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Keywords:

  • Trichoderma pseudokoningii SMF2;
  • peptaibols;
  • Trichokonins;
  • TMV resistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of peptaibols, which sheds light on the potential of peptaibols in plant viral disease control.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In past decades, attention has been paid to the development of biological control agents that are efficient, reliable and safe to the environment (Lyon & Newton, 1997). Among the biological control agents that have shown a satisfactory degree of control of pathogens, some Trichoderma spp. are well-known for their ability to reduce disease incidence by inhibiting growth and development of fungal and bacterial plant pathogens and inducing plant defense reactions (Yedidia et al., 1999; Segarra et al., 2009). Although the antimicrobial activity of Trichoderma spp. against fungi and bacteria and the involved mechanisms have been widely studied (Howell, 2003; Harman et al., 2004), the antiviral effect of Trichoderma spp. and the underlying biochemical and molecular mechanisms are still unknown.

Peptaibols, mainly identified from Trichoderma spp., play an important role in the antimicrobial activities of these biocontrol fungi (Daniel & Filho, 2007). At present, 316 peptaibols have been identified, >60% of which are from Trichoderma spp. (http://www.cryst.bbk.ac.uk/peptaibol/home.shtml). Most previous reports emphasized the analysis of their biosynthetic pathway, conformational properties, amino acid sequences and antimicrobial activity (Pandey et al., 1977; Rebuffat et al., 1995; Duval et al., 1997). Peptaibols have been shown to generally exhibit antimicrobial activity against Gram-positive bacteria and fungi (Jen et al., 1987). Only two peptaibols, Peptaivirins A and B from Sepedonium spp., were reported to have inhibitory activity against TMV infection to tobacco (Yun et al., 2000; Yeo et al., 2002). Trichokonins, a group of peptaibols produced by Trichoderma pseudokoningii SMF2, were demonstrated to exhibit antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens in vitro (Song et al., 2006). However, the antiviral activity of Trichokonins and the mechanism involved in plant resistance are still unknown.

Tobacco mosaic virus (TMV) is one of the most common causes of plant virus diseases and causes a serious loss of crops worldwide. TMV is known to infect >150 types of plants, including vegetables, flowers and weeds. Because of the high genetic variation of TMV, traditional chemical treatments have no stable effect to protect plants from virus infection. Moreover, the misuse of nonbiodegradable chemicals brings severe environmental pollution (Pfleger & Zeyen, 2008). Thus, it is important to study new biocontrol agents for plant viral disease. In this study, we tested the antiviral effect of Trichokonins against TMV infection to tobacco and analyzed the possible mechanism involved. Our results provided conclusive evidence that Trichokonins induced tobacco resistance against TMV infection through activation of multiple plant defense pathways.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and growth conditions

Tobacco (Nicotiana tabacum var. Samsun NN) seeds were sterilized by immersion in 70% ethanol for 2 min followed by 2.6% clorox for 7 min and thoroughly rinsed in sterile water. Seeds were germinated on Murashige and Skoog medium (Murashige & Skoog, 1962). Seedlings were uprooted and transferred into pots containing sterilized vermiculite at a density of one plantlet per pot. Seedlings were grown in a growth chamber [a photoperiod of 16/8 h (light/dark) (1.87 W m−2), 75–80% relative humidity, 25±1 °C] and were fertilized once a week with liquid Murashige and Skoog medium. Experiments were performed with plants at the 8–10-leaf stage.

Purification and preparation of Trichokonins

Trichokonins were prepared from solid-state fermented T. pseudokoningii SMF2 using the methods described previously (Song et al., 2006). The purified Trichokonins were dissolved in methanol to yield a 10 mM stock solution. Water (with 1% v/v Tween-80) was used for further dilution of Trichokonins in different experiments.

