NbHB1, Nicotiana benthamiana homeobox 1, is a jasmonic acid-dependent positive regulator of pathogen-induced plant cell death


  • Joonseon Yoon,

    1. Department of Biological Sciences, KAIST, Daejeon, 305-701, Republic of Korea
    2. Department of Plant Science, Plant Genomics and Breeding Institute and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea
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  • Won-Il Chung,

    1. Department of Biological Sciences, KAIST, Daejeon, 305-701, Republic of Korea
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  • Doil Choi

    1. Department of Plant Science, Plant Genomics and Breeding Institute and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea
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Authors for correspondence:
Doil Choi
Tel: +82 2 880 4568
Email: doil@snu.ac.kr
Won-Il Chung
Tel:+82 42 869 2624
Email: wichung@kaist.ac.kr


  • • Induction of cell death is an important component of plant defense against pathogens. There have been many reports on the role of phytohormones in pathogen-induced cell death, but jasmonic acid (JA) has not been implicated as a regulator of the response. Here, we report the function of NbHB1, Nicotiana benthamiana homeobox1, in pathogen-induced cell death in connection with JA signaling.
  • • Involvement of NbHB1 in cell death was analysed by gain- and loss-of-function studies using Agrobacterium-mediated transient overexpression and virus-induced gene silencing, respectively. Expression of NbHB1 following pathogen inoculations and various treatments was monitored by reverse transcription polymerase chain reaction.
  • • Transcript levels of NbHB1 were upregulated by infection with virulent and avirulent bacterial pathogens. Ectopic expression of NbHB1 accelerated cell death following treatment with darkness, methyl jasmonate, or pathogen inoculation. Conversely, when NbHB1 was silenced, pathogen-induced cell death was delayed. NbHB1-induced cell death was also delayed by silencing of NbCOI1, indicating a requirement for JA-mediated signaling. Overexpression of the domain-deleted proteins of NbHB1 revealed that the homeodomain, leucine zipper, and part of the variable N-terminal region were necessary for NbHB1 functionality.
  • • These results strongly suggest the role of NbHB1 in pathogen-induced plant cell death via the JA-mediated signaling pathway.


As sessile organisms, plants experience diverse environmental stresses such as drought, salt stress, freezing, and pathogen attack. As responses to those stresses, plants mount defense mechanisms to protect their growth (Hutcheson, 1998; Zhu, 2002). Pathogens actively attack plants, causing developmental defects, crop loss or even death of the entire plant body. To defend against pathogens, plants trigger signaling cascades to induce various defense responses, including the hypersensitive response (HR), a type of programmed cell death in plants (Hammond-Kosack & Jones, 1996). Through HR, plants restrict pathogens to the infected region, safeguarding the remaining plant body.

Many signaling components that regulate HR-like cell death have been identified. Mutants such as ‘accelerated cell death’ (Greenberg et al., 1994) and ‘lesions simulating disease resistance’ (Morel & Dangl, 1997) exhibit spontaneous cell death spreading out from the primary lesion. Some plant hormones are known to be involved in the regulation of cell death responses. Salicylic acid (SA), ethylene (ET), and jasmonic acid (JA) each have cooperative or antagonistic functions in different kinds of plant cell death (Rao et al., 2000; Overmyer et al., 2003; Tuominen et al., 2004). Among them, JA has been described as an antagonist of O3-induced cell death, while SA and ET are positive regulators (Rao et al., 2000; Tuominen et al., 2004). Recent reports indicate, however, that JA can also act as a positive regulator of plant cell death. Treatment with JA induces precocious senescence, and JA concentration increases during senescence in Arabidopsis (He et al., 2002). In coronatine insensitive 1 (coi1), a JA-insensitive mutant, the effect of JA on senescence is abolished, indicating that JA-induced senescence is genetically programmed (He et al., 2002). The JA signaling pathway is also known to be required for cell death induced by a fungal toxin, fumonisin B1 (Asai et al., 2000). In addition, JA accumulation following challenge of tobacco plants with an avirulent bacterial pathogen can synergistically induce cell death together with SA (Mur et al., 2006), suggesting the involvement of JA in pathogen-mediated cell death. Most recently, Zhang & Xing (2008) found that methyl jasmonate (MeJA) alone could induce cell death in Arabidopsis, and that cell death was followed by production of reactive oxygen species (ROS) and alterations in mitochondrial dynamics. Treatment of Taxus cell suspension cultures with MeJA also promotes rapid production of H2O2 with biphasic induction of ROS, a typical feature of elicitor-induced plant cell cultures (Wang & Wu, 2005). Together, these previous reports imply that JA may play a role in stress-induced plant cell death. To date, however, none of the downstream genetic elements involved in JA-mediated plant cell death have been identified.

Homeodomain-leucine zippers (HD-Zips) are transcription factors known to function in the regulation of developmental processes and adaptation to environmental stresses in plants (Ariel et al., 2007). The HD-Zip domain in the protein is essential for its function, binding to cis-elements and interacting with other HD-Zip proteins. The HD-Zip family of proteins are classified into four subfamilies (I–IV) by DNA-binding specificities, gene structures, additional conserved motifs, and their functions (Henriksson et al., 2005; Ariel et al., 2007). The genes in subfamily III and IV such as PHAVOLUTA and PHABULOSA in Arabidopsis are known to act in plant growth and development (McConnell et al., 2001; Izhaki & Bowman, 2007). Subfamily I and II genes participate in adaptation to environmental stresses such as drought, light, and salt (Ariel et al., 2007). H52, a member of the HD-Zip family in tomato, is involved in limiting cell death during plant–pathogen interactions (Mayda et al., 1999). When H52-silenced tomatoes are inoculated with a pathogen, cell death is induced and spreads to the uninfected region.

