The Role of Hydrogen Peroxide and Nitric Oxide in the Induction of Plant-Encoded RNA-Dependent RNA Polymerase 1 in the Basal Defense against Tobacco Mosaic Virus

Abstract

Plant RNA-dependent RNA Polymerase 1 (RDR1) is an important element of the RNA silencing pathway in the plant defense against viruses. RDR1 expression can be elicited by viral infection and salicylic acid (SA), but the mechanisms of signaling during this process remains undefined. The involvement of hydrogen peroxide (H2O2) and nitric oxide (NO) in RDR1 induction in the compatible interactions between Tobacco mosaic tobamovirus (TMV) and Nicotiana tabacum, Nicotiana benthamiana, and Arabidopsis thaliana was examined. TMV inoculation onto the lower leaves of N. tabacum induced the rapid accumulation of H2O2 and NO followed by the increased accumulation of RDR1 transcripts in the non-inoculated upper leaves. Pretreatment with exogenous H2O2 and NO on upper leaf led to increased RDR1 expression and systemic TMV resistance. Conversely, dimethylthiourea (an H2O2 scavenger) and 2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (an NO scavenger) partly blocked TMV- and SA-induced RDR1 expression and increased TMV susceptibility, whereas pretreatment with exogenous H2O2 and NO failed to diminish TMV infection in N. benthamiana plants with naturally occurring RDR1 loss-of-function. Furthermore, in N. tabacum and A. thaliana, TMV-induced H2O2 accumulation was NO-dependent, whereas NO generation was not affected by H2O2. These results suggest that, in response to TMV infection, H2O2 acts downstream of NO to mediate induction of RDR1, which plays a critical role in strengthening RNA silencing to restrict systemic viral infection.

About the Authors:

Yang-Wen-Ke Liao

Contributed equally to this work with: Yang-Wen-Ke Liao, Zeng-Hui Sun, Yan-Hong Zhou

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Zeng-Hui Sun

Contributed equally to this work with: Yang-Wen-Ke Liao, Zeng-Hui Sun, Yan-Hong Zhou

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Yan-Hong Zhou

Contributed equally to this work with: Yang-Wen-Ke Liao, Zeng-Hui Sun, Yan-Hong Zhou

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Kai Shi

* E-mail: [email protected]

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Xin Li

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Guan-Qun Zhang

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Xiao-Jian Xia

Affiliation: Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China

Zhi-Xiang Chen

Affiliations Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China, Department of Botany & Plant Pathology, Purdue University, West Lafayette, Indiana, United States of America

Jing-Quan Yu

Affiliations Department of Horticulture, Zhejiang University, Hangzhou, People’s Republic of China, Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Agricultural Ministry of China, Hangzhou, People’s Republic of China

Introduction

Most eukaryotes possess an RNA silencing system as a gene regulation and host defense mechanism. This system is activated in cells by double-stranded (ds) RNA, followed by the cleavage of the inducing RNA into short (21- to 24-nucleotide) fragments. These fragments, in turn, mediate multiple regulatory and defense functions in the cells [1], [2]. During this process, RNA-dependent RNA polymerases (RDRs) synthesize dsRNA intermediates, which can be cleaved by different Dicer-like proteins to produce different small interfering RNAs (siRNAs) [3]. Three functionally distinct RDRs have been characterized in tomato, tobacco, and Arabidopsis [4]–[6]. RDR1 has been found to be an element of the RNA silencing pathway in the plant defense against viruses and herbivores [6]–[9]. Several recent studies profiled virus-specific small interfering RNAs (vsRNAs) using next-generation sequencing platforms and compellingly implicated plant RDR1 in vsRNA biogenesis and vsRNA-mediated antiviral defense [10]–[12]. RDR1 activity and gene expression in Arabidopsis (AtRDR1) and Nicotiana tabacum (NtRDR1, the ortholog of AtRDR1) are elicited by viral infection and by salicylic acid (SA) treatment [7], [8]. After infection with a strain of Potato virus X (PVX), which does not spread in wild-type tobacco plants, transgenic NtRDR1 antisense plants accumulated the virus and developed symptoms locally in inoculated lower leaves and systemically in non-inoculated upper leaves [7]. A follow-up study also found that a T-DNA insertion AtRDR1 mutant exhibited markedly enhanced susceptibility to Tobacco mosaic tobamovirus (TMV) and Tobacco rattle virus [8]. Nicotiana benthamiana possesses an RDR1 gene that contains a 72-nucleotide insert with consecutive in-frame stop codons in the open reading frame and lacks active virus- and SA-inducible function [6]. This natural loss-of-function mutation was named NbRDR1m and was suggested to account for the plant’s hypersusceptibility to viruses. However, N. benthamiana transformed with the SA-inducible RDR1 gene from Medicago truncatula (MtRDR1) stilled showed enhanced resistance to tobamoviruses (e.g., TMV, Turnip vein-clearing virus, and Sunn hemp mosaic virus) but not to viruses outside of the tobamovirus genus (e.g., Cucumber mosaic virus (CMV) and PVX) [6]. These results strongly suggest that virus- and SA-inducible RDR1 plays an important role in the plant antiviral defense. However, the details of RDR1 induction and signal transduction during this process remain largely unknown.