TMV infection tests

When a tobacco plant was grown to the 8–10-leaf stage, 1 mL Trichokonins (50, 100 or 200 nM), or 1 mL ddH2O containing 1% (v/v) Tween-80 and 0.2% (v/v) methanol (control solution) was sprayed on the lower leaves (the fifth to seventh leaves from the top) of one plant. After 4 days, plants were inoculated with TMV (0.02 mg mL−1, 100 μL per leaf) by rubbing the untreated upper leaves (the second to fourth fully expanded leaves from the top) with carborundum (500 Mesh). After inoculation, leaves were rinsed with tap water. The number of visible viral lesions was counted and the diameter and area of lesions were measured 6 days after inoculation. For each treatment, six plants were used and each experiment was repeated three times.

Elicitor activity tests

The production of superoxide anion radical (O2) and peroxide (H2O2) in the leaves of the 8–10-leaf stage tobacco plants treated with Trichokonins or control solution were examined using the procedure of Fitzgerald et al. (2004). For detection of systemic responses, seedlings were cultured in MS-medium containing 100 nM Trichokonins or control solution for 4 days, after which the top leaf was harvested. For detection of local responses, Trichokonins (100 nM) or 2 μL control solution were placed on the adaxial surface of scratched leaves. Leaves were harvested and analyzed immediately. The leaves treated with Trichokonins or control solution were vacuum-infiltrated with nitrotetrazolium blue chloride (NBT) or 3,3-diaminobenzidine (DAB), incubated overnight at 28 °C, fixed and cleared in alcoholic lactophenol solution and examined for the formation of precipitates. Microscopic analysis was performed using an Olympus Stereoscope SZX-9 (Olympus America Inc., Melville, NY) at × 40 magnification. To test autofluorescence, 2 μL of 100 nM Trichokonins or 2 μL control solution were placed on the adaxial surface of scratched leaves. After 24 h incubation at 25±1 °C, the autofluorescence in the leaves was assessed (Fitzgerald et al., 2004). Microscopic analysis was performed using Olympus BX-51 fluorescent microscope (Olympus America Inc.) at × 200 magnification. The excitation wavelengths were 470–490 nm and the emission wavelengths were 510–550 nm.

Enzyme activity assays

Each tobacco plant at the 8–10-leaf stage was sprayed with 1 mL of 100 nM Trichokonins or 1 mL control solution. After 0, 1, 2, 3, 4, 5 or 6 days, the leaves of tobacco plants were harvested, ground to a fine powder in liquid nitrogen and stored at −80 °C until analysis. Phenylalanine Ammonia-Lyase (PAL, E.C.4.3.1.5) activity was determined as described by González-Aguilar et al. (2004). Peroxidases (POD, E.C.1.11.1.7) activity was determined as described by Rathmell & Sequeira (1974). Polyphenol oxidases (PPO, E.C. 1.14.18.1) activity was assayed using Flurkey's method (Flurkey, 1985). For each treatment, three tobacco plants were used and each experiment was repeated three times.

Reverse transcription (RT)-PCR analysis of the expression of tobacco defense-related genes

RT-PCR analysis was conducted to determine the expression of selected defense-related genes in Trichokonins-treated tobacco plants. Each tobacco plant at the 8–10-leaf stage was sprayed with 1 mL of 100 nM Trichokonins. Total RNA was extracted from treated tobacco leaves after 0, 1, 2, 4, 6, 9, 12, 24 or 48 h treatment. The quality of extracted RNA was tested by 1.0% agarose electrophoresis. The transcription levels of genes were detected by RT-PCR using a One Step RNA PCR Kit (TaKaRa, Japan). RT-PCR products were loaded on 1.0% agarose gel and the relative quantity of each band was estimated by a gel pro analyzer (GeneGenius). Nine genes involved in different plant defense pathways were selected: SOD (superoxide dismutase), CAT (catalase), APX (peroxidase ascorbate) and POX (peroxidase), NtPR1a (pathogenesis-related protein 1a), NtNPR3 (pathogenesis-related protein 3) and NtCOI1 (coronatine-insensitive 1) (Chen et al., 1993; Shoji et al., 2008). The actin gene was used as an internal control. Gene-specific primers of these genes are shown in Supporting Information Table S1.

