content of CBT-diols was 31.71–35.5 mg/g in CBTS1-OE plants, whose average was over 3.17-fold higher than that of control plants (11.20 mg/g). On the contrary, the content of CBT-ols was 0.04–0.07 mg/g in CBTS1-RI plants, whose average was less than 68% of that in control plants, and the content of CBT-diols was 2.57–5.00 mg/g in CBTS1-RI plants, which was less than 76% of that in control plants (Figure 2). Based on above data, the content ratio of CBT-ols to CBT-diols was about 1:37 in the CBTS1-OE plants and about 1:64 in the CBTS1-RI plants (Figure 2), showing that the ratios are different from that in control plants (1:80). These findings suggested that the change in CBTS1 transcription not only altered the contents of CBT-ols and CBT-diols in CBTS1-OE and CBTS1-RI plants, but also altered the content ratio of CBT-ols to CBT-diols in these plants.

2.3. Manipulating the Expression of CBTS1 Altered Tobacco Resistance to Insect Pest S. frugiperda

In order to evaluate the effect of the CBTS1 expression level changes on tobacco resistance to insect pests, the CBTS1-OE and CBTS1-RI plants were subjected to an insect pest challenge with S. frugiperda, and the empty vector transformed plants were used as controls. The second-instar larvae of S. frugiperda were continuously fed with tobacco leaf samples for 7 days, and the daily mortality of S. frugiperda were recorded for each experiment. After 4 days of feeding, 73% (22 of 30) of the larvae fed with tobacco leaves from the CBTS1-OE plants died, while only 43% (13 of 30) of those fed with tobacco leaves from control plants died (Figure 3A). The results showed that feeding with the leaves from CBTS1-OE plants significantly increased the mortality of the second-instar larvae of S. frugiperda. On the contrary, the larvae fed with tobacco leaves from CBTS1-RI plants had only 27% (8 of 30) of them died (Figure 3A). In the feeding preference test, measurement of the change in leaf area showed that the larvae feeding caused a leaf area loss of 0.16 cm² for the CBTS1-OE plants and a leaf area loss of 12.71 cm² for the CBTS1-RI plants, while it caused a leaf area loss of 4.64 cm² for the control plants (Figure 3C,D). Kaplan–Meier survival analysis showed that the median survival times for the larvae feeding on leaves from control, CBTS1-OE, and CBTS1-RI plants were 5, 3(−2), and 6(+1) days, respectively, showing significant differences in the survival rates. These tests evidenced that manipulation of CBTS1 expression altered tobacco resistance to the insect pest S. frugiperda.

2.4. Manipulating the Expression of CBTS1 Changed Tobacco Resistance to Oomycete Pathogen P. nicotianae

The CBTS1-OE plants, CBTS1-RI plants, and control plants were subjected to P. nicotianae challenge for determining the effects of changing CBTS1 expression on tobacco pathogen resistance. The leaves from CBTS1-OE, CBTS1-RI, and control plants were inoculated with 50 μL of P. nicotianae spore solution on the back. After 5 days of incubation in the dark, the disease symptoms on each leaf were inspected. The leaves of CBTS1-OE plants formed only very small necrotic lesions, while those of CBTS1-RI plants formed large infection patches that covered nearly 50% of the leaf area (Figure 4A). The infection patches on the leaves of control plants covered around 20% of the leaf area (Figure 4A). Further trypan blue staining revealed that the cell death area in the P. nicotianae-challenged leaves of CBTS1-RI plants was greatly larger than that in the leaves of control plants, and that only tiny cell death areas could be observed in the P. nicotianae-challenged leaves of CBTS1-OE plants (Figure 4B). These data suggested that manipulating the expression of CBTS1 changed tobacco resistance to the oomycete pathogen P. nicotianae.

