Introduction

Tomato (Solanum lycopersicum L.) is one of the most widely grown vegetables in the world and gray mold, which is caused by Botrytis cinerea Pers., is a serious disease resulting in large reductions in tomato production (Ma et al. 2018). Currently in China, gray mold is commonly controlled by spraying chemical fungicides, which has led to resistant strains of the pathogenic fungi and a certain degree of pesticide pollution owing to unreasonable application practices (Zhao et al. 2014; Liu et al. 2017a). The breeding of resistant varieties is an effective method for plant disease prevention and control, but there are limited reports on the breeding of tomato resistant to B. cinerea Pers. Transgenic breeding is a commonly used effective method. In Powell et al. (2000) study of the polygalacturonase inhibitor protein gene (PGIP) of pears (Pyrus spp.), transgenic pPGIP-expressing tomato was more resistant to gray mold than the control. Coego (2005) transformed an Arabidopsis thaliana transcription factor gene into tomato, resulting in a new tomato strain resistant to B. cinerea Pers. Study shown that the tomato calcineurin B-like gene CBL1 can regulate the resistance of tomato to gray mold by affecting resistance-related transcription factors (Wang et al. 2016). Our research group transformed the Eucommia ulmoides Oliver chitinase gene CHIT1 into tomato and found that the transgenic tomato had greater disease resistance than the untransformed control (Guo et al. 2016).

Laccase is a glycoprotein that has a wide range of substrates and a high catalytic activity (Wang et al. 2017). It can be divided into two groups, plant and fungal laccases, based on its source (Deng et al. 2017). Laccase has been reported in many plants, including A. thaliana, cotton (Gossypium spp.), tobacco (Nicotiana tabacum), rice (Oryza sativa), maize (Zea mays) and Populus trichocarpa (Kiefer-Meyer et al. 1996; Liang et al. 2006; Wang et al. 2008; Berthet et al. 2011; Cesarino et al. 2013; Cao 2016). Research on plant laccases has mainly focused on the polymerization of lignin (Liu et al. 2017b). In Arabidopsis, the AtlLAC4 and AtlLAC17 genes were found to contribute to constitutive lignification by studying the double mutants lac4-1 lac17 and lac4-2 lac17 (Berthet et al. 2011). Ranocha et al. (2002) researched three independent populations of antisense transgenic poplar plants and determined that the total soluble phenol content in the lac3AS line increased by two to three times. Moreover, they observed that the inhibition of lac3 led to dramatic alterations in xylem fiber cell walls. Laccases are also involved in lignin synthesis in the grass crop, Brachypodium distachyon, and the lignin content of a BdLAC5-misregulated Bd4442 mutant line was reduced (Wang et al. 2015). In addition, the laccase gene has been used to alter plant resistance to fungi, bacteria and insects. Transgenic tomatoes harboring the potato laccase gene show a significant increase in bacterial speck disease resistance compared with control tomatoes (Li and Steffens 2002). Wu discovered that the cotton (Gossypium hirsutum) laccase gene, GhLAC1, is involved in the lignin synthesis of cotton xylem and can accelerate the xylem lignification process, which is one reasons why this gene can enhance cotton’s resistance to diseases and insect pests (Wu 2014). Recently, GhLAC1 was shown to increase cotton resistance to Verticillium dahliae and cotton bollworm by strengthening lignification and mediating jasmonic acid (JA) biosynthesis (Hu et al. 2018). Zhang et al. (2019) determined that the cotton laccase gene LAC15 enhances Verticillium wilt resistance by increasing defense-induced lignification, as well as levels of arabinose, xylose and lignin components in the cell walls of plants. Thus, laccases participate in the synthesis of lignin in plants and strengthen plant defense systems.

In this study, the cotton laccase gene LAC1 was genetically transformed into tomato, and a new tomato germplasm resistant to gray mold was obtained. GhLAC1 enhanced tomato resistance to gray mold by increasing the cell wall’s lignin content, resulting in its improved strength. The results increase our knowledge of how plants strengthen disease defenses through lignification and provides theoretical support for future research on the detailed mechanisms of plant disease resistance.