Statistical analysis

Results were expressed as mean±SD. P-value <0.05 was considered statistically significant. All statistical analyses were performed using spss 11.5 for Windows.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Trichokonins-treatment induced resistance in tobacco plants against TMV infection

Initial results indicated that after a 4-day treatment with Trichokonins, tobacco plants achieved the highest resistance to TMV (data not shown). Therefore, a 4-day treatment was used in the following experiments. Trichokonins of various concentrations (50, 100 and 200 nM) were used to analyze their ability to induce tobacco resistance against TMV infection. Six days after inoculation with TMV, the number and diameter of lesions were measured. Trichokonin treatment led to a remarkable reduction in the number of lesions that appeared in the tobacco leaves compared with the control plants (Fig. 1a). The lesion number in tobacco pretreated with 50, 100 and 200 nM Trichokonins was 15%, 54% and 35% less, respectively, compared with the control. These results indicated that tobacco resistance against TMV was significantly improved after Trichokonins treatment, and that 100 nM Trichokonins was the most effective concentration (Fig. 1a). After treatment with 100 nM Trichokonins, the final lesion diameter in the inoculated leaves was 2.25±0.61 mm on average, which was much smaller than that of the control plants (5.22±0.79 mm) (Fig. 1b). The final lesion area of Trichokonin-treated tobacco was about 28.9% in average, which was 1.5-fold less than that in the control plants (41.4%) (Fig. 1c). Together, these results indicated that Trichokonin treatment induced tobacco resistance against TMV infection.

image

Figure 1.  (a) Lesion inhibition in tobacco caused by the Trichokonins from Trichoderma pseudokoningii SMF2. For resistance detection, the fifth to seventh leaves (from the top) of a tobacco plant at the 8–10-leaf stage were sprayed with 1 mL Trichokonins (50, 100 or 200 nM), or 1 mL control solution. TMV was inoculated on the second to fourth fully expanded leaves (from the top) after plants were treated for 4 days. Photographs depicting representative symptoms were taken at 3 days after challenge. Inhibition rate=(average lesion number of control tobacco−average lesion number of TKs pretreated tobacco)/average lesion number of control tobacco × 100%. (b) Average diameter of the final lesions on tobacco leaves caused by TMV infection. (c) Average area of the final lesions on tobacco leaves after TMV incubation. Average lesion area is shown as the percentage of the area of all the lesions on a leaf to the whole leaf area. Results are expressed as means±SD of triplicate experiments (each performed in duplicate). Asterisks indicate values significantly different from controls: *P<0.05.

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Trichokonin treatment enhanced pathogenesis-related reactive oxygen species and phenolic compounds in tobacco

Production of reactive oxygen species and accumulation of phenolic compounds are early responses in plant–pathogen or elicitor recognition (Hutcheson, 1998). We tested the ability of Trichokonins to elicit these responses. Compared with the control plants, higher levels of O2 and H2O2 were produced in tobacco leaves after tobacco plants were cultured in 100 nM Trichokonin solution for 4 days (Fig. 2a and c). In addition, 100 nM Trichokonins resulted in the production of O2 and H2O2 around the application area on leaves instantaneously (Fig. 2b and d). These results showed that Trichokonins induced the production of O2 and H2O2 locally and systemically in tobacco plants. Furthermore, the autofluorescence of phenolic compounds was tested. A high level of autofluorescence was detected in the Trichokonin-treated leaves, whereas only a very low level of autofluorescence was found in the control samples (Fig. 2e). Taken together, these results suggested that one possible mechanism of Trichokonin-induced resistance against TMV is the induction of early plant defense reactions.

image

Figure 2.  Effect of Trichokonins on the production of reactive oxygen species and accumulation of phenolic compounds in tobacco leaves. (a) Systemic detection of O2 in the seedlings cultured in Trichokonin solution (100 nM) or control solution. (b) Local detection of O2 in the control solution-treated (2 μL) leaf and the Trichokonin-treated (2 μL, 100 nM) leaf. (c) Systemic detection of H2O2 in the seedlings cultured in Trichokonin solution (100 nM) or control solution. (d) Local detection of H2O2 in the control solution-treated (2 μL) leaf and the Trichokonin-treated (2 μL, 100 nM) leaf. (e) Autofluorescence in the control solution-treated (2 μL) leaf and the Trichokonin-treated (2 μL, 100 nM) leaf. (a, b), In the presence of O2, NBT polymerized and led to formation of a royal purple precipitate in leaf vines (indicated by arrows). (c. d) In the presence of H2O2, DAB polymerized and led to formation of a dark red-brown precipitate in the treated tissues (indicated by arrows). (e) Micrographs centered on the region surrounding the application site (indicated by arrows).

