Correspondence: Dequan Li, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, 61 DaiZong Street, Tai'an, Shandong 271018, China. Tel.: +86 538 824 9137; fax: +86 538 824 9608; e-mail: email@example.com
Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) was the first pathogen to be demonstrated to infect Arabidopsis and to cause disease symptoms in the laboratory setting. However, the defense response to Pst DC3000 was unclear in tobacco. In this report, the expression profiles of twelve defense response–related genes were analyzed after treatment with salicylic acid (SA), jasmonic acid (JA), and pathogen Pst DC3000 by qRT-PCR. According to our results, it could be presented that the genes primarily induced by SA were also induced to higher levels after Pst DC3000 infection. SA accumulation could be induced to a higher level than that of JA after Pst DC3000 infection. In addition, SA could result in hypersensitive response (HR), which did not completely depend on accumulation of reactive oxygen species. These results indicated that tobacco mainly depended on SA signaling pathway rather than on JA signaling pathway in response to Pst DC3000. Further study demonstrated that JA could significantly inhibit the accumulation of SA and the generation of the HR induced by Pst DC3000.
Plants are sessile organisms, and thus, they have evolved efficient mechanisms to combat attacks from pathogens, including the basal immune systems and highly specific resistance (Jones & Dangl, 2006). One of the most effective defense mechanisms to against pathogens is the hypersensitive response (HR; Hammond-Kosack & Jones, 1996). The HR prevents pathogens from extracting nutrients from the host plant's healthy tissue. It is initiated as the plant develops necrotic lesions in the locally infected tissue, and is accompanied by the accumulation of salicylic acid (SA) and jasmonic acid (JA; Malamy et al., 1990; Metraux et al., 1990). Finally, some pathogenesis-related (PR) proteins become activated and participate in the HR (Bol et al., 1990; Ohshima et al., 1990; Seo et al., 1997).
The plant hormones SA and JA are thought to be involved in the regulation of signaling networks, including pathogen-associated molecular patterns (PAMP) responses and effector-triggered immunity (Bent & Mackey, 2007; Zipfel, 2009). Following the early signaling that occurs after a pathogen attack, plant-generated SA and JA usually act as secondary signaling molecules. So the accumulation of SA and JA has been widely used as a reliable marker of defense responses and is closely associated with redox homeostasis, hypersensitive cell death, and systemic acquired resistance (Dong, 2004; Song et al., 2004).
Plants defend themselves against a pathogen attack using two principally different mechanisms: (1) their existing defense faculties, such as physical barriers; and (2) inducible defense responses. When a pathogen breaks through a plant's physical barriers, the plant cells send signals alerting of the breach and then activate the inducible defense mechanisms. The inducible defense response often requires a large number of defense genes to be expressed, which produce many different types of proteins, such as cell wall proteins, hydrolytic enzymes (chitinases and β-1, 3-glucanases), and other PR proteins, WRKY transcriptional factors, protease inhibitors (PIs), signaling compounds (ethylene, JA, and SA), and enzymes involved in synthesizing lignin and phytoalexins also routinely to be expressed after a pathogen attack (Hammond-Kosack & Jones, 1996).
Pseudomonas syringae pv. Tomato strain DC3000 (Pst DC3000) is a Gram-negative, rod-shaped bacterium with polar flagella. This microorganism threatens a wide variety of plants with infection and disease. It regularly infects tomato plants and Arabidopsis thaliana, a small flowering plant commonly used as a model organism, with bacterial speck disease (Buell et al., 2003). Because the Pst DC3000 genome was completely sequenced in 2003, scientists have made tremendous advances in understanding the molecular interaction between this microorganism and its host plants. The pathogen Pst DC3000 has been well characterized as modern genomic technique as well as more traditional plant pathology approach.
Because of their suitability as model plants, A. thaliana and tobacco play important roles in studies directed at discovering gene function in plants. However, tobacco's defense response has not been studied in depth, and its specific defense mechanisms have not been fully characterized until now. In this study, we analyzed the expression patterns of 12 disease-related genes in tobacco under treatment conditions involving SA, JA, and Pst DC3000. We found that tobacco infected with Pst DC3000 primarily employed the SA signaling to initiate its defense response. Further investigation into the SA signaling pathway showed that SA resulted in the HR, which did not depend entirely on the accumulation of reactive oxygen species (ROS). However, the HR and the accumulation of SA induced by Pst DC3000 were significantly inhibited by the presence of JA.