In plants, one of the earliest host plant responses to pathogen invasion is an oxidative burst, in which the levels of reactive oxygen species (ROS) increase rapidly [13], [14]. ROS are believed to perform multiple roles during plant defense responses to microbial attack by acting directly in the initial defense and, possibly, serving as central cellular signaling molecules [15]. In particular, hydrogen peroxide (H2O2) is the most attractive candidate for a ROS signal because of its relatively long half-life and high permeability across membranes [16]. H2O2 may also play a role in the signaling cascade of virus- and SA-induced RDR1 activation in plants. This idea is supported by several lines of evidence. First, SA application and virus infection resulted in increased H2O2 concentration, and both are well known RDR1 elicitors [15], [17], [18]. Second, the first identified SA binding protein was a catalase (CAT, EC 1.11.1.6) from tobacco. CAT activity was inhibited by SA in vitro and in vivo, resulting in the generation of H2O2, which mediated the SA-induced defense response, whereas several antioxidants suppressed the SA-mediated activation of PR-1 expression and plant defense [15], [19]. Third, a recent study in Nicotiana glutinosa plants showed that exogenous H2O2 induced a significant increase in NgRDR1 transcripts [13].

In addition to H2O2, other reactive compounds are also involved in signaling in plants. Nitric oxide (NO) is an emerging essential regulatory molecule in plant immunity and has been well studied. Often, the production of NO and H2O2 overlaps both spatially and developmentally [20], [21]. Importantly, H2O2 and NO can react with each other and may influence the activities of enzymes that alter each other’s levels. NO donors caused rapid H2O2 accumulation in N. tabacum [22]. In contrast, a NO accumulation mutant noe1 was also identified that has a defect in a functional rice CAT, resulting in increased H2O2 in the leaves, which consequently promoted NO production via activation of nitrate reductase [23]. Similarly, in guard cells of Arabidopsis plants, NO synthesis and stomata closure in response to abscisic acid (ABA) were severely reduced in the H2O2-generation-related NADPH oxidase double mutant atrbohD/F [24]. Furthermore, a previous study also showed that virus infection and SA activated NO synthesis [25], [26]. Collectively, these studies suggest that H2O2 and NO generation may be related to virus- and SA-induced RDR1 induction in plants. However, the potential cross talk between H2O2 and NO, as well as its function in plant antiviral processes, has not yet been resolved.

In the present study, using pharmacological and biochemical approaches, we investigated the potential role of H2O2 and NO in virus- and SA-induced RDR1 activation and RDR1 function in the compatible interactions between TMV and the model host plants N. tabacum, N. benthamiana, and Arabidopsis. We found that RDR1 expression was elevated by H2O2 and NO donors, whereas TMV- and SA-induced RDR1 transcript levels were compromised by H2O2 and NO scavengers in all three plant species. Employing Arabidopsis mutants that were defective in H2O2 or NO synthesis and transgenic N. benthamiana plants transformed with SA-inducible MtRDR1 from M. truncatula [6], we showed that H2O2 may function downstream of NO and may mediate the induction of RDR1 in response to TMV challenge, thereby limiting the systemic infection and accumulation of the virus. These results provide initial insights into the signaling mechanisms underpinning virus- and SA-induced RDR1 activation in host plants.

Results

Changes of NtRDR1 expression and H2O2 and NO accumulation in N. tabacum after TMV inoculation

NtRDR1 relative expression and the quantity of H2O2 and NO were determined in the compatible interactions between TMV and N. tabacum plants (Figure 1). In non-inoculated upper leaves, NtRDR1 relative expression began to increase 2 days post inoculation (dpi) and reached a maximum induction of 3.7-fold at 4 dpi. Concomitant with the induction of NtRDR1 expression, we observed a rapid increase in the levels of H2O2 and NO signaling compounds, which resulted in significant differences over mock-treated plants at 1 and 0.5 dpi, respectively. Furthermore, the increased relative expression of NtRDR1 and increased concentration of H2O2 and NO resulting from TMV inoculation remained high until the end of the experiment. At 7 dpi, the relative expression of NtRDR1 and the concentration of H2O2 and NO were 2.2-, 1.0-, and 1.1-fold higher than those in mock-inoculated plants, respectively.

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Figure 1. Time course changes of NtRDR1 transcripts and concentrations of H2O2 and NO.