In this study, we isolated NbHB1, a homeobox (HB) gene from Nicotiana benthamiana, and investigated its role in pathogen-induced cell death. Through gain- and loss-of-function studies, NbHB1 was revealed to function as a positive regulator of pathogen-induced cell death. In addition, silencing of NbCOI1 delayed NbHB1-induced cell death. Together, these results strongly suggest that NbHB1 positively regulates pathogen-induced cell death, possibly via the JA-mediated signal transduction pathway.

Materials and Methods

Plant materials and treatments

Nicotiana benthamiana L. seeds were sown and grown in pots and maintained under a 16-h photoperiod at 26°C for 5–6 wk. For the induction of cell death after darkness treatment, NbHB1 was transiently overexpressed in the right half of fully expanded leaves. After 1 d of transient overexpression (TOE), they were detached, wrapped with aluminum foil, and kept in 26°C for 3 d (Pruzinskáet al., 2005). For phytohormone treatment, 125 µm SA or 25 µm MeJA was infiltrated by a needleless syringe in the fully expanded leaves during early daytime to harvest samples in light condition. Samples were then harvested for the RNA extraction 0, 12 or 24 h after treatment.

Measurement of ion leakage

For measurement of ion leakage, the assay was carried out as described in Mittler et al. (1999). After incubation of leaf discs (1 cm diameter) in 5 ml of distilled water for 3 h at room temperature, electrical conductivity was measured using an conductivity meter (model EC-470L; Istek, Seoul, Korea). To release total electrolytes from leaf discs, samples were then autoclaved, cooled to room temperature, and measured with a conductivity meter. Ion leakage was expressed as per cent leakage.

Staining with Trypan blue

Trypan blue (TB) staining was performed as described by Koch & Slusarenko (1990). Leaf discs (1 cm diameter) were infiltrated with lactophenol–TB (10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, 10 mg of TB, dissolved in 10 ml of distilled water) under vacuum. They were then boiled for 30 s and incubated overnight in covered box. And they were decolorized in chloral hydrate (2.5 g of chloral hydrate dissolved in 1 ml of distilled water) for at least 30 min, and mounted in chloral hydrate.

Staining H2O2 with 3,3′-diaminobenzidine (DAB)

Hydrogen peroxide accumulation was visualized with DAB as described in Thordal-Christensen et al. (1997) with some modifications. Leaf discs excised from leaves with a cork borer (1 cm diameter) were placed in 12-well plates containing DAB solution (1 mg ml−1). The DAB solution was infiltrated into leaf discs under vacuum for 15 min, and the samples were left under light for 30 min. They were then moved to an ethanol–lactophenol mixture and boiled for 1 min. Samples were rinsed with 50% ethanol twice and DAB staining was assessed visually.

Quantification of chlorophyll

The amounts of chlorophyll were measured according to Porra et al. (1989) with some modifications. Three leaf discs were collected by punching the leaves with a cork borer (1 cm diameter), and placed in conical tubes containing 5 ml of 80% acetone. Samples were stored overnight in the dark with shaking at room temperature. The concentration was determined by measuring the extinction of the extract at 663.6 nm (E663.6) for chlorophyll a and at 646.6 nm (E646.6) for chlorophyll b with a spectrophotometer (model DU 730; Beckman Coulter, Fullerton, CA, USA) and inserting these values into the equation, 19.54E646.6 + 8.29E663.6.

Plasmid construction

To construct TOE vectors, the PCR-amplified products of NbHB1, domain-deleted NbHB1, smGFP-NbHB1 and smGFP were ligated into pMBP-1 digested with BamHI and SacI. pTRV2-derived vectors were constructed for virus-induced gene silencing (VIGS). To clone the inserts, the first-strand cDNA of N. benthamiana was synthesized using an oligo (dT) primer, and cDNA fragments were amplified with gene-specific primers. Both PCR products and pTRV2 were digested and ligated together. Proper ligation of all cloned fragments was confirmed by DNA sequencing.

TOE and VIGS in N. benthamiana

For TOE, constructs in pMBP-1 were transformed into Agrobacterium tumefaciens strain GV2260 by freeze-thaw method (An et al., 1988). Agrobacterium tumefaciens containing each construct was grown in yeast extract peptone (YEP) medium overnight at 30°C with shaking, harvested by centrifugation and resuspended in 10 mm MgCl2. After the induction of virulence with 200 µm acetosyringone at 20°C by shaking for 2–4 h, cells were pressure-infiltrated with a needleless syringe into fully expanded leaves of N. benthamiana plants (Bendahmane et al., 2000). After 1 d of TOE, leaves were used for further experiments such as the treatment of pathogens, phytohormones or darkness.