Materials and Methods

Experimental material

Tomato (S. lycopersicum) ‘Micro-Tom’ was provided by our laboratory (Guizhou Key Laboratory of Agricultural Bioengineering). The seeds were sterilized and sown in germination medium [Murashige and Skoog (MS) + 20 g/L sucrose + 9 g/L agar powder], placed in a thermostatic plant tissue culture room, and cultured at 28°C with a Table 1: Primer sequences for PCR amplification

Primer	Sequence
Gh-Forward	5 ’-CCATTCCCAAAACCACAC-3’
Gh-Reverse	5 ’-CGCTACTAAATACTTGCCAGA-3’

Table 2: Primer sequences for qPCR

Primer	Sequence
CAC-F	5 ’-CCTCCGTTGTGATGTAACTGG-3’
CAC-R	5 ’-ATTGGTGGAAAGTAACATCATCG-3’
GhLAC-F	5 ’-AGGCTGTTGTCGGCATAG-3’
GhLAC-R	5 ’-TCACTGTTGGACTTGGGATT-3’

16-h light/8-h dark photocycle (Pan et al. 2010).

Construction of the plant overexpression vector

The sequence of the cotton laccase gene LAC1 (GenBank: KT290561.1) was retrieved from NCBI and pGM626 (Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University) was used as the initial vector to construct the Act1 promoter-driven GhLAC1-containing plant expression vector PGM626-Act1-GhLAC1. The construction of PGM626-Act1-GhLAC1 was completed by Shanghai Xuguan Company.

Tomato transformation and identification of transgenic plants

The plasmid pGM626-Act1-GhLAC1 was transformed into Agrobacterium using the freeze-thaw method and positive resulting colonies were identified by colony PCR. PCR amplification was carried out using the designed verification primers (Table 1), and the target amplicon size was 399 bp.

The Agrobacterium tumefaciens-mediated transformation method used was that of Guo et al. (2016). After the sterilized Micro-Tom seeds germinated on MS medium, the tomato cotyledons were cut and placed on the preculture medium (MS + 20.0 g/L sucrose + 9.0 g/L agar powder + 2.0 mg/L 6-BA + 0.5 mg/L IBA + 100 μmol/L AS), and cultured in the dark at 28°C for 2 d. Then, the cotyledon explants were subjected to Agrobacterium infection solution for 8 min and transferred to the co-cultivation medium (MS + 20.0 g/L sucrose + 9.0 g/L agar powder + 2.0 mg/L 6-BA + 0.5 mg/L IBA + 100 μmol/L AS) and cultured in the dark at 28°C dark for 3 d. After transformation, the cotyledon explants were placed on screening medium (MS + 20.0 g/L sucrose + 9.0 g/L agar powder + 2.0 mg/L 6-BA + 0.5 mg/L IBA + 0.5 mg/L Bar + 100 mg/L Tim) for regeneration. When the regenerated Basta-resistant seedlings grew to 1–2 cm, they were placed on rooting medium (1/2 MS + 10.0 g/L sucrose + 6.0 g/L agar powder + 0.2 mg/L IBA + 100 mg/L Tim) and cultured. Finally, the rooted plants were transplanted to a pre-treated mixed soil (2: 1: 1 loess : nutrient soil : perlite) after 2–3 d.

The DNA of Basta-resistant plants was extracted using the CTAB method for PCR identification.