Changes in the transcript levels of Nicotiana tabacum RNA-dependent RNA Polymerase 1 (NtRDR1, A), hydrogen peroxide (H2O2, B), and nitric oxide (NO, C) over time in the non-inoculated upper leaves of Nicotiana tabacum plants after TMV inoculation of the lower leaves. The results are expressed as the mean ± SD, n  =  4.

https://doi.org/10.1371/journal.pone.0076090.g001

TMV- and SA-induced RDR1 induction is enhanced by exogenous H2O2 and NO, and is reversed by scavengers

To explore the possible involvement of H2O2 and NO in RDR1 induction, we pretreated non-inoculated upper leaves of N. tabacum plants with dimethylthiourea (DMTU, a H2O2 and OH· scavenger) and 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a NO scavenger) 12 hours before TMV inoculation. TMV-induced NtRDR1 expression was compromised by DMTU and cPTIO application, showing a reduction of approximately 50% with both chemical modulators at 4 dpi (Figure 2A). Next, we applied the exogenous H2O2, the NO chemical donor sodium nitroprusside (SNP), and SA to N. tabacum leaves, and NtRDR1 expression was analyzed throughout a 2-day experiment. Exogenous H2O2 and SNP induced a significant increase in NtRDR1 transcript levels (Figure 2B). Furthermore, the SA-induced increase in NtRDR1 transcript levels was substantially suppressed by elimination of H2O2 and NO.

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Figure 2. Effects of TMV infection, SA, and different chemical pretreatments on NtRDR1 transcripts.

A. Changes in TMV-induced NtRDR1 transcript levels over time in non-inoculated upper leaves induced by hydrogen peroxide (H2O2) and nitric oxide (NO) scavengers. The non-inoculated upper leaves were subjected to DMTU (H2O2 scavenger) or cPTIO (NO scavenger) pretreatment 12 hours before TMV inoculation. B. Changes in SA-induced NtRDR1 transcript levels over time caused by exogenous H2O2, NO, and their scavengers. The results are expressed as the mean ± SD, n  =  4.

https://doi.org/10.1371/journal.pone.0076090.g002

To determine the involvement of H2O2 and NO in TMV- and SA-induced RDR1 in other plants, the same experiments were also performed with N. benthamiana and Arabidopsis plants. Similar to the results obtained with N. tabacum, DMTU and cPTIO significantly reduced TMV- and SA-induced NbRDR1m and AtRDR1 relative expression (Figure 3), whereas exogenous H2O2 and SNP application resulted in increased NbRDR1m and AtRDR1 transcript levels.

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Figure 3. Effects of TMV infection, SA, and different chemical pretreatment on transcripts of NbRDR1m and AtRDR1.

A and C. Changes in TMV-induced NbRDR1m (A) and AtRDR1 (C) transcript levels over time in non-inoculated upper leaves induced by scavengers of hydrogen peroxide (H2O2) and nitric oxide (NO). The non-inoculated upper leaves were subjected to DMTU (H2O2 scavenger) or cPTIO (NO scavenger) pretreatment 12 hours before TMV inoculation. B and D. Changes in SA-induced NbRDR1m (B) and AtRDR1 (D) transcript levels over time induced by exogenous H2O2, NO, and their scavengers. The results are expressed as the mean ± SD, n  =  4.

https://doi.org/10.1371/journal.pone.0076090.g003

TMV-induced H2O2 synthesis and associated RDR1 expression are reduced by NO elimination and reversed by exogenous NO application, but H2O2 does not influence TMV-induced NO evolution

To study the relationship between H2O2 and NO in RDR1 induction under TMV inoculation conditions, we treated the non-inoculated upper leaves of N. tabacum plants with DMTU and cPTIO, followed by inoculation with TMV inoculum on the lower leaves. At 2 dpi, a confocal laser scanning microscope (CLSM) was used to detect changes in H2O2 in the leaves. There was an increase in the green fluorescence H2O2 signal in TMV-inoculated plants compared to mock-inoculated plants (Figure 4A). The TMV-induced H2O2 accumulation was greatly suppressed by its scavenger DMTU. Interestingly, the NO scavenger cPTIO also completely inhibited H2O2 accumulation (Figure 4A). In contrast, NO quantification using Griess reagent showed that NO generation from TMV attack was only http://dict.cnki.net/dict_result.aspx?searchword=%e5%bd%bb%e5%ba%95&tjType=sentence&style=&t=thoroughlyblocked by cPTIO and not by H2O2 elimination (Figure 4B).

