The inhibitory effects of sulfated lentinan and lentinan against tobacco mosaic virus in tobacco seedlings and the underlying mechanism were investigated. sLNT and LNT significantly inhibited viral infection and TMV multiplication in tobacco plants. sLNT showed a higher inhibition effect against TMV than LNT treatment in a dose and time-dependent way. Moreover, sLNT induced a higher increase in the levels of transcription of pathogenesis-related (PR) protein genes [acidic PRs (PR-1a, PR-2, PR-3, PR-5) and basic PR-1] and defense-related enzymes [phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and 5-epi-aristolochene synthase (EAS, EC 2.5.1.35)] both locally and systemically, in correlation with the induction of resistance against tobacco mosaic virus. Furthermore, sLNT also induced accumulation of salicylic acid, SA 2-O-β-D-glucoside and H2O2. These results suggested that sLNT and LNT could control TMV incidence and the mechanism might attribute to activate the expression of a number of defense genes.
Keywords: Sulfated lentinan; Lentinan; Induced resistance; Tobacco mosaic virus
1. Introduction
Lentinan is a neutral polysaccharide extracted from the fruiting body of Lentinus edodes. It consists of a β-(1→3)-linked backbone of D-glucose residues, to which two β-(1→6)-D-glucosyl residues are attached for every five main-chain D-glucose residues (Saito, Ohki, Takasuka, & Sasaki, 1977, 1979). In addition to antimicrobial and antibacterial activity, lentinan can also inhibit viral infections of both naked and enveloped viruses, and the activity is primarily exerted during an early phase of the viral infection (Rincão et al., 2012). However, research has focused largely on Gram-negative bacteria and other species primarily involved in food spoilage, and on fungi and virus related to animal and human health (Markova, Kussovski, Radoucheva, Dilova, & Georgieva, 2002, 2003; Rincão et al., 2012). Although extracts from the mushrooms L. edodes have been shown to have the abilities in the control of tobacco mosaic virus infection (Kobayashi, Hiramatsu, & Akasuka, 1987), investigation on the ability of lentinan to control tobacco mosaic virus (TMV) appears limited.
Along with the increasing pursuit about manifold biological activities of polysaccharide, molecular modification and structure improvement of polysaccharide becomes an important research field (Liu & Sun, 2005). There are many methods about modification of polysaccharide, such as sulfating, oxydo-reduce-hydrolysis, enzyme-reducing, formaldehyde-reducing and so on. Sulfated polysaccharide, a kind of ones with sulfated group in its hydroxyl, has been very common in the study of biological activity of polysaccharide, where routes of sulfated modification have been very well established (Tian, Li, Song, Zheng, & Li, 1995). Moreover, many studies reported that sulfated modification could enhance the antiviral activity of polysaccharides against avian infectious bronchitis virus, infectious bursal disease virus, dengue virus, herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus (Huang, Wang, et al., 2008; Lu et al., 2008; Talarico et al., 2005; Zhang et al., 2004). However, whether sulfated modification could improve the biological activity of LNT against TMV or not? Furthermore, the systemic sulfated LNT (sLNT) responses and the underlying mechanism of the sLNT-mediated disease resistance against TMV have not been elucidated. Therefore, we used biochemical and molecular approaches to investigate the potential of sLNT and its possible mechanisms in controlling TMV in tobacco seedlings.
2. Materials and methods
2.1. Extraction and purification of polysaccharides
Lentinus edodes, bought from Fangge Company of Traditional Chinese Medicine, Zhejiang Province, was decocted with water into decoction. The crude total LNT was extracted from the decoction by ethanol precipitation whose content was 70% in the decoction. The crude total LNT was purified as follows: to remove protein by Sevag's method (Zhang & Lu, 1999), to remove pigment by active carbon adsorption, then through D101 macroaperture resin column and G-200 Sephadex column in turn (Zhao, 1994). At last the purified LNT was obtained. The polysaccharide contents (w/w) of LNT were 91%.