2.5. Expression of PR Genes were Changed in CBTS1-OE and CBTS1-RI Plants

Pathogenesis-related (PR) genes play important roles in mediating plant resistance to pathogens [27]. The altered resistance of CTBS1-OE and CBTS1-RI plants to P. nicotianae prompted us to investigate the transcriptional change of PR genes in these plants. The expression of four PR genes including PR-1a, PRB-1b, PR-4, and PR-5 in CTBS-OE, CBTS1-RI, and control plants was analyzed by qRT-PCR. The results showed that the expression of PR-1a, PRB-1b, and PR-5 was increased in the CBTS1-OE plants compared with that in control plants, especially for PR-1a whose expression level was up-regulated by over 200-fold (Figure 5). However, no obvious change in the expression of PR-1a, PRB-1b, PR-4, or PR-5 was observed in CBTS1-RI plants (Figure 5). These finding implied that changing the expression of CBTS1 gene or the correlated cembranoid content may have an effect on the expression of PR genes in tobacco plants.

3. Discussion

Tobacco cembranoids have excellent antimicrobial, insecticidal, and cytotoxic bioactivities [28]. These kinds of compounds are getting attention from scientists studying plant protection, biochemistry, etc., more than ever before [29,30]. In this study, we developed transgenic tobacco plants overexpressing the cembranoid biosynthetic gene CBTS1 and those with the expression of CBTS1 knocked down by RNAi-mediated gene silencing. Further studies found increased cembranoid contents and enhanced resistance to S. frugiperda and P. nicotianae in the CBTS1-overexpression plants and vice versa; these results are helpful in developing tobacco plants with improved resistance to both insect pests and oomycete pathogens.

CBTS1 is a known cembranoid synthase that catalyzes the biosynthesis of cembratrien-ols from GGPP [31]. The expression level of CBTS1 genes is a key factor controlling cembranoid biosynthesis in tobacco. Using the transgenic plants with altered CBTS1 expression, this work found that the content of CBT-ols was 0.95 mg/g in CBTS1-OE plants and 0.04 mg/g in CBTS1-RI plants, and that the content of CBT-diols was 35.5 mg/g in CBTS1-OE plants and 2.57 mg/g in CBTS1-RI plants. Compared with control plants, the contents of CBT-ols and CBT-diols were accentuated by the overexpression of CBTS1 and were attenuated by the knockdown of CBTS1. These findings showed that manipulation of CBTS1 expression altered the production of both CBT-ols and CBT-diols, supporting an important role of CBTS1 in their biosynthesis. On the other hand, the content ratio of CBT-ols to CBT-diols was about 1:37 in the CBTS1-OE plants and about 1:64 in the CBTS1-RI plants, which are different from that in control plants (1:80). These pieces of evidence indicate that the manipulations of CBTS1 expression also changed the content ratio of CBT-ols to CBT-diols in tobacco, implying that a change in CBTS1 activity may altered the dynamics of CBT-ols and CBT-diols conversion.

Tobacco CBT-ols are insecticidal compounds [32], and the altered content of CBT-ols in CBTS1-OE and CBTS1-RI plants should affect their resistance to insect pests. The insect pest resistance assay with second-instar larvae of S. frugiperda showed that 73% of the larvae fed with tobacco leaves from CBTS1-OE plants died, 43% of those fed with tobacco leaves from control plants died, and only 27% of those fed with tobacco leaves from CBTS1-RI plants died. Additionally, consistent results were observed in the measurement of leaf area loss after S. frugiperda feeding. Thes findings are consistent with the cembranoid contents in the corresponding transgenic plants and support that manipulation of CBTS1 expression could alter tobacco resistance to the insect pest S. frugiperda by altering the cembranoid production capability in tobacco. S. frugiperda is a polyphagous insect pest known as the fall armyworm and is invading the continents outside of the Americas [33]. This work not only suggests a potential application of cembranoids as chemicals for protecting plants from S. frugiperda attack, but may also provide a way for tobacco resistance breeding against this insect pest.