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Figure 4. Effects of scavengers of H2O2 and NO on TMV-induced H2O2 and NO accumulation in Nicotiana tabacum

. The non-inoculated upper leaves were subjected to DMTU (H2O2 scavenger) or cPTIO (NO scavenger) pretreatment 12 hours before TMV inoculation. A. Representative H2O2 visualization using H2DCF-DA and a confocal microscope in which H2O2 exhibited green fluorescence. All images are at the same magnification and the white scale bar indicates 75 µm. B. NO assayed with Griess reagent. The results are expressed as the mean ± SD, n = 4. The letters indicate significant differences between the treatments (P<0.05).

https://doi.org/10.1371/journal.pone.0076090.g004

To establish a more direct relationship between H2O2 and NO in TMV-induced RDR1 expression, we collected the Arabidopsis H2O2 synthetic enzymatic mutant atrbohD, the mutant atnoa1 that is indirectly impaired in NO synthesis, and the wild-type Columbia (Col-0) ecotype. We pretreated the plants with exogenous H2O2 and SNP on the non-inoculated upper leaves followed by TMV inoculation on the lower leaves. The in planta concentration of H2O2 and NO as well as RDR1 transcript levels in the non-inoculated upper leaves were analyzed at 2 dpi. In wild type Col-0 plants, pretreatment with either H2O2 or the NO donor SNP resulted in a significant increase in H2O2 histochemical staining compared to untreated plants (Figure 5), whereas the NO concentration was elevated by SNP pretreatment but not by exogenous H2O2. As expected, TMV-induced H2O2 accumulation was abolished in atrbohD http://www.arabidopsis.org/servlets/TairObject?id=132871&type=locusplants and was increased when exogenous H2O2 was applied. However, this effect was not observed in SNP-pretreated plants. At the same time, the NO concentration in atrbohD http://www.arabidopsis.org/servlets/TairObject?id=132871&type=locusplants was comparable to that in Col-0 plants, and no significant increase was observed after exogenous H2O2 treatment. In contrast, the H2O2 concentration in atnoa1 http://www.arabidopsis.org/servlets/TairObject?id=40002&type=locusplants was greatly reduced compared to that in Col-0 plants. However, the H2O2 concentration increased when either exogenous H2O2 or SNP was applied. The impaired NO accumulation in atnoa1 plants recovered to levels observed in the Col-0 control level after SNP application but showed no significant changes in response to exogenous H2O2 treatment. Furthermore, the changes of in planta H2O2 and NO concentration were closely related to RDR1 induction. In contrast to the further increase in TMV-induced AtRDR1 expression by exogenous H2O2 and SNP in Col-0 plants, AtRDR1 transcript levels in the leaves of atrbohD http://www.arabidopsis.org/servlets/TairObject?id=132871&type=locusand atnoa1 http://www.arabidopsis.org/servlets/TairObject?id=40002&type=locusplants showed a decrease of approximately 70% compared to the levels in Col-0 leaves. The lower AtRDR1 transcript levels in these mutants were greatly increased with exogenous H2O2 treatment, whereas SNP pretreatment only increased AtRDR1 transcript levels in atnoa1http://www.arabidopsis.org/servlets/TairObject?id=40002&type=locusplants and not atrbohD http://www.arabidopsis.org/servlets/TairObject?id=132871&type=locus plants.

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Figure 5. Relationship of H2O2, NO, and AtRDR1 transcripts in wild-type and mutant Arabidopsis plants.

Effects of TMV inoculation and exogenous application of hydrogen peroxide (H2O2) and nitric oxide (NO) on endogenous H2O2 and NO concentrations and on transcript levels of RNA-dependent RNA Polymerase 1 in non-inoculated upper leaves of wild-type and mutant Arabidopsis plants (AtRDR1). The non-inoculated upper leaves were pretreated with H2O2 or SNP 12 hours before TMV inoculation, and leaf samples were collected at 2 days post inoculation. A. H2O2 detection using DAB histochemical staining. B. NO concentration. C. AtRDR1 transcripts. The results are expressed as the mean ± SD, n  =  4. The letters indicate significant differences between the treatments (P< 0.05).

https://doi.org/10.1371/journal.pone.0076090.g005

Function of H2O2- and NO-associated RDR1 induction in the basal antiviral defense

We then tested whether the H2O2- and NO-associated RDR1 induction functions in the basal defense against TMV inoculation. Exogenous H2O2 and NO chemical modulators were applied on the non-inoculated upper leaves of N. tabacum plants 12 hours prior to TMV inoculation. The symptoms of systemic leaves were then photographed at 27 dpi (Figure 6A). The TMV infection clearly resulted in visible damage with necrotic lesions and crinkling of leaves. Pretreatment with H2O2 or SNP significantly decreased TMV susceptibility, and these leaves did not show any visible systemic symptoms. In contrast, DMTU or cPTIO pretreatment substantially increased the TMV susceptibility and led to the development of enhanced necrotic lesions and crinkling of leaves. Moreover, the levels of TMV-CP mRNA in non-inoculated upper leaves analyzed by RT-PCR at 7 dpi correlated highly with the observed symptoms (Figure 6B). Compared to plants that received the TMV challenge alone, H2O2 and SNP pretreatment reduced TMV-CP mRNA by 75% and 98%, respectively, whereas DMTU and cPTIO pretreatment markedly increased these levels.