2.2 Sulfated modification of LNT
LNT was sulfated by the chlorosulfonic acid-pyridine method and the modified conditions were based on the preparative experiment (Chen, Wu, & Wang, 2005). In brief: The chlorosuLNTonic acid-pyridine complex (1:4) was prepared in ice bath. Then, 400 mg LNT was added, respectively, stirred for 4 h at temperature 60 °C, dissolved in 100 mL ice-cold water, cooled to room temperature, neutralized with saturated NaOH solution and precipitated with 95% ethanol (EtOH). The sediments were re-dissolved with water. The solution was dialyzed against tap water for 48 h and distilled water for 12 h in turn, then, lyophilized to obtain three sLNTs. Their degrees of substitution (DS) were determined by Antonopoulos' method (Zhang, Li, & Fan, 2002). The DS of sLNT was 0.98.
2.3 Plant culture and treatments
Tobacco plants (Nicotiana tabacum var. sam sun NN) were grown from seeds in a greenhouse and were used at the 6-leaf stage after 2 months in culture. The plants were kept in a growth chamber at 23 ± 1 °C with light/dark period of 16 h/8 h and 70-80% relative humidity for several days before treatments.
Tobacco mosaic virus (TMV) that came from our collection was multiplied in N. tabacum. TMV was extracted from infected leaves of systemically infected plants by homogenization in 0.05 M H3PO4 buffer (0.05 M KH2PO4, 0.05 M Na2HPO4 pH 5.5) with subsequent clarification of the extract by centrifugation at 2000 g for 6 min. The supernatant extract was used for mechanical inoculation.
The bioactivity assay for protection and inactivation and cure effect was assessed according to the method described by Wang et al. (2010). For the inactivation assay by half-leaf method, leaves of N. tabacum were split into two halves of left and right from the midrib with scissors and kept in wet absorbent paper in porcelaneous dishes before use. sLNT and LNT (25, 50, 75, 100 μg/mL), the blank control, and Ningnanmycin (500 μg/mL, reference agent) were mixed with an equal volume of TMV solution (10.19 μg/mL final concentration), and left standing for 30 min and mechanically inoculate the left half leaves of. N. tabacum, whereas the right halves were treated with the blank control and TMV solution containing the same solvent as a control, using 500-mesh carborundum as abrasive. After inoculation, leaves were washed immediately with distilled water. The number of local lesion was recorded 3-4 days after inoculation. The inhibition level of viral infection was recorded and calculated according to the formula:
inhibition rate (%)= (1-T/C) Ã-100
Where T is the average mean lesion number of treated half-leaves and C is the average lesion number of the control halves.
For the protection assay, leaves of N. tabacum were sprayed with sLNT and LNT at different concentrations, respectively, and the control plants were sprayed with water and Ningnanmycin (500 μg/mL). At 48 h after sLNT and LNT application, plants were inoculated mechanically with TMV. The inoculated plants were maintained at 25 ± 2 °C under cool-white fluorescent lamps. The disease index was investigated as previously described at 5 d after inoculation (Zhao, She, Du, & Liang, 2007). For the detection of effects of sLNT or LNT on TMV multiplication, the tobacco leaves were sprayed with sLNT and LNT (100 μg/mL), respectively. At 48 h after LNT or sLNT treatment, the leaves were inoculated with TMV. One-gram leaves inoculated with TMV were collected at 8, 12 and 24 h after inoculation. The fold changes in TMV coat protein (TMV-CP) gene expression using RTqPCR were determined.
For the cure assay by leaf-disc method, the TMV suspension of 10.19 μg/mL was inoculated on leaves of N. tabacum. Growing leaves of N. tabacum were mechanically inoculated with TMV (10.19 μg/mL). After 6 h, 12-mm diameter leaf discs that were smooth and thin and without major veins were cut from the leaf surface. The leaf discs were floated on the solution of each sample and then incubated at 25 ± 2 °C for 48 h. The discs were treated with solvent only as the positive control, while discs of healthy leaves were used as the negative control. After 48 h, leaf discs were ground in coating buffer, and their viral concentration was assessed by ELISA. Indirect ELISA was mainly performed as described by Zhou et al. (2004). The inhibition rate of viral replication was calculated according to the formula:
inhibition rate (%)= (1-C/C0) Ã-100
Where C is the viral concentration in the treated leaf discs and C0 is the viral concentration in the positive control (French & Towers, 1992). TMV concentration was calculated by the standard curve with the A405 value of TMV at concentrations of 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/mL. All assays were performed in triplicate with at least five tobacco seedlings per replicate.