For VIGS, pTRV2-derived vectors were transformed into A. tumefaciens strain GV2260. Agrobacterium cultures containing pTRV1 or pTRV2 constructs were grown, harvested and resuspended in 10 mm MgCl2. They were then virulence-induced by adding 200 µm acetosyringone with shaking, mixed in a 1 : 1 ratio, and infiltrated into the leaves of N. benthamiana when the plants were at the four-leaf stage (Liu et al., 2002). After 3 wk of Agro-inoculation, the silenced plants were used for further experiments.

Subcellular localization of smGFP-NbHB1 or smGFP

The pMBP-1:smGFP-NbHB1 or pMBP-1:smGFP construct was transformed into A. tumefaciens strain GV2260 using a freeze–thaw method (An et al., 1988). The transformed cells were infiltrated into N. benthamiana leaves for TOE of each protein. Two days after inoculation, the abaxial epidermal cell layer was peeled off and observed using a fluorescence microscope (Axiophot; Zeiss). For confirmation of subcellular localization of NbHB1 protein, 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei.

Analysis of transcript levels by reverse transcription polymerase chain reaction (RT-PCR)

For semiquantitative RT-PCR, total RNA was extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA) following the manufacturer's protocol. For N. benthamiana genes, first-strand cDNA was synthesized using an oligo (dT) primer and 1 µg of total RNA with master premix (ELPIS, Daejeon, Korea), followed by RT-PCR with gene-specific primers. Quantitative PCRs were carried out using a MJ-Cromo 4 apparatus (Bio-Rad) in a 20 µl final volume containing 1 µl EvaGreen (20×), 10 pmol of each primer, 2 µl reaction buffer (10×), 0.5 µl dNTP mixture (10 mm each), 2 µl of the reverse transcription reaction and 0.2 µl h-Taq DNA polymerase (Solgent, Daejeon, Korea). Fluorescence was measured at 72°C during 40 cycles. RNA levels were normalized to NbACTIN RNA levels. The PCR products were electrophoresed in agarose gels to quantify the amplified DNA. Primer sequences for RT-PCR and for each gene are listed in the Supporting Information Table S1 and Table S2, respectively.

Pathogen inoculation

For inoculation of Pseudomonas syringae pv. tomato (Pst) T1, Pseudomonas syringae (Ps.) pv. tabaci (avrPto) and Pseudomonas tabaci in N. benthamiana plants, bacterial pathogens were cultured in YEP medium overnight at 30°C with shaking at 200 rpm, centrifuged at 3577 g for 10 min, and resuspended in 10 mm MgCl2. For the analysis of NbHB1 expression during plant–pathogen interaction, P. tabaci and P. tabaci (avrPto) were diluted to 0.5 of OD600 value. For the analysis of pathogen-induced cell death, Pst T1 or P. tabaci was diluted serially. The bacterial suspensions were pressure-infiltrated into fully expanded N. benthamiana leaves with a needleless syringe (Lee et al., 2004).


Isolation of NbHB1

To identify genes involved in plant–microbe interactions, we referred to a previous microarray analysis carried out with pepper cDNA after infection of pepper plants with a nonhost pathogen, Xanthomonas axonopodis pv. glycines 8ra (Lee et al., 2004). Among the genes that showed altered expression in the pepper microarray experiments, we identified an HD-Zip family gene, Capsicum annuum homeobox 1 (CaHB1). In N. benthamiana, another solanaceous plant and a well-known model plant for pathogen–plant interaction, a CaHB1 ortholog was cloned and designated as NbHB1 (Fig. S7). NbHB1 encoded a 263 amino acid protein and belonged to subfamily II in the HD-Zip family, with an N-terminal variable region and C-terminal conserved CPSCE motif (Fig. 1a). The HD and associated Zip of NbHB1 were well conserved with matching proteins of other species, but there was no significant homology in the N-terminal variable region or the C-terminal region except for the CPSCE motif. Phylogenetic analysis with other HD-Zips from various species indicated that NbHB1 belonged to subfamily II of HD-Zip proteins, but had low similarity with other subfamily HD-Zip proteins such as H52 and LeHB1 (Fig. 1b).

Figure 1.

Sequence analysis of NbHB1 and corresponding phylogenetic tree. (a) Comparison of amino acid sequences with NbHB1 and orthologs from Arabidopsis, poplar, and tomato. The black bar above the sequences represents the homeodomain-leucine zipper (HD-Zip) domain and asterisks (*) indicate the conserved CPSCE motif. (b) Phylogenetic tree with NbHB1 and HD-Zip family genes from various plants. The numbers of each node indicate the percentage bootstrap support. The scale bar indicates the genetic distance based on branch lengths. NP_179865, NP_196289 and NP_195493, Arabidopsis thaliana; CAN60988, Vitis vinifera; CAA06728, Craterostigma plantagineum; AAO00684 and ABK95369, Populus trichocarpa; AAZ23784, Medicago sativa; H52 and LeHB1, Solanum lycopersicum; HAHB4, Helianthus annuus.