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Figure 6. Effects of chemical pretreatments on TMV infection in Nicotiana tabacum plants

. Effects of different chemical pretreatments and TMV infection on TMV symptom development and TMV-CP transcription in the non-inoculated upper leaves of Nicotiana tabacum plants. The non-inoculated upper leaves were subjected to application of hydrogen peroxide (H2O2) or nitric oxide (NO) chemical modulators 12 hours before TMV inoculation. A. Symptoms were photographed at 27 days after inoculation. B. TMV-CP transcript levels were analyzed by real-time reverse-transcription polymerase chain reaction at 7 days after inoculation. The results are expressed as the mean ± SD, n  =  4. The letters indicate significant differences between the treatments (P< 0.05).

https://doi.org/10.1371/journal.pone.0076090.g006

To test whether H2O2- and NO-associated RDR1 has a general function in different species, we also applied exogenous H2O2 and NO to transgenic N. benthamiana plants transformed with the SA-inducible MtRDR1 gene from M. truncatula or an empty vector (EV) as a control. A chlorophyll fluorescence imaging method was used to analyze the response of the maximum photochemical efficiency of PSII in the dark-adapted state (Fv/Fm) to the H2O2 and NO chemical modulators in TMV-infected N. benthamiana plants. At 7 dpi, TMV inoculation resulted in a significantly lower Fv/Fm in the central leaves of EV control plants relative to their MtRDR1 transgenic counterpart (Figure 7A), which indicated that TMV damaged EV plants more severely than it damaged MtRDR1-transformed plants. Furthermore, there were no evident effects of H2O2 or NO on TMV susceptibility in EV and transgenic plants. TMV-CP transcripts assayed at the same time showed the same pattern as the chlorophyll fluorescence imaging (Figure 7B), which showed that without the significant effects of H2O2 or SNP, the TMV-CP mRNA level in non-inoculated upper leaves of EV plants was approximately 2-fold higher than that in leaves of MtRDR1 transformed plant.

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Figure 7. Effects of exogenous H2O2 and NO on TMV infection in Nicotiana benthamiana plants.

Effects of exogenous application of hydrogen peroxide (H2O2) and nitric oxide (NO) and TMV inoculation on the maximum photochemical efficiency of photo system II (Fv/Fm) and TMV-CP gene transcription in the non-inoculated upper leaves of empty vector (EV)-transformed and MtRDR1-transformed Nicotiana benthamiana plants. The non-inoculated upper leaves were pretreated with hydrogen peroxide (H2O2) or nitric oxide (NO) 12 hours before TMV inoculation. A. Chlorophyll fluorescence images of the maximum quantum yield of PSII (Fv/Fm) at 7 days post inoculation. The color gradient scale at the top indicates the magnitude of the fluorescence signal represented by each color. B. TMV-CP transcript levels analyzed by real-time reverse-transcription polymerase chain reaction at 7 days post inoculation. The results are expressed as the mean ± SD, n  =  4. The letters indicate significant differences between the treatments (P< 0.05).

https://doi.org/10.1371/journal.pone.0076090.g007

Wild-type Col-0 Arabidopsis was also pretreated with H2O2 and SNP followed by inoculation with crucifer tobamovirus, which is related to TMV (TMV-cg, a strain of Turnipvein-clearing virus). As shown in Figure 8, in non-inoculated upper leaves, H2O2 and SNP pretreatment greatly reduced TMV-cg mRNA levels at 4 and 6 dpi compared to untreated plants that received TMV inoculation alone.

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Figure 8. Effects of exogenous H2O2 and NO on TMV infection in Arabidopsis plants.

Effects of exogenous application of hydrogen peroxide (H2O2) and nitric oxide (NO) and TMV inoculation on TMV-cg transcription in non-inoculated upper leaves of Arabidopsis plants. The non-inoculated upper leaves were pretreated with H2O2 or SNP 12 hours before TMV inoculation. TMV-cg transcript levels were analyzed by real-time reverse-transcription polymerase chain reaction at 4 and 6 days post inoculation (dpi). The results are expressed as the mean ± SD, n  =  4. The letters indicate significant differences between the treatments (P< 0.05).

https://doi.org/10.1371/journal.pone.0076090.g008

Discussion

RDR1, a key enzyme in viral resistance, is known to be induced by viruses and SA [6], [8], [27], [28]. In the present study, we showed that RDR1 transcript activation was accompanied by H2O2 and NO accumulation after TMV challenge in N. tabacum, N. benthamiana, and Arabidopsis plants. Chemical scavenging of H2O2 and NO partly blocked TMV- and SA-induced RDR1 expression and increased TMV susceptibility. Furthermore, TMV-induced H2O2 synthesis and the associated RDR1 expression were reduced by NO elimination and reversed by exogenous NO application, but H2O2 removal had no effect on TMV-induced NO evolution. Therefore, our results indicate that H2O2 may act downstream of NO to up-regulate RDR1, which contributes to the restriction of systemic infection and virus accumulation. This study provides initial insights into the signaling mechanisms underpinning virus- and SA-induced RDR1 activation in host plants.