To determine the effects of sLNT and LNT on the amounts of H2O2, SA and SA 2-O-β-D-glucoside (SAG) and the transcript levels of defense related genes in tobacco seedlings, tobacco seedlings were sprayed with 75 μg/mL sLNT or LNT, until drops began to fall from the leaves. At various times after the treatment, leaf samples were collected, and aliquots normalized by their fresh weight (approximately 1 g) were taken from the treated leaves, and the first upper untreated leaf. The leaves were immediately frozen in liquid nitrogen and stored at - 80 °C.
2.4 H2O2 measurements
H2O2 was measured according to the method of Mukherjee & Choudhuri (1983), with some modifications. One gram of fresh leaf tissue was homogenized with 5 mL ice-cold acetone and centrifuged at 10, 000 g for 10 min at 4 °C. Then 1 mL of the supernatant was added to 0.1 mL 20% TrisCl4-HCl solution and 0.2 mL ammonia solution and then centrifuged at 10, 000 g for 10 min. The residue was washed 5 times with acetone and then dissolved in 3 mL 1 M H2SO4. The absorbance was measured at 410 nm. The same protocol was used to make a standard curve for H2O2 and this was used to calculate the amount of H2O2. Each treatment had three replicates with at least 5 tobacco seedlings per replicate.
2.5 SA and SAG measurements
The amounts of SA and SAG extracted from tobacco leaves were measured by HPLC according to the method described by Verberne et al. (2002). HPLC analysis of SA was performed using an ATvp HPLC (Shimadzu, Japan) with a chromatographic column (Hypersil ODS (C18), 5 mm, 250 Ã- 4.6 mm). The eluent was 0.2 M sodium acetate buffer pH 5.5 (90%) with methanol (10%) at a flow rate of 0.8 mL/min. The column temperature was 40 °C. The RF-10Az spectrofluorometric detector operated at an emission wavelength of 407 nm and an excitation wavelength of 305 nm. Each treatment contained three replicates with at least 5 tobacco seedlings per replicate.
2.6 RNA isolation
Total RNA was extracted by Trizol Reagent (Invitrogen, USA) according to manufacturer's instruction. Isolated RNA was dissolved in 20 µL of RNase free H2O, quantified by spectrophotometry and stored at −80 °C.
2.7 Real-time quantitative PCR (RT-qPCR)
For RT experiments 0.5 µg of total RNA was used for reverse transcription. The reaction was performed using an RT-PCR kit (TOYOBO, Japan) according to the manufacturer's instruction. The relative level of transcripts coding for TMV-CP, PR-1a, Basic PR-1, PR-2, PR-3, PR-5, PAL, and EAS was determined using EF-1a as internal control. The reaction mixture was incubated for 20 min at 42 °C and terminated by 99 °C for 5 min. The specific genes were amplified using gene specific primers designed from coding sequences of each gene using Primer Express 2.0 software (Applied Biosystems, United States) (Table 1). RT-qPCR using the PTC-200 Real-Time PCR system and SYBR Green Master mix (BIO-RAD, United States) was performed using primers at a final concentration of 0.25 mM each and 2 mL of cDNA as template in a 25 mL reaction. PCR-cycling conditions comprised an initial polymerase activation step at 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 50 s. After each run, a dissociation curve was designed to confirm specificity of the product and avoid production of primers-dimers. The gene for EF-1α was used as a control. Calculation of relative amounts of amplification productswas completed with the comparative 2-ΔΔCÑ‚ method (Livak & Schmittgen, 2001). All reactions were performed in triplicate and each sample was further amplified without reverse transcription to confirm there was no DNA contamination in the sample.
2.8 Statistical analysis
All statistical analyses were performed with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was carried out to determine the effects of the treatments, and those means were compared by Duncan's multiple range tests (P<0.05). Analysis between sLNT and LNT treatment group was performed with a Student's t-test, and differences were considered significant at P≤0.05 or P≤0.01. Data presented in this paper were pooled across three independent repeated experiments.
3. Results
3.1 Preliminary antiviral activity assay
To make a judgment of the antiviral potency of sLNT and LNT, the commercial plant virucide Ningnanmycin was used as the control.