qRT-PCR analysis

The GhLAC1 expression level in transgenic tomato plants was assessed by real-time PCR using gene-specific primers, and the Clathrin adaptor complex (CAC) gene served as the internal reference (PCR primers CAC-F and CAC-R; Table 2). Real-time qRT-PCR was performed using a SYBR Green I Dye Kit (Applied Biosystems Inc., Foster, CA, USA) and the CFX Connect^TM Real-Time System (Applied Biosystems Inc.). Total RNA was isolated from the leaves of transgenic and control Micro-Tom, using the RNAiso Plus and Fruit-mate for RNA purification (TaKaRa, Dalian, China) and reverse-transcribed into cDNA by Reverse Transcriptase M-MLV (RNase H) (TaKaRa). The amplification cocktail was as follows: 3 µL template RNA, 1 µL oligo (dT)12–18 primer (50 µM) and 2 µL RNase free ddH₂O. This was placed at 70°C for 10 min and then on ice for 3 min. Afterward, 2 µL 5× M-MLV Buffer, 0.5 µL dNTP Mixture (10 mM), 0.25 µL RNase Inhibitor (40 U/µL), 0.25 µL RTase M-MLV (RNase H 200 U/µL) and RNase free ddH₂O were added up to 10 µL. The PCR cycles were 42°C for 1 h and 70°C for 15 min. Samples were then placed on ice for 3 min. The RT-PCR reaction system was as follows: 10 µL Power SYBR Green PCR Master, 0.2 µL forward primer, 0.2 µL reverse primer, 1 µL cDNA and 4.2 μL ddH₂O. The reaction conditions were 35 cycles of 98°C for 10 min, 58°C for 30 s and 72°C for 1 min, followed by a 12°C hold. All the experiments were repeated three times.

Analysis of the laccase activity

The laccase activity was determined using the ABTS method (Zhang 2007; Wang et al. 2008), and wild-type tomato was used as the control. Briefly, 0.1 g amples of the fifth leaf from the top of each tomato plant were taken. This was repeated three times. Samples were placed in liquid nitrogen and fully ground. The resulting powder was transferred into protein extraction buffer containing 25 mmol/L MOPS and 200 mmol/L CaCl₂, placed at 4°C for 4 min and centrifuged for 10 min at 10,000 ×g. The supernatant is the crude protein extract and it was added to 1 mL of the newly prepared 1 mmol/L ABTS solution. The absorbance A₁ was measured at 420 nm using a microplate reader. After 30 min of reaction at 30°C in a constant temperature water bath, the absorbance A₂ was measured at 420 nm. The laccase activity was calculated according to the following formula: enzyme activity = 1,000 × 0.1844 × (A₂−A₁) × 60 × 1,000 × dilution factor, the unit of enzyme activity is nmol/L/min, or U/g. A laccase activity unit (U) is defined as the amount of product produced by converting 1 nmol of ABTS substrate per gram of fresh tissue at 30°C for 1 min.

Analysis of pathogen resistance

The in vitro leaf inoculation method (Kovacs et al. 2013) was used. Wild-type plants were used as controls. Transgenic GhLAC1-expressing tomato having the same physiological state at the 5–6 leaf stage was used. After the leaf blade was cut from the petiole, the petiole was wrapped with sterilized cotton, and the cotton was moistened by adding an appropriate amount of sterile water. A 100-μL pipette was used to draw 50 μL of a Botrytis spores spore suspension at a 5 × 10⁵ cfu/mL concentration. This was dropped on the front of the selected leaves and the leaves were cultured at 20°C.

Inoculation with sterile water was used as the blank control and the degree of gray mold infection was determined by measuring the diameters of leaf lesions after 5 d (Liu et al. 2016).

Determination of the lignin content

The lignin content was determined using the acetyl bromide method (Morrison 1972). Briefly, 1.0 g samples of tomato leaves from the top to the bottom of the fifth branch were taken, as was 1.0 g of a stem segment at 5 cm from the ground. Each sample was homogenized in 95% ethanol, centrifuged at 4,500 rpm for 5 min at room temperature, washed three times with 95% ethanol, and washed twice with a 1:2 (v/v) solution of ethanol: n-hexane. The precipitate was collected and dried at 60°C. Then, 2 mL of 25% bromoacetyl glacial acetic acid [1:3 (v/v) bromoacetyl: glacial acetic acid] solution was added to the dried precipitate. The dissolved precipitate was placed in a water bath at 70°C for 30 min and 0.9 mL of 2 mol/L NaOH solution was added. Then, 2 mL of glacial acetic acid and 0.1 mL of 7.5 mol/L hydroxylamine hydrochloride were added to the reaction solution to terminate the reaction and the volume was adjusted to 5 mL using glacial acetic acid. The sample was then centrifuge at 4,500 rpm for 5 min at room temperature. The supernatant was aspirated and the absorbance at 280 nm measured using a microplate reader. The lignin content is directly proportional to the optical density value at 280 nm and the relative absorbance of lignin per gram of fresh weight (FW) was expressed as the absorbance at 280 nm.