Materials and methods
Plant materials and growth conditions
The seeds of WT tobacco (Nicotiana tabacum cv NC 89) were treated with 70% ethanol for 30 s and with 2.6% bleach for 10 min and then were washed six times with sterile water; after that, the seeds were plated on MS medium (Murashige & Skoog, 1962) under light/dark cycle conditions of 16/8 h at 25 °C.
Pathogen inoculation and disease development
Pseudomonas syringae pv. Tomato DC3000 was cultured on King'S B (KB) medium (Liu et al., 2010) containing 50 μg mL−1 rifampicin. Overnight, log-phase cultures were grown to an optical density at OD600 nm of 0.6 to 0.8 (OD 0.1 = 108 cfu mL−1) and diluted with 10 mM MgCl2 to the concentrations of 107 cfu mL−1 before inoculation. Control was performed with 10 mM MgCl2. The bacterial suspensions were infiltrated into the abaxial surface of a leaf using a 1-mL syringe without a needle, and the HR was detected by trypan blue staining as described (Koch & Slusarenko, 1990; Katagiri et al., 2002).
The expression profiling analysis of different genes using real-time PCR under SA, JA, and Pst DC3000 treatment conditions
The tobacco seedlings were incubated with Hoagland's solution for 6 weeks, and uniformly sized plants at similar growth stage were treated with SA, JA, and Pst DC3000. For SA and JA treatment, plants were sprayed with 100 μM SA, 100 μM JA, or water (control). For Pst DC3000 treatment, plants were sprayed with a bacterial suspension of 1 × 107 cfu mL−1 supplemented with 0.05% Silwet L-77 or 10 mM MgCl2 (control). The leaves were collected at specific time points; the materials were immediately frozen in liquid nitrogen and stored at −80 °C. Total RNA was extracted using the RNeasy plant mini kit (Tiangen) according to the manufacturer's instruction. The first-strand cDNA synthesis was performed using 5 μg of total RNA, Oligo (dT)12–18 primer, and ThermoScript Reverse Transcriptase (Fermentas) according to the manufacturer's instruction. The synthetic cDNAs were used as templates in qRT-PCRs performed using primers (Supporting Information, Table S1) and SYBR Green SuperMix (TransGene, China). Tobacco actin gene (X69885.1) was amplified using primers (Ntactin-S and Ntactin-A) along with gene to allow gene expression normalization and subsequent quantification. All qRT-PCRs were carried out using Bio-Rad CFX96 Real-Time System (Bio-Rad). Cycling parameters for all qRT-PCRs were 94 °C for 10 min initially followed by 40 cycles, each comprising of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s.
Histochemical detection of ROS
Reactive oxygen species was detected using nitroblue tetrazolium (NBT) staining methods. The seedlings were infiltrated with 0.5 mg mL−1 NBT for 20 h in the dark to detect ROS. Then, the seedlings were decolorized by boiling in ethanol (96%) for 10 min. After cooling, the leaves were extracted at room temperature with fresh ethanol and were photographed using stereomicroscope. The quantitative detection of ROS was performed as well as described by Jiang & Zhang (2001).
Quantitative analysis of endogenous JA and SA
Six-week-old tobacco plant leaves were injected with MgCl2 (control) or Pst DC3000 strains (1 × 107 cfu mL−1) alone or supplemented with 100 μM SA or JA. The extraction and quantitative analysis of JA and SA followed the protocol of Engelberth et al. (2002) and Huang et al. (2003).
Results and discussion
Phylogenetic analysis of disease-related genes
Plants experience various environmental stresses in nature. To manage these stresses, plants are capable of differentially activating distinct defense pathways that depend on the SA and JA signaling networks. Many studies have demonstrated that defense-related proteins, such as PR proteins, WRKY transcriptional factors, and PIs, play important roles in defense processes (Hammond-Kosack & Jones, 1996). In this study, we quantified the transcript accumulation of 12 disease-related genes under different conditions in tobacco: they consist of genes from the PR family (PR1a, PR2, PR4, PR5, PR10), the PI family (PI1, PI2), and the WRKY family (WRKY1, WRKY12), as well as the NPR1, PAL, and CHS genes. We inferred the phylogenetic relationships between these genes and the evolutionary history of the gene families by constructing a combined phylogenetic tree using aligned protein sequences. Except for PR5, all proteins displayed higher homology with proteins in the same family, indicating a close evolutionary relationship between these proteins (Fig. S1).