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Trichokonin treatment increased activities of selected pathogenesis-related (PR) enzymes

To find out the mechanism involved in Trichokonin-induced resistance against TMV in tobacco, the activities of PAL, POD and PPO were analyzed. These PR enzymes play key roles in tobacco resistance against TMV (Chen et al., 2009). As shown in Fig. 3a and b, the activities of PAL and POD increased after Trichokonin treatment. On the fourth day of treatment, both PAL and POD reached their maximum activity, with the peak values of 8.4-fold (PAL) and 5.2-fold (POD) higher than in the control plants, respectively. After a 4-day treatment, the activities of these two enzymes began to decrease and showed a drastic decrease after a 5-day treatment. PPO activity showed a slight increase during a 6-day treatment with Trichokonins (Fig. 3c). Apparently, Trichokonin treatment could differentially influence the activities of PR enzymes.

image

Figure 3.  Effect of Trichokonin treatment on the activities of some pathogenesis-related enzymes in tobacco. Each tobacco plant at the 8–10-leaf stage was sprayed with 1 mL of 100 nM Trichokonins or 1 mL control solution. Activities of PAL (a), POD (b) and PPO (c) of the leaves were analyzed during a 6-day treatment. Results are expressed as means±SD of triplicate experiments (each performed in duplicate). Asterisks indicates values significantly different from controls: *P<0.05.

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Trichokonin treatment induced the expression of plant defense genes in tobacco

To gain further insight into the mechanism involved in Trichokonin-induced resistance against TMV, the transcription levels of selected plant defense genes were analyzed. As shown in Fig. 4, seven genes involved in plant defense response were studied. A gene expression level that upregulated >1.5-fold (P<0.05) was considered a significant difference between control and Trichokonin treatment. SOD, CAT, APX and POX are known to be associated with the reactive oxygen intermediate (ROI)-mediated signaling pathway (Baker et al., 1997). Trichokonin treatment led to about 1.8-fold upregulation of SOD and CAT, 2.5-fold of APX and 2.3-fold of POX genes, compared with the controls (Fig. 4a). Trichokonin treatment also upregulated the expressions of NtPR1a, a marker gene of the SA-mediated defense pathway (1.9-fold) (Fig. 4b). The expression of NtPR3, a marker of the ethylene-mediated defense pathway, was increased by 1.9-fold, 9 h after Trichokonin treatment (Fig. 4b). NtCOI1, required for JA response in tobacco, was also induced by Trichokonin treatment, the expression of which was increased by 1.8-fold after a 6-h treatment. These results suggested the involvement of multiple defense pathways in Trichokonins-induced tobacco resistance against TMV.

image

Figure 4.  Gene expression profiling upon Trichokonin treatment in tobacco. The internal control was the actin gene. Lanes from left to right show the RT-PCR results of gene expression at 0, 1, 2, 4, 6, 9, 12, 24 and 48 h after Trichokonin treatment. The column is the schematic mode corresponding to the panels by gel pro analyzer. Three parallel experiments generated similar results, and representative pictures are shown. Results are expressed as means±SD of triplicate experiments (each performed in duplicate). Asterisks indicate values significantly different from various treatment times, *P<0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Several peptaibols isolated from Trichoderma spp. have been reported to have antimicrobial activity against Gram-positive bacterial and fungal phytopathogens (Daniel & Filho, 2007). Peptaivirins A and B from Sepedonium spp. are the only two peptaibols known to have antiviral activity against TMV, with an inhibitory effect of 74% and 79%, respectively, at concentration of 10 μg mL−1 (Yun et al., 2000). The Trichokonins isolated from T. pseudokoningii SMF2 have been shown to exhibit antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens with a concentration of 20 μg mL−1in vitro (Song et al., 2006). In this study, we found that these Trichokonins showed antiviral activity against TMV at very low concentration. Treatment with 100 nM (200 ng mL−1) Trichokonins led to 54% lesion inhibition in tobacco (Fig. 1). Although Peptaivirins A and B showed TMV inhibitory activity in tobacco, the mechanism involved in this antiviral activity was not studied (Yun et al., 2000; Yeo et al., 2002). Thus, this report represents the first study on the mechanism of peptaibols against plant virus.