CBT-diols of tobacco plants are antimicrobial compounds and have been demonstrated to possess bioactivities against multiple pathogens [34]. The pathogen resistance assay with CBTS1-OE, CBTS1-RI, and control plants revealed that the leaves of CBTS1-OE plants formed very small necrotic lesions, those of CBTS1-RI plants formed infection patches that covered ~50% of the leaf area, and those of control plants formed infection patches covering ~20% of the leaf area. Similar results were observed in the trypan blue staining of the P. nicotianae-infected leaves. These findings are also consistent with the cembranoid contents in the corresponding transgenic plants and support that alteration in CBTS1 expression could change tobacco resistance to the oomycete pathogen P. nicotianae by altering the cembranoid production. Intriguingly, investigation of the transcriptional change of PR genes in CBTS1-OE, CBTS1-RI, and control plants discovered that the expression of PR-1a, PRB-1b, and PR-5 was increased in the CBTS1-OE plants, and the expression level of PR-1a was enhanced by over 200-fold. However, no obvious change in the expression of these PR genes were observed in the CBTS1-RI plants. These results implied that increases or changes in cembranoid contents may have effects on the expression of PR genes in tobacco. Previous intensive studies showed that plant resistance to pathogens is highly correlated with the transcription of PR genes [35]. The findings of this work suggest that manipulation of cembranoid production may change the pathogen resistance of tobacco plants by regulating the expression of PR genes. Previous studies showed that cembranoids also exhibited inhibitory activities on other pathogens, such as Aspergillus niger, Alternaria alternata, and Candida albicans [8]. The findings of this study may also provide cues for investigating the mechanisms underlying plant resistance to these pathogens.

Taken together, this work developed transgenic plants that have the transcripts of CBTS1 enhanced by the gene overexpression method or knocked down by the RNAi-mediated gene silencing method. Subsequent studies demonstrated that changing the expression levels of CBTS1 altered the cembranoid contents in tobacco and affected tobacco resistance to both the insect pest S. frugiperda and the oomycete pathogen P. nicotiana. Therefore, this work is helpful in establishing an integrative method for manipulating cembranoid biosynthesis in tobacco and for improving tobacco resistance to insect pests and oomycete pathogens via both chemical and biological approaches.

4. Materials and Methods

4.1. Growth Conditions for Tobacco Plants

The plants of tobacco (Nicotiana tabacum L.) cultivar TN90 was cultivated in a greenhouse at 25 °C with a photoperiod of 14 h light/10 h dark. Tobacco plants about 5 cm in height were used for genetic transformation. The transgenic plants for further analyses were cultivated under the same condition.

4.2. Vectors Construction and Transgenic Plant Development

The full-length coding region of CBTS1 (GenBank: AAS46038.1) was amplified from the cDNA of tobacco plants, cloned into the pENTR-D-TOPO^® vector (Invitrogen, Carlsbad, CA, USA), and then integrated into the pMDC-attR-G11 (Flag) vector (Curtis and Grossniklaus, 2003) by LR recombination with Gateway^® LR Clonase™ (Thermo, Waltham, MA, USA) to obtain the gene overexpression vector pMDC-CBTS1-FLAG (Figure 6A). To construct the gene-silencing vector, a 392 bp fragment of the CBTS1 gene was amplified and integrated into pHZPRi-Hyg vector [36] that was modified from pMDC32 [37] to obtain the gene silencing vector pMDC-CBTS1-RI (Figure 6B). The pMDC-CBTS1-FLAG and pMDC-CBTS1-RI vectors were introduced into Agrobacterium tumefaciens strain LBA4404 and transformed into tobacco by the leaf-disk transformation method [38]. All primers used for vector construction are listed in Supplementary Table S1.

4.3. PCR and qRT-PCR

The PCR experiments were performed using the Phire Plant Direct PCR Master Mix (Thermo, Waltham, MA, USA) rapid amplification system using transgenic leaf discs with a diameter of 0.3 mm as a template according to the manufacturer’s instructions. The PCR products were separated on 1% agarose gel and visualized by ethidium bromide staining.

For qRT-PCR analyses, the total RNA was extracted by the TRIzol method from the leaves of tobacco plants, and the cDNA was prepared by reverse transcription using the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The expression of the CBTS1 gene in transgenic tobacco plants was determined using the BlasTaqTM 2 × qPCR Master Mix kit (Vazyme, Nanjing, China) and QuantStudio^® 5 real-time fluorescence quantitative PCR (Thermo, Waltham, MA, USA) with a preliminary denaturation at 95 °C for 10 s, and 40 cycles of amplification consisting of 95 °C for 5 s and 63 °C for 30 s. NtActin gene expression was used as an internal control. The primers for PCR and qRT-PCR are shown in Supplementary Table S1.