Expression of NbHB1 during plant–pathogen interactions

To investigate the function of NbHB1 in defense against pathogens, we monitored its expression patterns during plant–pathogen interactions using semiquantitative RT-PCR. To monitor the expression of NbHB1 in both incompatible and compatible interactions, Pto transgenic N. benthamiana plants were inoculated with P. tabaci (avrPto), an incompatible pathogen, and P. tabaci, a compatible pathogen. This pathosystem originated from a tomato against Pst and was transferred successfully to N. benthamiana (Martin et al., 1993; del Pozo et al., 2004). The maximum amount of NbHB1 transcript was detected at 6 h postinoculation (hpi) in the Pto-mediated incompatible interaction and at 12 hpi in the compatible interaction with P. tabaci (Fig. 2; Fig. S2). Under the same conditions, transcripts of pathogenesis-related 1a (PR1a), a marker gene of pathogen infection and SA signaling, were detected at 12 hpi and 24 hpi for the incompatible condition, indicating the accumulation of SA during the Pto-mediated resistance response (Fig. 2a), but were not detectable in the compatible interaction within 24 hpi (Fig. 2b). To monitor the effect of defense-related hormones on the transcript level of NbHB1, N. benthamiana leaves were treated with SA or JA. As shown in Fig. S1, SA significantly induced transcription of NbHB1, while JA had only a moderate effect.

Figure 2.

Expression of NbHB1 transcripts during plant–pathogen interactions. (a) Expression of NbHB1 during an incompatible interaction. Leaves of Nicotiana benthamiana Pto transgenic plants were infiltrated with Pseudomonas syringae tabaci (avrPto). NbPR1a was used as an salicylic acid (SA)-inducible marker gene. (b) Expression of NbHB1 during a compatible interaction. Leaves of N. benthamiana Pto transgenic plants were infiltrated with Ptabaci. Samples were harvested during the time course, and reverse transcription polymerase chain reaction (RT-PCR) was carried out to quantify the amount of transcripts.

NbHB1 protein is localized to the nucleus

The HD-Zip family of proteins are known as transcription factors localized in the nucleus. To determine the nuclear localization of the NbHB1 protein, smGFP was fused to the N-terminus of NbHB1 (Fig. 3a). The fused construct was cloned into pMBP-1 to overexpress smGFP-NbHB1 under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. Two days after TOE of this construct in N. benthamiana, green fluorescent protein (GFP) fluorescence was consistently localized in the nuclei (Fig. 3b). The DAPI staining, which specifically stains DNA, was colocalized with the green fluorescence, confirming nuclear localization of the NbHB1 protein (Fig. 3b). As a control, smGFP alone was localized in nucleus and cytoplasm (Fig. 3b).

Figure 3.

Localization of NbHB1 in the nucleus. (a) Vector construction for overexpression of smGFP-fused NbHB1. NbHB1 was fused to the C-terminal region of smGFP, which was constitutively expressed under the control of the CaMV 35S promoter. (b) Localization of smGFP-NbHB1 protein in the nucleus. Agro-infiltrated leaves were observed using a fluorescence microscope for detection of green fluorescence which indicates NbHB1 protein accumulation. Localization in the nucleus was confirmed by staining cells with 4′,6-diamidino-2-phenylindole (DAPI).

Overexpression of NbHB1 accelerates cell death upon stresses

To investigate the functional role of NbHB1 in plants, A. tumefaciens transformed with the overexpression construct of NbHB1 was infiltrated into N. benthamiana leaves (Fischer et al., 1999; Bendahmane et al., 2000). When NbHB1 was overexpressed in N. benthamiana leaves, dead cell spots were occasionally observed on the infiltrated leaves, whereas empty vector-containing Agrobacterium or buffer-infiltrated leaves showed no cell death (data not shown). Overexpression of NbHB1 could apparently induce microscopic cell death, but was not sufficient for full induction of plant cell death. We speculated that additional stresses on top of NbHB1 expression were necessary for induction of plant cell death.

As NbHB1 was upregulated by pathogen inoculation, the role of NbHB1 expression in pathogen-induced cell death was examined. After the TOE of empty vector control and NbHB1 on matched halves of N. benthamiana leaves, the leaves were inoculated with Pst T1, an avirulent pathogen of tobacco, or P. tabaci, a pathogen of tobacco, in tenfold serial dilutions. Two days after inoculation, the extent of cell death induced by either Pst T1 or P. tabaci was significantly greater in the leaf halves with NbHB1-overexpression than in the empty vector-infiltrated controls (Fig. 4a; Fig. S3a,b).

Figure 4.

Induction of cell death by NbHB1 overexpression under various stresses. (a) Acceleration of pathogen-induced cell death by transient overexpression (TOE) of NbHB1. Cell death was induced by infiltration of Pst T1 or Pseudomonas tabaci. Each pathogen was serially diluted from OD600  = 0.1 to OD600  = 0.0001, and infiltrated into each half of a leaf after TOE of pMBP-1 and NbHB1. Photographs were taken 2 d post inoculation (dpi). (b) Induction of cell death by TOE of NbHB1 after treatment of methyl jasmonate (MeJA) in the right half of a leaf. Photographs were taken 5 d after treatment. Arrows indicate the spreading region of cell death over the midvein. (c) Induction of cell death by TOE of NbHB1 after treatment of darkness. pMBP-1 or NbHB1 was overexpressed in the right half of leaves, and darkness was applied. Photographs were taken after 3 d of darkness treatment. M, mature leaf; Y, young leaf. (d) Spreading of cell death induced by darkness. Cell death occurred on the right half of a leaf after 3 d of darkness treatment and spread to the left region of the leaf over the midvein. These experiments were repeated at least three times. (e) PR1a expression after TOE of NbHB1 and salicylic acid (SA) treatment. (f) NbTPI and NbACO expression after TOE of NbHB1 and methyl jasmonate (MeJA) treatment. For the confirmation of TOE of NbHB1 transcripts, reverse transcription polymerase chain reaction (RT-PCR) was performed showing much higher amounts of transcripts compared with control. Numbers indicate hours after treatment with MeJA or SA.