The anti-TMV bioassay indicated that the inhibitory effect of sLNT or LNT on TMV was positively related to the concentration used (Table 2). Inactivation effects of sLNT and LNT were 85.6% and 81.4% at 100 μg/mL, respectively. Moreover, no obvious differences were obtained between sLNT, LNT and Ningnanmycin (97.0%, 500 μg/mL) (P<0.01). In addition, sLNT and LNT exhibited potential protection bioactivities, with values of 75.2% and 69.6% at 100 μg/mL, respectively. Furthermore, the protection effect of sLNT was higher than that of Ningnanmycin (72.0%). Compared with the inactivation and protection activities, sLNT and LNT possessed relatively lower curative activities, with values of 62.0% and 59.0% at 100 μg/mL, respectively. However, the curative effects were not different significantly between sLNT, LNT and Ningnanmycin (56.0%, 500 μg/mL) (P<0.05). Interestingly, the antiviral activities of sLNT and LNT were not different significantly at 75 and 100 μg/mL. Hence, we choose the concentration of 75 μg/mL for the following assays.
In order to detect whether sLNT and LNT had systemic protection against TMV, we measured the levels of transcription of the TMV-CP gene using RT-qPCR. The results indicated that treated tobacco leaves with sLNT and LNT 48 h before inoculation with TMV significantly inhibited TMV multiplication (P<0.05). The level of TMV-CP transcripts in DW treated leaves was about 6.9 and 3.85-fold higher than that in the sLNT and LNT treated leaves 24 h after inoculation respectively (Fig. 1). In addition, about 5 d later, the leaves treated with DW had the typical mosaic pattern, while the leaves treated with sLNT did not (data not shown). These results suggested that both sLNT and LNT treatments improved the level of resistance to TMV.
Table 2
Fig. 1
3.2 Determination of changes in the amounts of H2O2 in tobacco leaves
As shown in Fig. 2, rapid generation of H2O2 in sLNT treated tobacco leaves was detected, which reached the highest values at the 9 h time point after the initiation of treatment. The amount of H2O2 in sLNT treated tobacco leaves was about 2.4-fold higher than that in the LNT treated leaves at the 9 h time point. Moreover, significant differences in the production of H2O2 were obtained between the sLNT and LNT treatment since 3 h time point. Nevertheless, LNT did not lead to significant changes of H2O2 in tobacco leaves during the whole experiment. Interestingly, both sLNT and LNT did not lead to tissue necrosis in tobacco leaves (data not shown).
Fig. 2
3.3 sLNT locally and systemically caused an increase in SA and SAG in tobacco
Treatment with sLNT locally and systemically induced a rapid accumulation of SA and SAG in tobacco leaves (Fig. 3). In treated leaves (Fig. 3A), the maximum levels of SA and SAG were obtained at 12 h. The level of SA was about 5.5-fold higher than that in LNT treatment at the same time point, while SAG was 6.45-fold higher. In the untreated upper leaves (Fig. 3B), SA and SAG both reached their peaks at 24 h. SA was approximately 2.6-fold higher than that in LNT treatment, and SAG was about 3.5-fold higher. The LNT treated leaves maintained low levels of SA and SAG throughout the experiment.
Fig. 3
3.4 sLNT locally and systemically induced expression of PR protein genes in tobacco
The increased transcript levels of acidic PRs (including PR-1a, PR-2, PR-3 and PR-5) and basic PR-1 were detected in the sLNT and LNT treated leaves (Fig. 4). In treated leaves, transcripts of the basic PR-1, PR-2 and PR-3 genes reached their maximal levels 48 h after sLNT treatment. The level of basic PR-1 was approximately 5.2-fold higher than the LNT treated leaves, while PR-2 was 3.1-fold higher and PR-3 was 5.6-fold higher. Meanwhile, the PR-1a and PR-5 gene transcripts reached their maximum levels 24 h after sLNT treatment. The level of PR-1a was about 4.9-fold higher than the LNT treated leaves while PR-5 was 3.2-fold higher. In the untreated upper leaves, transcripts of PR-1a, basic PR-1, PR-2, PR-3 and PR-5 genes all reached their highest values 48 h after sLNT treatment. The transcript levels of PR-1a were about 2.9-fold higher than the LNT treatment, basic PR-1 levels were 3.3-fold higher, and PR-2 levels were 1.9-fold higher. The transcript levels of PR-3 and PR-5 were 2.8 and 2.1-fold higher, respectively, than the LNT treated leaves at the 48 h time point.