4.4. Western Blotting Analysis

The total protein was extracted from the leaves of tobacco plants, and was then concentrated by ultracentrifugation and precipitated with acetone to effectively remove the phenolic compounds in the leaves. The protein and markers were transferred onto a PVDF membrane by electroblotting. The membrane was blocked with 5% skim milk at 4 °C overnight. For the detection of Flag-tagged CBTS1 protein, the monoclonal mouse anti-FLAG antibody (Sigma-Aldrich, St Louis, MO, USA) and HRP-labeled goat anti-mouse IgG (H + L) were used, and the signal was captured by fluorescence image analysis system (Tanon 5200, Shanghai, China) using the Pierce^® ECL Western Blotting Substrate (Thermo, Waltham, MA, USA).

4.5. Extraction and Determination of Cembranoid Diterpenes from Tobacco

The cembranoid content of CBTS1-OE and CBTS1-RI plants was determined as described previously [26]. Briefly, the same amount of tobacco leaves collected at the indicated growth stage and leaf position were immersed into 150 mL of dichloromethane for cembranoid extraction, and then extracted for another two repeats in the same way. The total 450 mL of combined extract was evaporated by a rotary evaporator, dissolved in 10 mL dichloromethane, and dried under nitrogen flow. The dried sample was redissolved in 1 mL of 60% acetonitrile and filtered through a 0.22 μm filter. The sample was subjected to cembranoid quantification using an ultra-performance liquid chromatography (UPLC) machine equipped with a C18 chromatographic column (ACQUITY UPLC BEHC18, 1.7 μm, 150 × 2.1 mm) at a column temperature of 40 °C, flow rate of 0.3 mL/min, and injection volume of 5 μL. CBT-ol and CBT-diol were detected at 208 nm.

4.6. Insect Toxicity and Feeding Preference Test

The toxicity of tobacco plants to S. frugiperda was evaluated using the plant feeding test. The larvae of our lab-maintained S. frugiperda stock were fed with a fruit fly diet at 28 °C with a photoperiod of 16 h light/8 h dark. Detached leaves from the tobacco plants were randomly selected and placed on moist paper in a Petri dish. Ten second-instar larvae of S. frugiperda were placed on the detached leaves and incubated for 7 days at room temperature [39]. The area loss of tobacco leaves after feeding was recorded at the indicated times. In order to test the feeding preferences of S. frugiperda, six second-instar larvae were placed between the leaves of different plants, so that the larvae had equal opportunity to feed on the leaves of CBTS1-OE plants, CBTS1-RI plants, or control plants. The area loss of the tobacco leaves was recorded after 4 h of feeding. Each experiment was tested for three independent replicates.

4.7. Oomycete Pathogen Challenge of Tobacco Leaves

The lab-maintained oomycete pathogen P. nicotianae was cultured on oatmeal agar (OA) in Petri dishes for 21 days at 25 °C. The spore suspension of P. nicotianae was prepared by serial dilution to the final concentration of 1 × 10⁵ spores per milliliter. Then, the leaves of 5-week-old tobacco plants with similar sizes were collected and inoculated with 50 μL of the spore suspension of P. nicotianae on both sides of the leaf back. The inoculated leaves were placed in a moisture box with a relative humidity of 95% at 25 °C in the dark. A set of leaves inoculated with sterile water were used as control. Five days later, the leaves were stained with trypan blue and the percentage of the infected area of tobacco leaves was recorded with the help of a leaf area meter. Each sample was tested with three independent replicates.

4.8. Data Analysis

The quantitative data were expressed as mean ± standard deviation (SD). A one-way analysis of variance was performed using IBM SPSS Statistics 26.0.0.0 software. Kaplan–Meier survival analysis and log rank test were used for survival rate comparisons. The differences between the values were assessed using the indicated test method, and p < 0.05 was considered to be statistically significant.