Involvement of H2O2 and NO in TMV- and SA-induced RDR1 induction

H2O2 and NO are secondary molecules induced by pathogen invasion to activate defense expression and mediate SA defense responses in processes called oxidative bursting and NO bursting, respectively [18], [20], [29]. In this study, we observed a rapid and significant accumulation of H2O2 and NO in N. tabacum plants (Figure 1). Interestingly, this TMV-induced generation of NO and H2O2 was followed by NtRDR1 transcript induction in short succession (Figure 1). These results indicate a possible connection between TMV-induced H2O2 and NO production and the induction of RDR1 expression. Furthermore, in agreement with a previous study on N. glutinosa plants where exogenous H2O2 induced transcript expression of NgRDR1 [13], we observed that exogenous H2O2 and NO induced NtRDR1 transcript expression in N. tabacum, and TMV-induced NtRDR1 expression was strongly suppressed by DMTU and cPTIO application. As the important plant signaling molecule involved in defense responses to pathogen attack, SA has also been found to elicit plant RDR1 expression [6]–[8], and in this study, the expression of SA-induced NtRDR1 was also compromised by H2O2 and NO scavengers. These results highlight the indispensable roles of H2O2 and NO in TMV- and SA-induced RDR1 induction in N. tabacum plants. However, NtRDR1 induction was not fully abolished by DMTU or cPTIO treatment (Figure 2); thus, additional unknown H2O2- and NO-independent pathways in the RDR1 signaling pathway may also exist.

The characteristics of H2O2 and NO involvement in TMV- and SA-induced NtRDR1 expression in N. tabacum are similar in N. benthamiana and Arabidopsis plants (Figure 3), although the magnitude of induction/suppression by exogenous H2O2, NO, and their scavengers differed among species. In Arabidopsis, AtRDR1 expression in atrbohD and atnoa1 mutants was only 20% of the expression in Col-0 plants in response to TMV challenge (Figure 5C), which further emphasizes the role of H2O2 and NO in RDR1 induction. Collectively, H2O2 and NO mediate TMV- and SA-induced RDR1 activation in all three model host plants, and this may be a general mechanism in all plants.

Relationship between H2O2 and NO in the RDR1 induction signaling pathway

In N. tabacum, the NO scavenger cPTIO completely inhibited H2O2 accumulation by TMV inoculation (Figure 4). Conversely, H2O2 elimination did not affect TMV-induced NO generation. These observations suggest a possible crosstalk between H2O2 and NO, and NO may act upstream of H2O2 in modulating TMV-induced RDR1 activation. This possibility was also supported by time-course data showing that NO generation preceded H2O2 induction in N. tabacum plants in response to TMV infection (Figure 1). This signaling pathway also seems to exist in Arabidopsis plants, as H2O2 accumulation was sensitive to NO, whereas NO generation was not affected by H2O2 under TMV-inoculated conditions using http://www.arabidopsis.org/servlets/TairObject?id=40002&type=locusatrbohD and atnoa1 http://www.arabidopsis.org/servlets/TairObject?id=132871&type=locusmutants (Figure 5). Furthermore, in planta H2O2 concentration changes were closely related to AtRDR1 induction, and lower AtRDR1 transcript levels in both atrbohD http://www.arabidopsis.org/servlets/TairObject?id=132871&type=locusand atnoa1 mutants were greatly increased by exogenous H2O2 treatment. In contrast, SNP pretreatment only increased AtRDR1 transcript levels in atnoa1http://www.arabidopsis.org/servlets/TairObject?id=40002&type=locus plants and not in atrbohD plants. Therefore, TMV-induced NO likely triggers H2O2 accumulation, which plays a role in inducing plant RDR1 activation.

Exactly how NO cooperates with H2O2 to induce RDR1 remains unknown. NO inhibits CAT and ascorbate peroxidase (APX) activity to increase the level of H2O2 [30]. It has been proposed that periodic H2O2 accumulation can be attributed to the inhibition of CAT and APX by a strong early NO burst, which is accompanied by a following wave of secondary NO generation to enhance defense responses to pathogen stress [31]. H2O2 may function as a downstream amplifier to spread the signal and induce the defense response. Thus far, it is not known whether a direct interaction between H2O2 and RDR1 occurs. Alternatively, H2O2 may activate downstream signal transduction such as the mitogen-activated protein kinase (MAPK) cascade and transcription factors to modify RDR1 expression [32], [33]. Nevertheless, H2O2 and NO have been reported to interact in a variety of patterns, and although some studies have shown that NO treatment can induce the production of H2O2 [22], other studies have shown opposite results. For example, in cucumber plants, NO acted downstream of H2O2 in brassinosteroid-induced abiotic stress tolerance [21]. Thus, the relationship between H2O2 and NO in signal transduction may be more complicated than the simple linear manner in which H2O2 induces NO or vice versa [21]. Thus, the relationship between H2O2 and NO in signal transduction may be more complicated than a simple linear relationship in which H2O2 induces NO or vice versa [21]. In addition, H2O2 and NO may crosstalk differently under different stresses, species, and plant status. Further experiments are still needed to fully establish the relationship between H2O2 and NO and its possible function in RDR1 induction in different plants.