The protein PR5 was found to be highly similar to ribosomal protein S9 from the plants Ricinus communis, A. thaliana, and Solanum demissum. These proteins were previously found to be involved in transcription and translation, or act in signal transduction pathways (Wang et al., 2000).
The genes PR1a, PR2, and PR10 belonged to antifungal protein biosynthesis genes, which may work to inhibit the growth of pathogens. The PR4 gene encodes hevein-like precursor protein, which is known to have antifungal activity (Sels et al., 2008).
PI1 and PI2 are PIs, which perform the general functions of binding proteases and controlling protease activity. Because these general functions are associated with many biochemical processes, PIs may have multiple functions in plants (Haq et al., 2004; Valueva & Mosolov, 2004; Christeller & Laing, 2005).
The WRKY DNA-binding transcriptional factors are involved in plant's responses to biotic and abiotic stresses. It had been previously shown that Arabidopsis WRKY3 and WRKY4 were expressed in response to both pathogen infection and SA treatment. Another WRKY protein, Arabidopsis WRKY70, was found to control the balance between JA- and SA-dependent defense pathways (Li et al., 2006).
l-Phenylalanine ammonia-lyase (PAL), the first enzyme involved in the phenylpropanoid biosynthesis pathway, is often encoded by multigene families in plants. The phenylpropanoid pathway produces a wide range of compounds including flavonoids, hydroxycinnamic acids, coumarins, stilbenes, lignin, and condensed tannins, which collectively function as phytoalexins, signaling molecules, and structural components of plants (Dixon et al., 2002).
Chalcone synthase (CHS) is a key enzyme in the biosynthetic pathway that produces flavonoids. Flavonoids have many important biological functions in plants, such as protecting the organism from UV damage and disease (Ferrer et al., 1999).
Different expression pattern of disease-related genes in response to SA and JA
SA and JA are known to play major roles in regulating plant defense responses against various pathogens (Bari & Jones, 2009). Previous studies have shown that the SA-dependent and JA-dependent pathways act antagonistically. Consequently, SA is a potent inhibitor of JA-dependent defense responses (Penninckx et al., 1996; Bowling et al., 1997; Niki et al., 1998; Van Wees et al., 1999). There is some evidence that JA inhibits SA-dependent activities as well (Prithiviraj et al., 2007). Huang et al. (2003) demonstrated that induced volatile emissions from tobacco plants in response to P. syringae are not linked with the changes of JA.
To further confirm how disease resistance genes respond to SA and JA exposure, the expression patterns of these genes were analyzed using qRT-PCR after exposing tobacco samples to 100 μM SA or JA for different periods of time. The results showed that PR1, PR2, WRKY12, PI1, and PAL were primarily expressed in response to SA exposure rather than JA exposure. Unlike PI1, PI2 was mainly expressed in response to JA exposure. JA and SA have a similar influence on the expression of PR4, PR5, PR10, WRKY1, and NPR1. Of note, we found that JA significantly inhibited the transcription of PAL. The expression of CHS was also stifled in response to SA and JA treatment (Fig. 1).
In this study, although no significant inhibition was observed after treating tobacco samples with JA and SA, the expression of PR1, PR2, PI1, and WRKY12 increased to a higher level in response to SA than JA. However, the expression of PI2 was higher in response to JA than SA. The genes PR4, PR5, PR10, WRKY1, and NPR1 were expressed at similarly low levels after exposure to SA and JA, but had higher levels of expression than PAL and CHS.
Tobacco primarily employs the SA signaling pathway in response to Pst DC3000
As a result of the many previous studies that have used A. thaliana as a model plant, the Pst DC3000 pathogen is well characterized as a traditional plant pathology approach. To better understand the signaling pathways in tobacco plants following exposure to Pst DC3000, we determined the transcriptional levels of the 12 defense-related genes at different time points after being exposed to Pst DC3000. As shown in Fig. 2, the transcriptional levels of all genes except CHS increased after pathogen infection. However, the genes that were primarily induced by SA exposure rose to higher transcriptional levels than the other genes examined in this study. The expression of PAL increased after exposure to SA and Pst DC3000, but was significantly inhibited by JA. Similar to its response after exposure to SA and JA, the expression of CHS was inhibited by Pst DC3000. Further study indicated that the SA accumulation could be induced to a higher level in response to Pst DC3000 (Fig. 4c). However, the content of JA did not have significantly change (data not shown). According to these results, we could conclude that the tobacco depends primarily on the SA signaling pathway to combat with the Pst DC3000 pathogen.