In addition, treatment of NbHB1-overexpressed leaves with methyl jasmonate (MeJA), a methyl ester of JA, induced cell death, while no cell death was observed in the control (Fig. 4b; Fig. S4a). Staining with TB specifically stained the NbHB1-overexpressed tissues indicating that cell death occurred. The cell death induced by NbHB1 expression and MeJA treatment gradually spread to the part of the leaves that did not overexpress NbHB1. By contrast, SA, which induced the transcription of NbHB1 by external application, did not promote cell death in NbHB1 TOE leaves (data not shown).

Expression of CaHB1, an ortholog of NbHB1, was dramatically increased during senescence (data not shown), and so we tested treatment with darkness to see if overexpression of NbHB1 affected the senescence of N. benthamiana. Unexpectedly, we observed dramatic cell death in NbHB1-overexpressed leaves at 3 d after treatment with darkness, but only weak chlorosis in the control leaves (Fig. 4c; Fig. S4b). The NbHB1- and dark-induced cell death occurred not only in mature leaves, but also in young leaves. At 5 d after the darkness treatment, cell death had further spread over the midvein, where the overexpression construct was not infiltrated (Fig. 4d). For the quantification of cell death, the amount of chlorophyll was measured after treatment of MeJA or darkness. However, there was minor difference between control and NbHB1-overexpressing leaves (Fig. S4e,f), implying that the experimental period for the cell death induction was not enough for the chlorophyll degradation.

In many cases, the cell death triggered by stress is linked to hormonal regulation. Among such hormones, SA, ET and JA have been recognized as the main regulators of stress-induced cell death in plants (Asai et al., 2000; Overmyer et al., 2000; O'Donnell et al., 2001). Because NbHB1 positively regulated plant cell death, we assessed the effect of NbHB1 expression on hormonal responses by monitoring the transcript levels of marker genes such as PR1a, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and trypsin proteinase inhibitor (TPI) for SA-, ET- and JA-responsive expression, respectively. After treatment with SA, NbPR1a was upregulated in both control and NbHB1-overexpressed leaves, although levels were lower in the NbHB1-overexpressed leaves (Fig. 4e; Fig. S5a). The MeJA treatment of the NbHB1 overexpressed plants almost abolished the MeJA-mediated induction of NbTPI (Fig. 4f, Fig. S5a). Enhanced expression of an ethylene-responsive NbACO was also observed in the NbHB1-overexpressed plants following treatment with MeJA (Fig. 4f; Fig. S5a). Together, these results indicate that the overexpression of NbHB1 in N. benthamiana accelerated pathogen-induced cell death, possibly via modulation of phytohormone-mediated signal transductions.

Silencing of NbHB1 delays cell death induced by P. tabaci

Because overexpression of NbHB1 accelerated pathogen-induced cell death, we examined the silencing effect of NbHB1 on plant cell death by cloning the C-terminal region of NbHB1 (including the 3′-UTR) into a pTRV2 vector and performing VIGS in N. benthamiana. Three weeks after NbHB1 silencing, significantly less NbHB1 transcript was detected in silenced plants compared with GFP-silenced control plants (Fig. 5a). No visible phenotypic differences were observed between NbHB1-silenced and control plants. To evaluate the effects of silencing on pathogen-induced cell death, plants were inoculated with Pst T1 or P. tabaci at a density of OD600 = 0.1 or 0.01. The cell death induced by compatible P. tabaci was significantly delayed in NbHB1-silenced plants at 2 d postinoculation (dpi) (Fig. 5b; Fig. S3c). There was, however, no significant temporal difference in the hypersensitive cell death induced by the incompatible pathogen Pst T1 (data not shown).

Figure 5.

Effect of NbHB1 silencing on pathogen-induced cell death. (a) NbHB1 was silenced by virus-induced gene silencing (VIGS). Silencing of NbHB1 transcripts was confirmed by reverse transcription polymerase chain reaction (RT-PCR) showing the presence of fewer amounts of transcripts. The picture was taken 3 wk after Agrobacterium infiltration. (b) After VIGS of NbHB1, cell death was evaluated by inoculation of Pseudomonas tabaci at a density of OD600 = 0.1 or OD600 = 0.01. Pictures were taken 2 d postinoculation (dpi). Experiments were repeated three times. (c) PR1a expression after VIGS of NbHB1 and salicylic acid (SA) treatment. (d) NbTPI and NbACO expression after VIGS of NbHB1 and methyl jasmonate (MeJA) treatment. For the confirmation of transient overexpression (TOE) of NbHB1 transcripts, RT-PCR was performed showing the presence of much less amounts of transcripts compared with control. Numbers indicate hours after treatment with SA or MeJA.