Electron microscope observations of tomato stem cross sections

The cross sections of the stem segments of wild-type and transgenic tomato plants were taken, and the samples were submerged in 2.5% glutaraldehyde fixative. After fixing for 12 h in a refrigerator at 4°C, the samples were thoroughly washed with buffer at room temperature. The buffers were a stepwise gradient concentration of ethanol as follows: 30, 50, 70, 80, 95 and 100% and each wash time was 15 to 20 min. Then, the sample was freeze dried and plated with using a Hitachi e-1010 ion sputtering apparatus. Then, the xylem cell wall morphology of the tomato stem section was observed using a Hitachi S3400 scanning electron microscope and photographed.

Statistical analysis

All experiments were repeated three times. The experimental data were statistically analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). In all these experiments, the quantitative differences between the compared data groups were statistically significant (P < 0.005). The 2 ^-ΔΔCt method was used to analyze qRT-PCR data.

Results

Identification of transgenic tomato

The plant expression vector PGM626-Act1-GhLAC1 (Fig. 1) was transformed into Agrobacterium, and positive colonies were selected by colony PCR for tomato genetic transformation experiments (Fig. 2). The positive colonies had a target band of 399 bp (Fig. 3). Using wild-type and Basta-resistant tomato DNA as a template for PCR identification of transgenic plants, the results shown that the 399 bp specific band was amplified from transgenic tomato DNA, but not from wild-type (Fig. 4), indicating that GhLAC1 was integrated into the tomato genome.

Relative expression of GhLAC1 in transgenic tomato plants

The relative expression levels of GhLAC1 were analyzed by quantitative reverse transcription real-time (RT)-PCR (Fig. 5). The expression of GhLAC1 was highest in transgenic plant 12 (TP12), while the expression level in transgenic plant 9 (TP9) was much lower. The expression levels of GhLAC1 in TP12 and transgenic plant 7 (TP7) were 4.71 and 4.54 times that of TP9, respectively. The difference in the relative expression between TP7 and TP9 is extremely.

Laccase activity in tomato

The laccase activities in three independent transgenic lines (TP7, TP9 and TP12) were determined and wild-type tomato was used as the control. The laccase activity levels of transgenic tomato plants overexpressing GhLAC1 were exceedingly significantly higher than that of wild-type (Fig. 6). The laccase activities in TP9 (4,589.19 U/g), TP7 (6,773.50 U/g) and TP12 (6,879.96 U/g) were 1.47, 2.05 and 2.08 times that of the wild-type (3,307.52 U/g). The laccase activity of TP9 was significantly lower than those of TP7 and TP12, which had higher GhLAC1 expression levels. The results supported that the increase in laccase activity in transgenic tomato plants is caused by the expression of GhLAC1, and they indicate that expressing the cotton laccase LAC1 gene significantly increased the laccase activity in tomato.

Disease resistance assay with Botrytis cinerea Pers

Fig. 1: Diagram of expression vector of PGM626-Act1-GhLAC1.

RB: right border; LB: left border; Bar: Basta resistance marker; GUS: β-glucuronidase;

NOS: 3 'signal of nopaline synthase; Act1:Actin1 promoter

Fig. 2: The tomato genetic transformation process

A: co-culture; B: screening culture; C: differentiation; D: rooting

Fig. 3: Colony PCR for Agrobacterium strains harboring pGM626-Act1-GhLAC1

M: DNA marker DL 2000; 1-2: Positive colonies

Fig. 4: PCR identification for transgenic tomato

M: DNA marker DL 2000; TP1-TP6: transgenic plants 1–6; WT: wild-type plant

Fig. 7: The inoculation of wild-type and transgenic GhLAC1-expressing tomato plants leaves with B. cinerea

WT: wild-type plant; TP: transgenic GhLAC1-expressing plant; scale = 1 cm;

A: Not inoculated with B. cinerea; B: Three days after inoculation with B. cinerea; C: Five days after inoculation with B. cinerea