Fig. 4
3.5 sLNT locally and systemically induced expression of defense-related enzyme genes in tobacco
In treated leaves, Maximum induction of PAL (Fig. 5A) occurred at 24 h after sLNT treatment, and the level was about 2.6-fold higher than that in LNT treated leaves. Expression of EAS (Fig. 5B) was strongly induced and reached its maximal level at 48 h after treatment with about 5.1-fold increase. In the untreated upper leaves, the expression of PAL (Fig. 5A) and EAS (Fig. 5B) was also enhanced by SLNT, and both reached their peaks at 48 h, with a relative increase of about 2.2 and 2.8-fold, respectively.
Fig. 5
4. Discussion
In the present study, both sLNT and LNT exhibited a significant inhibition effect on viral infection (Table 2) and TMV multiplication (Fig. 1) in tobacco plants in greenhouse, especially inactivation and protection activity. Moreover, sLNT showed a higher inhibitory effect than LNT treatment against TMV in a dose-dependent way in the preliminary antiviral activity assay, which were the same as the previous studies that sulfated polysaccharides exhibited a stronger antiviral activity than native ones in a dose-dependent manner (Zhang, Peter, Vincent, & Lina, 2004). These confirmed that sulfated modification could enhance anti-viral activity of LNT.
Sulfated polysaccharides has been reported to combine with positive charge on the surface of recipient cells or combine with virus molecule thereby obstruct the virus adsorption or inhibit the reverse transcriptase of virus (Talarico et al., 2005; Wallace, 1990). Therefore, we supposed that both sLNT and LNT could decrease the incidence of TMV disease by interacting with viral particles and preventing the entry of virus into the host cell. In order to test whether sLNT and LNT had the ability to influence the formation of normal virus particle, we determined the levels of transcription of the TMV-CP gene using RT-qPCR.
TMV CP possessed the ability to protect TMV RNA from digestion by ribonuclease and then help the reverse transcriptase of virus (Berlutti et al., 2011). The levels of transcription of the TMV-CP gene in sLNT and LNT treated leaves decreased obviously in our study, which inferred that the antiviral activities of sLNT and LNT might be associated with affinity towards TMV CP. Moreover, using ultraviolet-vis spectroscopic and fluorescence spectroscopic methods, we found that sLNT and LNT had affinity to TMV CP 4S and 20S protein by induction to red shift and fluorescence quenching phenomenon, but not to TMV RNA. These results will be published in Crop Protection (under review).
The antiviral activity of sLNT and LNT is considered to involve several mechanisms. It may involve a direct virucidal activity and elicitation effect on hosts (Ma, Guo, Wang, Hu, & Shen, 2010; Wang, Guo, et al., 2010). In order to test whether sLNT had the ability to induce systemic resistance against TMV, we measured the level of H2O2 in response to various treatments. H2O2 has been described as key roles in resistance responses against pathogens. H2O2 is took account into involvement in phytoalexin production, lipid peroxidation and defense related genes expression, etc. (Aziz et al., 2003). Previous studies have shown that treatment with laminarin, oligosaccharide and chitosan elicitors, binding to their receptors on the cellular membranes, could induce rapid generation of ROS and increase diseases resistance against plant pathogens in various plant seedlings (Aziz et al., 2003; Yin, Zhao, & Du, 2010; Zhao, She, Du, & Liang, 2007). In our research, we got similar results that sLNT could also induce rapid generation of H2O2 (Fig. 2) and increase resistance against TMV in tobacco seedlings.
Systemic acquired resistance (SAR) is accompanied by an increased level of salicylic acid (SA) both locally and systemically and by the coordinated upregulation of a specific set of genes encoding pathogenesis-related (PR) proteins, which are thought to contribute to disease resistance (Edreva, 2005; Van Loon, Rep, & Pieterse, 2006). In the present study, sLNT could promote the production of significant amounts of SA and SAG (Fig. 3) in treated leaves and untreated younger leaves. Salicylic acid as an endogenous plant hormone could be induced in pathogen-inoculated leaves, correlated with the induction of PRs and resistance. In addition, SA has also been reported as a signal molecule, necessary for generation of SAR (Edreva, 2005; Van Loon, Rep, & Pieterse, 2006). Taken together with our results, sLNT might induce SAR, possibly be mediated by the SA pathway in tobacco plants.