Oxidative burst and phenolic compounds accumulation are early responses in plant defense system (Hutcheson, 1998). Reactive oxygen species control multiple cellular functions in plants, including the oxidative cross-linking of cell-wall proteins, alteration of the redox status to regulate specific plant transcription factors and direct antimicrobial activity (Mittler et al., 2004). Trichokonins induced production of O2 and H2O2, both locally and systemically (Fig. 2a–d), and accumulation of phenolic compounds at the application site (Fig. 2e). Hence, Trichokonins induced TMV resistance in tobacco plants by priming elicitor-like cellular defense response.

PAL, POD and PPO are important defense-related enzymes in plants (Sticher et al., 1997). PAL catalyzes the first step of the phenylpropanoid-metabolic pathway, which results in an increased lignin biosynthesis in tobacco and Arabidopsis (Gális et al., 2006; Pauwels et al., 2008). Trichokonin treatment led to a significant increase in PAL activity in tobacco (Fig. 3a). POD catalyzes the reduction of H2O2 via the transport of electrons to various donor molecules, which is implicated in a broad range of physiological processes, including lignification, suberization, auxin metabolism, the cross-linking of cell wall proteins and defense against pathogenic attack (Passardi et al., 2005). Trichokonin treatment also resulted in a significant increase in the activity of POD (Fig. 3b). PPO catalyzes the O2-dependent oxidation of phenolics to quinines, which is proposed as a component of elaborate plant defense mechanisms (Li & Steffens, 2002). In tomato, PPO plays a critical role in disease resistance to Pseudomonas syringae pv. tomato (Thipyapong et al., 2004). Trichokonin treatment also caused a slight increase of PPO activity in tobacco (Fig. 3c). Therefore, Trichokonins probably induce PAL-, POD- and PPO-involved defense responses in tobacco against TMV.

Antioxidant enzymes are involved in the plant defense signal transduction pathway by leading to the production of ROIs. ROIs may directly trigger a hypersensitive response or programmed cell death and the subsequent induction of defense-related genes (Baker et al., 1997). The upregulation of antioxidative enzyme genes, such as APX and POX, in tobacco after Trichokonin treatment indicated that the ROI-mediated signaling pathway is involved in Trichokonin-induced tobacco resistance against TMV (Fig. 4a). It has been demonstrated that plant defense responses are regulated by a network of interconnected signal transduction pathways in which SA, JA and ET play central roles (Robert-Seilaniantz et al., 2007). These signaling pathways do not function independently but influence each other through a complex network of synergistic and antagonistic interactions (Koornneef & Pieterse, 2008). Trichokonins upregulated the expression of SA-responsive PR gene acidic NtPR1a, ethylene-responsive gene basic NtPR3 and the key player in activating the JA signaling pathway, NtCOI1 (Fig. 4b). These results suggested that multiple defense pathways are involved in Trichokonin-induced resistance in tobacco against TMV. Likely, cross-talk between the different defense pathways occurs.

In summary, we studied the antiviral effect of Trichokonins against TMV infection and the mechanism involved. Trichokonins from T. pseudokoningii SMF2 can induce tobacco systemic resistance against TMV via activation of multiple plant defense pathways. The results imply the potential of peptaibols in plant viral disease control.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by Hi-Tech Research and Development program of China (2007AA091504), National Natural Science Foundation of China (30870047) and Foundation of State Key Lab of Microbial Technology, Shandong University, China.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1. Primers used for RT-PCR analysis in tobacco plants.

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.