Fig. 5: Relative expression of GhLAC1 in transgenic tomato plants

Error bars indicate standard error (SE); * and ** indicate significant differences at P < 0.05 and P < 0.01, respectively

Fig. 6: Laccase activity in tomato

WT: wild-type plant; TP: transgenic GhLAC1-expressing plants

Error bars indicate standard error (SE); Letters on bars show whether the values are significant or not. Means having the same letter are not statistically significant (P < 0.01) according to least significant difference test

To determine whether expressing the GhLAC1 improved tomato disease resistance, the gray mold inoculation experiment (Fig. 7A-C) was performed on the leaves of wild-type tomato and the transgenic tomato line having the highest GhLAC1 expression level (TP12). At 3 d after inoculation with B. cinerea Pers., the wild-type tomato leaves showed obvious round lesions, while the transgenic tomato leaves only showed spotted lesions (Fig. 7B). At 5 d after inoculation, the lesions of wild-type tomato leaves increased significantly and started to spread to other uninfected leaflets; however, the lesions on leaves of transgenic tomato overexpressing GhLAC1 were much smaller and did not spread (Fig. 7C). In addition, the diameters of the leaf lesions were statistically analyzed at 5 d after inoculation (Fig. 8) and the average diameter of wild-type leaf lesions was 0.765 cm, while that of transgenic tomato leaves was 0.585 cm, which was an extremely significant 23.5% smaller than that of wild-type. This result indicated that expression of GhLAC1 in tomato plants effectively increased tomato resistance to gray mold.

Lignin content and electron microscope observations of the tomato cell wall

Fig. 8: Mean diameters of necrotic lesion in B. cinerea-inoculated tomato blades at 5 d after inoculation.

WT: wild-type plant; TP: transgenic GhLAC1-expressing plant

Error bars indicate standard error (SE); ** indicate significant differences at P < 0.01

Fig. 9: Lignin contents in leaves and stems of wild-type and transgenic GhLAC1-expressing tomato plants.

Error bars indicate standard error (SE); * and ** indicate significant differences at P < 0.05 and P < 0.01, respectively

Fig. 10: Electron microscopic observations of stems of wild-type and transgenic GhLAC1-expressing tomato plants.

WT: wild-type plant; TP: transgenic GhLAC1-expressing plant; magnification 1,000×

A thicker cell wall can improve plant resistance to external stresses, and lignin is an important component of the cell wall. Consequently, the lignin content is closely correlated with disease resistance. The laccase gene is involved in the regulation of lignin synthesis; therefore, we hypothesized that the increased resistance of transgenic tomato expressing GhLAC1 to gray mold was caused by cell wall thickening. Therefore, the lignin contents of leaves and stems in wild-type and transgenic tomatoes were analyzed. The average lignin contents in the leaves and stems of wild-type tomato were 2.13 (OD₂₈₀/g FW) and 2.36 (OD₂₈₀/g FW), respectively, while those of transgenic tomato were 2.55 (OD₂₈₀/g FW) and 2.89 (OD₂₈₀/g FW), respectively, which were 19.7 and 22.5% higher than those of wild-type tomato, respectively (Fig. 9). The result suggested that the lignin contents in leaves and stems of transgenic tomato were significantly higher than those in wild-type, and the difference in the lignin contents of stems between wild-type and transgenic tomato plants was greater than that between leaves. Thus, over the expression of GhLAC1 in tomato significantly increased the lignin content. Because the lignin content in the stem of the transgenic tomato was extremely significantly increased compared with the wild-type, further observations of the cross sections of wild-type and TP12 stems by scanning electron microscopy were performed. The cell wall near the xylem of the transgenic tomato was thicker and more contoured than that of the wild-type (Fig. 10). Clearly, GhLAC1 enhanced the cell wall of tomato. The correlation analysis between lignin content and lesion diameter revealed a high correlation. Higher the content of the lignin, the smaller was the lesion diameter (Table 3). Therefore, it provides evidence supporting the hypothesis that transgenic tomato overexpressing GhLAC1 has a higher gray mold resistance than wild-type because the increase in the lignin content leads to the thickening of plant cell walls.