The HR induced by SA does not depend entirely on the accumulation of ROS
The accumulation of SA, JA, and ROS has often been widely used as a reliable marker to signal-elevated defense responses in plants and is closely associated with redox homeostasis, hypersensitive cell death, and systemic acquired resistance (Dong, 2004; Song et al., 2004). However, the relationship between SA, JA, and ROS was not clear in tobacco plants. In this study, we reported that SA induced the accumulation of ROS and that this accumulation could be inhibited by the scavenger dimethylthiourea (DMTU). We also found that tobacco plants exposed to JA did not result in the accumulation of ROS (Fig. 3).
When plants are invaded by a pathogen, a zone of dead cells forms at the infection site. This zone of dead tissue limits the speed at which the pathogen grows, because the pathogen cannot obtain nutrients from the dead host cells. This response is referred to as the HR, and it is frequently associated with resistance to further multiplication and spreading of the pathogen (Shirano et al., 2002; Pike et al., 2005; Nemchinov et al., 2008).
SA, JA, and ROS are important factors that influence the HR. To determine the roles of SA, JA, and H2O2 in the defense response of tobacco, we analyzed the HR after injecting SA, JA, and H2O2 (with or without DMTU) in tobacco leaves. We found that injecting SA and H2O2 resulted in HR, but injecting JA did not. To determine the relationship between H2O2 and SA in plant defense responses, the tobacco plants were also treated with SA and H2O2 with DMTU. After treatment, the HR symptom that was induced by H2O2 was significantly inhibited, and the HR symptom that was induced by SA was also alleviated (Fig. 3). These results indicated that the HR induced by SA does not depend entirely on the accumulation of ROS.
JA may negatively regulate the HR and the accumulation of SA in response to Pst DC3000 in tobacco
Pst DC3000 is an important model pathogen for plant–pathogen interaction in A. thaliana. Plants can induce the HR when pathogen Pst DC3000 enters a plant's leaves through stoma (Hirano & Upper, 2000). Previous studies have assumed that plant-induced SA and JA signaling pathways act linearly and independently and trigger distinct plant defense responses including cell death. However, some studies have revealed that the SA and JA signaling pathways do not act independently in A. thaliana and could mediate the interactions between these diverse signaling pathways (Dong, 1998; Genoud & Metraux, 1999; Clarke et al., 2000; Greenberg et al., 2000).
To verify the roles of SA and JA in response to Pst DC3000, the Pst DC3000 cell suspension was injected into tobacco leaves with SA or JA. After inoculation with Pst DC3000 6 h, the tobacco leaves developed HR symptoms (identified by trypan blue staining). When the Pst DC3000 cell suspension was supplemented with 100 μM SA and then injected into tobacco leaves, the HR was enhanced. However, when the Pst DC3000 cell suspension was supplemented with 100 μM JA, the HR symptoms were inhibited seriously. When the Pst DC3000 cell suspension was supplemented with 100 μM of both JA and SA, the HR occurred again (Fig. 4a). We found that pathogen-induced cell death was suppressed by 100 μM JA, but then induced again by an additional 100 μM SA (Fig. 4b). These results indicate that the HR induced by Pst DC3000 arises primarily through the SA signaling pathway, while the JA signaling pathway suppresses the HR in tobacco after exposure to Pst DC3000. Further study indicated that the SA accumulation could be induced to a higher level in response to Pst DC3000, and this role could be inhibited by JA (Fig. 4c).
Although it is routinely used as a model plant, the tobacco's defense mechanisms are not well understood. In this study, we identified the expression profiles of 12 genes that contribute to the defense response after treating tobacco plants with SA, JA, and the pathogen Pst DC3000. According to our results, the genes identified here are differentially inducible in response to SA, JA, and Pst DC3000. Tobacco accumulated higher-level SA content rather than JA in response to Pst DC3000. In addition, we found that SA, but not JA, was able to induce the HR, and the HR response was not completely dependent on the accumulation of ROS. Furthermore, we showed that JA had an antagonistic effect on the Pst DC3000-induced HR and the accumulation of SA in tobacco. Finally, we concluded that tobacco's defense response to Pst DC3000 was conducted primarily through the SA signaling pathway.
This work was supported by the Grants from the Nation Natural Science Foundation of China (Nos 31071337, 31271633) and the State Key Basic Research and Development Plan of China (No. 2009CB118500).