Pepper mitochondrial FORMATE DEHYDROGENASE1 regulates cell death and defense responses against bacterial pathogens.

Pepper Mitochondrial FORMATE DEHYDROGENASE1 Regulates Cell Death and Defense Responses against Bacterial Pathogens1[C][W][OPEN] Du Seok Choi 2, Nak Hyun Kim, and Byung Kook Hwang* Laboratory of Molecular Plant Pathology, College of Life Sciences and Biotechnology, Korea University, Seoul 136–713, Republic of Korea

Formate dehydrogenase (FDH; EC 1.2.1.2) is an NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide. Here, we report the identification and characterization of pepper (Capsicum annuum) mitochondrial FDH1 as a positive regulator of cell death and defense responses. Transient expression of FDH1 caused hypersensitive response (HR)-like cell death in pepper and Nicotiana benthamiana leaves. The D-isomer-specific 2-hydroxyacid dehydrogenase signatures of FDH1 were required for the induction of HR-like cell death and FDH activity. FDH1 contained a mitochondrial targeting sequence at the N-terminal region; however, mitochondrial localization of FDH1 was not essential for the induction of HR-like cell death and FDH activity. FDH1 silencing in pepper significantly attenuated the cell death response and salicylic acid levels but stimulated growth of Xanthomonas campestris pv vesicatoria. By contrast, transgenic Arabidopsis (Arabidopsis thaliana) overexpressing FDH1 exhibited greater resistance to Pseudomonas syringae pv tomato in a salicylic acid-dependent manner. Arabidopsis transfer DNA insertion mutant analysis indicated that AtFDH1 expression is required for basal defense and resistance gene-mediated resistance to P. syringae pv tomato infection. Taken together, these data suggest that FDH1 has an important role in HR-like cell death and defense responses to bacterial pathogens.

Microbial pathogens require host machinery or nutrients for their development, growth, and reproduction. Plants lack the specialized immune systems of animals that can directly attack and inhibit pathogens (Lam et al., 2001). However, plants and mammals share an innate immune system and common putative analogs (Nishimura and Dangl, 2010). One common defense strategy is programmed cell death (PCD) at the site of pathogen infection in plants, which resembles apoptosis of animals. In plants, PCD induces a localized and rapid cell death response in the vicinity of pathogen-challenged sites; this process is often called the hypersensitive response (HR). A number of resistance (R) genes are involved in triggering the HR signaling pathway. Representative examples are avirulence (avr)-R gene pairs in the gene-for-gene hypothesis (Flor, 1971). For

1 This work was supported by the Cooperative Research Program for Agriculture Science and Technology (Project no. PJ00802701), Rural Development Administration, Republic of Korea. 2 Present address: Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521. * Address correspondence to [email protected]. The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Byung Kook Hwang ([email protected]). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OPEN] Articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.114.246736

example, two Arabidopsis (Arabidopsis thaliana) R genes (Resistance to Pseudomonas syringae2 [RPS2] and Resistance to Pseudomonas syringae pv maculicola1 [RPM1]) recognize two bacterial effectors (avrRpt2 and avrRpm1, respectively) to activate downstream HR signaling cascades (Mackey et al., 2003). The Nicotiana tabacum N gene, which encodes a Toll-interleukin-1 receptor/nucleotide-binding site/Leu-rich repeat protein, contributes to the HR necrotic lesion that occurs during tobacco mosaic virus infection by recognition of virus replicase proteins (Whitham et al., 1996; Liu et al., 2002b). Disruption of these R genes in loss-of-function mutants mediated by virus-induced gene silencing (VIGS) leads to a failure to trigger HR and consequently, renders the mutant plants susceptible to pathogens. The mitochondrion is a membrane-enclosed organelle that supplies cellular energy by producing ATP (McBride et al., 2006). Mitochondria are associated with the cell cycle, signaling, differentiation, growth, and cell death (McBride et al., 2006). It has been suggested that plant mitochondria play a crucial role in the regulation of PCD during tracheary element formation (Yu et al., 2002). There is evidence for a key role for plant mitochondria in PCD in that important apoptosis regulators, such as B-cell chronic lymphocytic leukemia/lymphoma (BCL)2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (proapoptotic) and Bcl-2 and Bcl-xl (antiapoptotic), are present in the mitochondrial membrane (Collazo et al., 2006). In animals, mitochondrial outer membrane permeabilization is a pivotal event in the mitochondrial apoptosis pathway (Green and Kroemer, 2004).

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Formate Dehydrogenase in Cell Death and Defense