H2O2- and NO-associated RDR1 induction functions in the basal defense against TMV infection

In this study, H2O2 and NO assisted in defending against viral attack in N. tabacum and Arabidopsis, and DMTU and cPTIO significantly increased TMV susceptibility in N. tabacum (Figures 6 and 8). These results imply that H2O2- and NO-associated RDR1 induction may play a key role in the anti-TMV defense. Furthermore, TMV attack damaged EV plants more severely than it damaged MtRDR1-transformed N. benthamiana plants (Figure 7), and the natural loss of RDR1 function in N. benthamiana did not allow H2O2 and NO to diminish TMV infection in these plants, even though NbRDR1m was induced by exogenous H2O2 and NO. There were also no evident effects of H2O2 and NO on TMV susceptibility in transgenic N. benthamiana plants, as the MtRDR1 gene was constitutively expressed under the 35S promoter in the transgenic plants (Figure 7). These results highlight the primary function of H2O2- and NO-associated RDR1 in the antiviral mechanism, which is contingent on functional RDR1. RDR1 was found to be involved in separate but overlapping viral resistance and post-transcriptional gene silencing mechanisms in plants [34]. A recent study using siRNA deep sequencing revealed that rdr1-1 plants contained dramatically fewer viral siRNAs in a short time after the inoculation of TMV-cg, which clearly suggests that RDR1 contributes to the biosynthesis of TMV siRNAs [11]. AtRDR1-dependent production of CMV siRNA has also been observed when the silencing suppressor of CMV is absent [10]. In the present study, H2O2 and NO pretreatment induced a higher level of the initial RDR1 transcript, which may have induced stronger RNA silencing and contributed to the basal antiviral defense in N. tabacum and Arabidopsis. Furthermore, N. benthamiana naturally loses RDR1 function to maintain a higher level of RDR6-dependent antiviral defense [34]. However, in this study, MtRDR1-transformed plants showed a higher constant resistance to TMV than EV N. benthamiana plants (Figure 7), and this compensation mechanism may not be sufficiently effective to compensate for the loss of RDR1.

In summary, we have addressed the role of H2O2, NO, and the relationship between them in TMV-induced RDR1 activation and the antiviral defense response in the model host plants N. tabacum, N. benthamiana, and Arabidopsis. The results strongly imply that TMV-induced NO accumulation functions upstream of H2O2 to mediate RDR1 induction, which plays a critical role in strengthening RNA silencing to restrict virus systemic infection and accumulation (Figure 9). This signaling pathway may be a general pathway common to plants.

[Figure omitted. See PDF.]

Figure 9. A hypothetical model for the role of H2O2 and NO in the induction of RDR1.

Black dashed lines indicate positive interactions; red solid line indicates negative regulation.

https://doi.org/10.1371/journal.pone.0076090.g009

Materials and Methods

Plants, viruses, and chemical treatments

Transgenic homozygous N. benthamiana plants with the binary vector pCAMBIA 2300 containing the RDR1 gene from Medicago truncatula (R15-1) or empty vector (EV) not containing the RDR1 transgene (V16-2) were generously provided by Richard S. Nelson (Samuel Robert Noble Foundation, Ardmore, OK). The leaf disc method was used for Agrobacterium tumefaciens-mediated transformation of N. benthamiana harboring the 35S: MtRDR1 transgene, which was detailed in a previous study [6]. These N. benthamiana and N. tabacum plants were cultivated in a controlled growth chamber at 24°C with a 16-hour photoperiod and 50% humidity. For Arabidopsis, seeds of wild-type ecotype Col-0 and mutants atrbohD (AT5G47910) and atnoa1 (AT3G47450) were sown on autoclaved soil and vernalized at 4°C for 3 days. Then, the plants were germinated and grown at 23 to 25°C with 16 hours of light and 8 hours of darkness. Approximately 3 to 4 weeks after germination, the N. tabacum, N. benthamiana, and Arabidopsis plants were chemically treated in the experiments. The plants leaves were sprayed with water or fresh solutions of 5 mM H2O2, 0.2 mM SNP, 5 mM DMTU, 0.2 mM cPTIO, 2 mM SA, 5 mM DMTU plus 2 mM SA, or 0.2 mM cPTIO plus 2 mM SA. In the experiments with a combination treatment of chemical modulator and TMV inoculation, chemical pretreatments were only applied on the upper leaves, and the lower 2–3 fully developed leaves were used for inoculation with TMV 12 hours after chemical pretreatment. Specifically, leaves of N. tabacum and N. benthamiana were inoculated with TMV (U1 strain) suspensions using cotton tips on adaxial surfaces previously dusted with carborundum powder. The viral inocula (prepared in 5 mM sodium phosphate, pH 7.5) of N. tabacum and N. benthamiana were applied at concentrations of 10 µg/mL and 1 µg/mL, respectively. The inoculation of Arabidopsis plants was performed using a fine sable paintbrush to apply a suspension of the TMV-cg strain at 5 µg/mL in 5 mM sodium phosphate, pH 7.5 onto leaves sprinkled with carborundum. Mock inoculations were performed with phosphate buffer only. Plants were randomly assigned to the treatments, each with four replicates. The upper, newly developed systemic leaves were collected at different time points as indicated for physiological and molecular measurements. One biological sample was obtained by pooling the leaves from three to four plants, and four biological repeats were analyzed for each treatment. The experiments were independently performed three times.