Discussion

Lignin is a phenolic biopolymer derived from the phenylpropane pathway. It is an important component and can increase the strength of plant cell walls (Ding et al. 2016). There are three main types of monomers that make up lignin, coumarinol, coniferyl alcohol, and glucosinolate. They are oxidized and polymerized into p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin, respectively (Cao 2019). Laccase is thought to be involved in the polymerization of lignin monomers, which can oxidize lignin monomers and increase lignin deposition (Chou et al. 2018; Tobimatsu and Schuetz 2019). The laccase gene is involved in lignin synthesis in Arabidopsis, Populus tomentosa, and cotton (Cao 2016; Hu 2018; Chou et al. 2018). Here, the expression of the GhLAC1 gene in tomato increased laccase activity, and the lignin contents in leaves and stems of transgenic tomato were significantly higher than those of wild-type. This is consistent with previous findings; therefore, we believe that an increase in laccase activity leads to an increase in the lignin content of transgenic plants. Lignin monomers are synthesized in the cytoplasm, secreted into the cell wall and then polymerized into lignin by oxidases, such as laccase and peroxidase, to increase the mechanical strength of the cell wall (Vanholme et al. 2010; Barros et al. 2015). Cheng et al. (2019) transformed the pear laccase gene LAC1 into Arabidopsis and found that the transgenic plant had increased lignin content and thickened cell walls in interfascicular fibers and xylem cells. Scanning electron microscopy results in this study showed that the xylem cell walls of the transgenic plants were significantly thicker than those of the wild-type, and the outlines were more pronounced. We hypothesized that this was caused by the increase in lignin. Lignin can improve disease resistance in plants (Xia et al. 2015; Ma et al. 2017).

After a plant is infected with a fungus, lignin can induce plants to produce antitoxins or act as defense signaling molecules in the form of phenylpropane compounds (Mcfadden et al. 2001; Dixon et al. 2002; Naoumkina et al. 2010). Previous study found that the CCoAOMT of maize is related to the resistance of pathogens, and it may affect plant resistance by participating in the phenylpropanin metabolism pathway (Yang et al. 2017). Yang et al. (2018) found that phenylalanine metabolism is involved in BcGs1-induced tomato defense responses to gray mold. Additionally, lignin improves plant disease resistance by strengthening the lignification of cell walls. Lignin provides a physical barrier that limits the colonization capabilities of pathogenic fungi, thereby increasing plant resistance (Bonello and Blodgett 2003; Zhang et al. 2017). In resistant cotton, the greater lignin content increases resistance to fungal diseases (Pomar et al. 2004). Gayoso et al. (2010) found that lignin synthesis plays a key role in the defense mechanism of tomato against Verticillium wilt. In wheat, a higher S-lignin content is regarded as a cell wall biochemical trait related to Fusarium resistance (Lionetti et al. 2015). High lignin content contribute to the basic defence response in tobacco (Ma et al. 2017). Here, we showed that the GhLAC1 gene increased tomato resistance to gray mold, and we speculate that this may be correlated with the higher lignin content and cell wall enhancement in transgenic tomatoes.

In summary, we believe that the increased resistance of GhLAC1-expressing transgenic tomatoes to gray mold may result from the increase in lignin caused by the increase in laccase activity. Whether the mechanism of disease resistance results from the effects of phenylpropane compounds or the thickening of the cell wall caused by the enhancement of lignification remains to be studied further. In this study, the cotton laccase gene GhLAC1 was genetically transformed into tomato for the first time and tomato plants with significant resistance to gray mold were obtained, which provides a new idea for tomato breeding against gray mold.

Conclusion

This study reported that expressing the GhLAC1 gene in tomato increased its lignin content and resistance to gray mold. The results indicated that GhLAC1 is a potential candidate gene for genetic engineering to develop crops with gray mold resistance which raises a possibility of improving plant defense. The detailed disease resistance mechanism of GhLAC1-expressing transgenic plants needs further research.

Acknowledgements

This work was supported by the National Key R & D Plan “Cotton Quality, Stress-Resistant Functional Genomics and Recombination Networks” (Project number: 2016YFD0101006) and the Major Project for the Cultivation of New Varieties of GMOs (Project number: 2016ZX08010003). We thank Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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