After establishing that overexpression of NbHB1 influenced hormonal responses, we investigated the silencing effect of NbHB1 on the expression of hormone-responsive genes. Treatment with SA induced the expression of NbPR1a in both control and NbHB1-silenced plants, but a greater amount of NbPR1a transcripts was detected in NbHB1-silenced plants than in controls (Fig. 5c; Fig. S5c). In contrast to the effects of NbHB1 overexpression on NbACO and NbTPI, significantly reduced amounts of NbACO transcripts and earlier induction of NbTPI transcripts were observed in MeJA-treated NbHB1-silenced plants compared with controls (Fig. 5d, Fig. S5d). These results clearly demonstrate the opposite responses of hormone-responsive gene expression in NbHB1-silenced and NbHB1-overexpressed plants. These findings further suggest that NbHB1 is a positive regulator of pathogen-induced cell death with integration of phytohormonal responses.

JA signaling is required for NbHB1-mediated cell death

Many signaling molecules that affect cell death have been described in plants, and their genetic relationships have been studied by epistatic analysis (Lorrain et al., 2003). Because SA, ET and JA are known to be associated with the regulation of plant cell death (Overmyer et al., 2003) and NbHB1 affected responses to those chemicals, we examined their involvement in NbHB1-mediated cell death using VIGS. ISOCHORISMATE SYNTHASE 1 (ICS1), an enzyme for SA biosynthesis, ETHYLENE INSENSITIVE 2 (EIN2), or COI1, a JA signaling component (Alonso et al., 1999; Wildermuth et al., 2001; Xu et al., 2002), were independently silenced in N. benthamiana, and NbHB1 was transiently overexpressed in the silenced plants. A RT-PCR analysis of NbICS1, NbEIN2 or NbCOI1 transcripts confirmed that the levels were downregulated by VIGS (Fig. 6a). Darkness-induced cell death was moderately accelerated in NbICS1-silenced plants, but was not much different in NbEIN2-silenced plants (Fig 6b). A significant delay of cell death was observed in NbCOI1-silenced plants (Fig. 6b). These results correlated with MeJA-induced cell death after TOE of NbHB1, suggesting that JA-, rather than SA- or ET-mediated signaling, is required for the function of NbHB1 in NbHB1-mediated plant cell death.

Figure 6.

Downstream signaling components required for cell death induced by NbHB1 and darkness. Evidence that each signaling component was silenced by virus-induced gene silencing (VIGS). Polymerase chain reaction was performed with 34 cycles for ICS1 and 40 cycles for EIN2 and COI1. Lower amounts of transcripts were detected in silenced plants confirming that VIGS was carried out properly. (b) Signaling components that affected NbHB1-induced cell death. ICS1, EIN2 or COI1 was silenced and NbHB1 was transiently overexpressed by agro-infiltration in the right half of leaves. Photographs were taken to evaluate the occurrence of cell death after 3 d of darkness treatment. Experiments were repeated at least three times.

NbHB1 is involved in the detoxification of ROS

In many cases, plant cell death is tightly related to ROS generation and its scavenging capacity to modulate the level of the response. If the ROS scavenging system does not function properly, accumulated ROS cannot be removed and cells regard ROS as the death signal (Apel & Hirt, 2004). While searching for the genes regulated by NbHB1 during NbHB1-mediated cell death, we identified the differential expression of NbCAT1, a homolog of the hydrogen peroxide scavenging gene NtCAT1 with 99% amino acid identity, in NbHB1-overexpressed leaves. NbCAT1 expression was dramatically suppressed following treatment with MeJA (Fig. 7; Fig. S6), suggesting that NbHB1-mediated cell death might be caused by the loss of ROS scavenging activity. However, transcripts levels of ASCOBATE PEROXIDASE (APX) genes such as NbAPX1, NbAPX2, NbAPXT1 and NbAPXT2 did not significantly change under the same conditions (Fig. 7; Fig. S6), indicating that NbCAT1 could be specifically regulated by NbHB1. A RT-PCR analysis of NbHB1 and NbACTIN transcripts was used for evidence of TOE (Fig. 7). These results may indicate that NbHB1 renders cells more sensitive to the death signal by regulating the cellular redox state possibly through suppression of catalase activity.

Figure 7.

Effect of NbHB1 transient overexpression (TOE) on the expression of reactive oxygen species (ROS) scavenging genes after treatment of methyl jasmonate (MeJA). Reverse transcription polymerase chain reaction (RT-PCR) analyses of NbCAT1, NbAPX1, NbAPX2, NbAPXT1 and NbAPXT2 expression were carried out using MeJA-treated samples where vector or NbHB1 was transiently overexpressed. Larger amounts of NbHB1 transcripts confirmed the TOE of NbHB1. Numbers indicate hours after MeJA treatment.