H2O2 and NO detection and quantification

H2O2 detection using a CLSM system was conducted as described in previous studies with minor modifications [35]. Leaf sections (0.5 by 0.5 cm) were placed into a loading buffer with 50 mM Tris-KCl (pH 7.2) containing 100 µM of H2DCF-DA. Before further experiments were performed, the peels were preincubated in the dark for 1 hour and immediately examined under a CLSM system (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany). The sections were excited using the 488-nm line of an argon laser, and dye emissions were recorded using a 505- to 530-nm band-pass filter.

H2O2 in situ detection was performed using a 3,3-diaminobenzidine (DAB) staining method [36]. H2O2 quantification was performed using a spectrophotometric assay at OD412 [37], [38]. The minor modifications to these experiments have been previously described [39]. The NO concentration was determined using Griess reagent (Sigma-Aldrich) [40].

RNA extraction and transcript level estimation by real-time quantitative PCR

One biological sample was obtained by pooling the leaves from five plants, and four biological repeats were analyzed. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s specifications. Genomic DNA was removed using a purifying column. Total RNA (1 µg) was reverse-transcribed using 0.5 mg of oligo(dT)12–18 (Invitrogen, Carlsbad, CA, USA) and 200 units of Superscript II (Invitrogen) following the manufacturer’s instruction. Gene-specific primers were designed based on the mRNA sequence for analyzing transcript levels of RDR1 in each species. TMV-specific primers were also designed according to the sequence encoding the TMV-CP (U1 strain) and TMV genome (cg strain). The primers used are listed in Table S1.

Quantitative real time-PCR was performed using an iCycler IQTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). PCR was performed using SYBR Green PCR Master Mix. PCR cycling conditions were as follows: 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 30 seconds. Fluorescent signals were collected during the 58°C step. To verify the amplification of a single product, a dissociation curve was generated at the end of the PCR cycles using software provided with the iCycler iQTM real-time PCR detection system. The NtActin, NbActin, and AtActin genes were used as internal controls, relative gene expression was calculated [41].

Chlorophyll fluorescence imaging

Chlorophyll fluorescence was determined using PAM imaging (IMAG-MAXI, Heinz Walz, Effeltrich, Germany). Plants were placed in darkness for 30 s to measure Fv/Fm. Minimal fluorescence (F0) was measured during the weak measuring pulses, and maximal fluorescence (Fm) was measured by a 0.8-second pulse of light at approximately 4000 µmol/(m2s).

Statistical methods

Four independent replicates were applied in each determination. The data were statistically analyzed using analysis of variance and tested for significance (P<0.05) using Tukey’s test.

Supporting Information

[Figure omitted. See PDF.]

Table S1.

Primers used for real time reverse-transcription polymerase chain reaction assays. F: forward; R: reverse.

https://doi.org/10.1371/journal.pone.0076090.s001

(DOCX)

Acknowledgments

We thank Dr. Richard S. Nelson (Samuel Robert Noble Foundation, Ardmore, OK) for providing MtRDR1-expressing N. benthamiana and the transformed control line.

Author Contributions

Conceived and designed the experiments: KS ZXC JQY. Performed the experiments: YWKL ZHS XL GQZ. Analyzed the data: YWKL KS. Contributed reagents/materials/analysis tools: XJX YHZ. Wrote the paper: YWKL KS.

Citation: Liao Y-W-K, Sun Z-H, Zhou Y-H, Shi K, Li X, Zhang G-Q, et al. (2013) The Role of Hydrogen Peroxide and Nitric Oxide in the Induction of Plant-Encoded RNA-Dependent RNA Polymerase 1 in the Basal Defense against Tobacco Mosaic Virus. PLoS ONE 8(9): e76090. https://doi.org/10.1371/journal.pone.0076090

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