PRs can be induced by different stress stimuli and play an important role in plant defense against pathogenic constraints, and in general adaptation to stressful environments (Edreva, 2005). Of the PR protein families, PR-1 proteins are the most abundant after pathogen infection and PR-1a may constitute about 1% of the soluble protein in tobacco 7 d after infection. The increased expression of acidic PR-1a is usually used as a marker of SAR, but the precise function of PR-1a is still not clear (Liu, Du, & Wan, 2005). The group of PR-2 proteins could catalyze endo-type hydrolytic cleavage of the β-1, 3-D-glucosidic linkages in β-1, 3-glucans. The group of PR-3 proteins is endo-chitinases that catalyze the hydrolysis of β-1, 4-N-acetylglucosamine linkages, so they can cleave fungal cell walls in situ and play a major role in disease resistance. Transgenic tobacco over-expressing PR-2 and PR-3 had improved resistance to Cercospora nicotianae (Zhu, Maher, Masoud, Dixon, & Lamb, 1994). PR-5 proteins are a class of thaumatin-like proteins, and have strong antifungal activity (Pierpoint, Tatham, & Pappin, 1987). Consistent with previous findings that a number of different elicitors (chitosan oligosaccharide, chitosan, SA, oxalic acid, calcium chloride) significantly induced expression of PR-1a, PR-2 and PR-3 in plants (Yin, Zhao, & Du, 2010; Zhao, She, Du, & Liang, 2007), we observed that sLNT significantly increased the transcript levels of PR-1a, PR-2, PR-3 and PR-5 in treated tobacco leaves. Interestingly, sLNT also induced the increase of the above PR protein genes systemically (Fig. 4). These results suggest that the sLNT not only could induce a dose and time-dependent resistance in treated leaves but also long-term systemic protection against TMV in plant tissues distant from the primary inoculation.
Previous studies have reported that SA, which is synthesized by the phenyl-propanoid pathway from trans-cinnamic acid and benzoic acid, regulates the expression of genes for acidic PR proteins and induces defense against biotrophic pathogens that feed and reproduce on live host cells, whereas jasmonic acid (JA) or ethylene (ET) regulates the expression of genes for basic PR proteins and activates defense against necrotrophic pathogens that kill host cells for nutrition and reproduction (Bostock, 2005; Glazebrook, 2005). In our study, besides the increased transcript levels of acidic PRs [PR-1a, PR-2, PR-3, PR-5], sLNT also affected the accumulation of basic PR-1 gene expression. The reason that causes this phenomenon probably is a mutually synergistic interaction between the SA and JA pathways. Such cross-talk provides the means by which plants can regulate their responses to maximize defense (Bostock, 2005; Glazebrook, 2005).
PAL is a key enzyme of the phenyl-propanoid pathway, contributing to the synthesis of phenolic compounds, phytoalexin and salicylic acid (SA) (Dixon, & Paiva, 1995). Previous studies have shown that laminarin and sulphated laminarin, a β-1,3 glucan and β-1,3 glucan sulfate, which has the same structure as LNT and sLNT, induced a transient increase in PAL activity (Klarzynski, et al., 2000; Ménard, et al., 2004). Our results also found that sLNT significantly induced expression of the PAL gene systemically and locally in tobacco leaves (Fig. 5 A), compared with LNT. Moreover, 5-epi-aristolochene synthase (EAS), an important enzyme in the phytoalexins synthesis pathway was also significantly induced systemically and locally in tobacco leaves (Fig. 5 B). Therefore, the sLNT induced increases in TMV resistance in tobacco seedlings was probably correlated with increases in PAL and EAS mRNA levels.
In conclusion, sLNT and LNT exhibited a potential antiviral activity against TMV. This inhibitory effect might be made by binding to tobacco cell receptors, or viral particles or both and inhibiting viral adhesion and entry into host cells, but also attribute to activate the expression of a number of defense genes.
Acknowledgement
This work was supported by the National Key Technology R&D Program of China (201003004).