HD-Zip domain is necessary for the induction of cell death

After determining that the TOE of NbHB1 in N. benthamiana consistently induced cell death after treatment with darkness, we attempted to define the domains required for NbHB1-mediated cell death. We monitored darkness-induced cell death following overexpression of domain-deleted NbHB1 constructs in N. benthamiana leaves (Fig. 8). Whereas the full NbHB1 induced cell death in the dark, proteins lacking either the HD or Zip domain failed to induce cell death. Expression of proteins lacking residues from the 77th to the 111th amino acid of NbHB1 also did not induce cell death following darkness treatment. By contrast, dark-induced cell death occurred when truncated proteins lacking the 1st to the 76th amino acid residues or the C-terminal region was overexpressed. Because the CPSCE motif in the sunflower HD-Zip gene is known to be involved in homodimerization of the protein (Tron et al., 2002), we evaluated whether the CPSCE motif of NbHB1 was necessary for NbHB1function in vivo by substituting the cysteine residues in the CPSCE motif. When the construct was overexpressed, cell death still occurred 3 d after darkness treatment. These results indicate that the HD-Zip domain and part of the N-terminal variable region, but not the N- or C-terminal region, were required for dark-induced cell death in NbHB1-overexpressed plants.

Figure 8.

Identification of domains necessary for NbHB1 function. Constructs containing regions (a–o) of NbHB1 were transiently overexpressed, and darkness was applied for 3 d. a, Full length of NbHB1 and b, empty vector (and they were used for positive and negative controls, respectively). Construct n. contained the site-directed mutant for CPSCE motif. Red-boxed pictures indicate the constructs that induced cell death. HD, Zip, and the amino acids near the HD were required for cell death. Numbers indicate the position of amino acid. aa, amino acids.


In this study, we isolated NbHB1, a member of the plant HD-Zip family of transcription factors, and characterized its roles in pathogen-induced cell death. Overexpression of NbHB1 induced cell death after treatment with stresses, and cell death was delayed by silencing of COI1. These results delineate NbHB1 as a new regulator of cell death functioning via JA signaling.

Regulation of cell death by NbHB1 via JA signaling

Jasmonic acid is an important signaling molecule in the regulation of male sterility, defense against pathogens and herbivores, and stomatal closure (Stintzi & Browse, 2000; Stintzi et al., 2001; Kang et al., 2006; Munemasa et al., 2007). It has also been repeatedly linked to the acceleration of senescence and induction of cell death (He et al., 2002; Mur et al., 2006; Zhang & Xing, 2008). These reports imply that JA signaling could be involved in the regulation of different types of cell death processes in plants, but none of the components of the JA-mediated plant cell death signaling pathway have been identified.

Our findings indicate that NbHB1 could be one of the components of the JA-mediated pathway, at least in pathogen-induced cell death. First, NbHB1 was upregulated by pathogens that induced plant cell death. Second, TOE of NbHB1 induced cell death after treatment with darkness or MeJA, and accelerated pathogen-induced cell death. These results show that NbHB1 positively influences cell death, which is possibly regulated by JA. Third, darkness-induced cell death in NbHB1-overexpressed plants was significantly delayed in NbCOI1-silenced plants, supporting the mediation of cell death by JA signaling. Finally, silencing of NbHB1 delayed cell death induced by P. tabaci. These findings suggest that NbHB1 is a regulator of pathogen-induced cell death that functions at least partly via JA signaling.

Although SA has been described as a positive regulator of cell death in many reports (Hunt et al., 1997; Aviv et al., 2002; Brodersen et al., 2005), it seemed to have a negative effect on NbHB1-mediated cell death in this study. Salicylic acid treatment did not induce cell death in NbHB1-overexpressed leaves. Furthermore, silencing of NbICS1, an enzyme necessary for SA biosynthesis, did not abolish NbHB1-mediated cell death, but rather slightly accelerated cell death instead (Fig. 6b). This result indicates that SA may function in the delay of NbHB1-mediated cell death, possibly through alterations in the antioxidant capacity of SA under certain conditions (Rao & Davis, 1999). Depletion of SA exacerbates plant cell death in several mutants, including acd2 (Mach et al., 2001), lsd2 and lsd4 (Hunt et al., 1997).

Plants initiate cell death as part of developmental processes or stress responses (Greenberg, 1996; Huh et al., 2002), raising the possibility that NbHB1 could be involved in not only pathogen-induced cell death, but also other types of plant cell death. When we examined the expression of NbHB1 during senescence or after treatment with salt stress, however, there was no difference in transcription level (data not shown), suggesting that NbHB1 may be involved primarily in pathogen-induced cell death.

We did not anticipate the observed induction of cell death by treatment with darkness or MeJA. Although darkness treatment has been used to induce senescence, it takes a relatively long time to induce complete death (Weaver & Amasino, 2001; Liu et al., 2005). In our experiments, the dying process of regions that overexpressed NbHB1 was completed after 3 d of darkness treatment, possibly indicating that NbHB1 accelerates cell death by a certain signal induced by darkness. Furthermore, MeJA treatment of NbHB1-overexpressed leaves induced cell death, hinting that JA signaling was involved in the cell death phenotype. It is possible that the elevation of JA during senescence (He et al., 2002) may induce cell death after treatment with darkness. In agreement with these findings, CaHB1, the closest ortholog of NbHB1, was dramatically upregulated during senescence (data not shown), suggesting that NbHB1 orthologs in plants may be involved in cell death regulation mediated by JA signaling.

The possible mode of regulation of cell death by NbHB1

NbHB1 encodes a protein belonging to the HD-Zip family, which is known to participate in stress adaptation and development in plants. The HD-Zip protein, H52, has been implicated in pathogen-induced cell death in tomato as a negative regulator of cell death (Mayda et al., 1999). When H52 is silenced, cell death develops spontaneously and spreads out of the pathogen-inoculated region.

NbHB1 accelerated pathogen-induced cell death. Interestingly, cell death was induced by TOE of NbHB1 together with MeJA or darkness treatment and then spread to the nonoverexpressed regions (Fig. 4b,d). Spreading of cell death may be caused by ET, because NbACO, an enzyme required for ET biosynthesis, was upregulated by NbHB1 overexpression (Fig. 4f). This postulation is supported by several reports that ET functions as a positive regulator of cell death propagation. For example, the ET concentration was highly elevated when H52 was silenced in tomato (Mayda et al., 1999). In the rcd1 mutant, ET promoted the inline image accumulation that drives lesion propagation (Overmyer et al., 2000). It also propagated cell death in concert with SA in an Arabidopsis lesion mimic mutant, vad1 (Bouchez et al., 2007).

Transcription of NbHB1 was upregulated during plant–pathogen interactions and then dramatically decreased to the basal level by 24 hpi (Fig. 2a,b). It is possible that NbHB1 induces the spreading of cell death, as transcripts were significantly down-regulated at the final step of cell death. This expression pattern and the phenotypic similarity between NbHB1 overexpression and H52 silencing suggest that NbHB1, together with a H52 homolog in N. benthamiana, may function to regulate cell death and its limitation. As NbHB1 and H52 both belong to HD-Zip family, it is possible that NbHB1 controls downstream genes at least partly shared by a H52 homolog. Alternatively, NbHB1 may interact directly with the H52 homolog, repressing the DNA-binding capacity of the NbHB1 protein because Zip domains are known to interact with other Zip domains (Ariel et al., 2007; Kim et al., 2008).

Salicylic acid-induced NbHB1 expression also provides a mechanism for the control of cell death by NbHB1 during plant–pathogen interactions. The accumulated SA in plants attacked by pathogens induces the expression of NbHB1, rendering the cells ready to initiate cell death by accumulating death signals such as JA (Mur et al., 2006). The mutual antagonism between SA and JA signaling has been well described (Kunkel & Brooks, 2002; Li et al., 2004). Experiments with several mutant lines such as NPR1 (Spoel et al., 2003), WRKY70 (Li et al., 2004), and EDS1 (Xing & Chen, 2006) have substantiated that SA negatively affects JA signaling. There is also evidence that JA antagonizes SA signaling (Kunkel & Brooks, 2002). Based on these studies, antagonism between SA and JA signaling appears to be genetically controlled. Because SA induced NbHB1 expression (Fig. S1a) and NbHB1 suppressed JA-inducible NbTPI expression, it is possible that NbHB1 is involved in the antagonistic effect of SA on JA signaling in N. benthamiana. If so, NbHB1 has two distinct features: the mediation of cell death induced by stress, and the suppression of JA-mediated defense-related genes specifying the SA-mediated defense response.

Because overexpression of NbHB1 alone was insufficient to induce cell death, NbHB1 may potentiate cell death, priming cells to respond to even a weak death signal. Cells that become more vulnerable to death signaling because of NbHB1 expression may still have the capacity to resist the death process, but even a weak signal such as JA could cause them to induce cell death. This hypothesis is supported by our expression analysis of ROS-scavenging enzymes (Fig. 7). Induction of cell death by MeJA may be caused by the failure to detoxify accumulated ROS produced by MeJA treatment (Zhang & Xing, 2008). Treatment with either MeJA or darkness induced H2O2 accumulation in NbHB1 TOE plants (Fig. S4a,b), and NbHB1 overexpression caused a significant repression of NbCAT1 transcripts after treatment with MeJA (Fig. 7). Reactive oxygen species play roles in signaling cascades or exacerbation of damage under stresses (Apel & Hirt, 2004). Because ROS production can damage cells, the cellular level of ROS requires strict control (Yang & Poovaiah, 2002). Catalase acts as one of the major ROS scavenging mechanisms, decomposing H2O2 to water and oxygen. Transgenic plants with antisense NtCAT1, the closest ortholog of NbCAT1, exhibit a > 90% reduction in overall catalase activity, leading the tobacco plants to develop chlorosis and necrosis (Takahashi et al., 1997). Because there was no change in the transcription level of cytosolic or chloroplastic APX genes by TOE of NbHB1, the reduction of NbCAT1 might be critical for the loss of H2O2 scavenging activity. Based on these findings, it seems that TOE of NbHB1 alone does not induce cell death, but rather potentiates plant cells for death when stress is received.

In conclusion, we provide evidence that NbHB1, a member of the HD-Zip family of transcription factors, is involved in the potentiation and modulation of plant cell death and possibly cooperates with JA to induce cell death. Identification of NbHB1 target genes and closer examination of potential genetic or physical interactions with a possible H52 homolog could provide insight on the role of NbHB1 in the regulation of pathogen-induced cell death in plants.


We are grateful to G. B. Martin for Pto transgenic N. benthamiana, P. tabaci (avrPto), and P. tabaci. This work was financially supported by grants from CFGC (CG1132) and PDRC (PF06300-00) of the 21st Century Frontier Research Program, funded by MEST of the Korean government.