Recognition of intracellular death signals in mitochondria leads to changes in calcium ion levels, cellular pH, and levels of several metabolites. Mitochondria may then initiate apoptosis in response to the cellular changes (Lam et al., 2001). Accumulating evidence suggests that mitochondria are involved in HR-associated PCD in plants. In N. benthamiana cells, the bacterial effector harpin alters mitochondrial functions and induces HR-like cell death (Xie and Chen, 2000). Transient expression of Bax in N. benthamiana induces HR-like cell death and defense gene expression in mitochondria, which support similar cell death processes in plants and animals (Lacomme and Santa Cruz, 1999). Alternative oxidase regulates the generation of reactive oxygen species (ROS) in mitochondria during HR activation, suggesting the involvement of mitochondria in HR-induced cell death (Lam et al., 1999). A representative common event for PCD in both animals and plants is the release of cytochrome c from mitochondria (Balk et al., 1999; Balk and Leaver, 2001). Blocking the release of cytochrome c from mitochondria inhibits apoptosis in animal cells. Cytochrome c release is an early event of PCD in plants (Balk et al., 1999); however, there is evidence that cytochrome c relocation is insufficient to trigger cell death in zinnia (Zinnia elegans) mesophyll cells (Yu et al., 2002). NAD-dependent dehydrogenases and NADPdependent dehydrogenases catalyze a number of major metabolite pathways. Representative examples are alcohol dehydrogenase (EC 1.1.1.1; Shiraishi et al., 2000), lactate dehydrogenase (EC 1.1.1.27; O’Carra and Mulcahy, 1996), malate dehydrogenase (EC 1.1.1.37; Trevanion et al., 1997), and glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12; Tarze et al., 2007). Malate dehydrogenase catalyzes the conversion of malate to oxaloacetate in the mitochondrial citric acid cycle; the product oxaloacetate is converted to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. Glyceraldehyde-3-P dehydrogenase is implicated in the initiation of apoptosis (Tarze et al., 2007). NAD-dependent isocitrate dehydrogenase (EC 1.1.1.42) is localized to mitochondria, but its physiological function remains unclear (Gálvez et al., 1998). Formate dehydrogenase (FDH; EC 1.2.1.2) is an NAD(P)-dependent dehydrogenase that oxidizes formate to carbon dioxide in the presence of NAD (Hourton-Cabassa et al., 1998). Some prokaryotic FDH containing molybdenum or tungsten cofactors acts as the electron donor to drive carbon dioxide reduction (Reda et al., 2008). In plants, FDHs have been reported in stress responses. FDH transcript levels are higher in potato (Solanum tuberosum) and Arabidopsis leaves in response to continuous darkness and environmental stresses, such as drought, wounding, chilling, and heat (Hourton-Cabassa et al., 1998; Herman et al., 2002). Several mitochondrial FDHs contain the predicted signal peptide for targeting to mitochondria (AmbardBretteville et al., 2003), and this signal peptide is usually removed after import into the mitochondria (Neupert, 1997). FDH transcript levels in barley (Hordeum vulgare)

are induced by various stimuli (Suzuki et al., 1998), and respond to treatments with methanol and formate in potato leaves (Hourton-Cabassa et al., 1998). Bean (Phasuolus vulgaris genotype BAT93) FDH is upregulated by dark treatment and incompatible Colletotrichum lindemuthianum C531 infection (David et al., 2010). However, the mechanisms regulating FDHs during defense response to pathogen challenge are not known. Here, we functionally characterized pepper (Capsicum annuum) mitochondrial FDH1, which was isolated from a pepper complementary DNA (cDNA) library using macroarray-based differential hybridization (Jung and Hwang, 2000). Agrobacterium tumefaciens-mediated transient expression of FDH1 in pepper and N. benthamiana leaves and VIGS assays in pepper leaves (Choi et al., 2012) were used to investigate the role of FDH1 during HR-associated PCD. We introduced pepper FDH1 into Arabidopsis Columbia-0 (Col-0) to generate gain-of-function transgenic plants overexpressing FDH1. We used the transfer DNA (T-DNA) insertion mutant fdh1 for analysis of FDH1 loss of function in Arabidopsis. FDH1 silencing in pepper enhanced susceptibility to Xanthomonas campestris pv vesicatoria (Xcv) infection, leading to a reduction in cell death response. Overexpression (OX) of FDH1 in Arabidopsis conferred greater resistance to Pseudomonas syringae pv tomato (Pst) in a salicylic acid (SA)-dependent manner. Arabidopsis T-DNA insertion mutant analysis showed that AtFDH1 expression was required for basal defense and R gene-mediated resistance to Pst infection. Taken together, the results of this study suggest that pepper FDH1 may be involved in the cell death signaling pathway, defense-related hormone regulation, and defense gene activation, ultimately leading to hypersensitive cell death and defense response to bacterial pathogens. RESULTS Isolation and Sequence Analysis of FDH1

We isolated pepper defense-related genes that were differentially induced during incompatible interactions between pepper and Xcv strain Bv5-4a using the macroarray-based differential hybridization technique (Jung and Hwang, 2000). We selected and fully sequenced the pepper FDH1 cDNA, which was cloned into the pBluscript vector. The full-length cDNA sequence and the deduced amino acid sequence of FDH1 are shown in Supplemental Figure S1. The predicted amino acid sequence has a D-isomer-specific 2-hydroxyacid dehydrogenase NAD-binding signature (approximately 197–224 amino acids) and two D-isomer-specific 2-hydroxyacid dehydrogenase signatures (approximately 247–269 and 276–292 amino acids) as predicted by PROSITE (http://prosite.expasy.org/ scanprosite/), a database of protein families and domains. FDH1 shares 96% identity with the amino acid sequences of both the potato mitochondrial formate dehydrogenase (MFDH) and the tomato (Solanum

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lycopersicum) FDH. FDH1 encodes a 1,143-nucleotide open reading frame; FDH1 protein contains 381 amino acids with a predicted molecular mass of 42 kD and isoelectric point of 6.84. Sequence analysis by SignalP server 3.0 (http://www.cbs.dtu.dk/services/SignalP/) indicates that pepper FDH1 contains a signal peptide of 15 amino acids (MAMRRVASTAARAFA) and a putative cleavage site between Ala-28 and Ser-29, which was observed in the orthologs of potato, barley, and rice (Oryza sativa; Tishkov and Popov, 2004; Supplemental Fig. S2). The other putative cleavage site between Lys-33 and Lys-34 was identified using the MitoProt program (http://ihg.gsf.de/ihg/mitoprot.html). The mitochondrial targeting sequence (TRQLQA) is conserved in the N-terminal region (Supplemental Fig. S2). The computational analysis tool for predicting subcellular localization, PSORT (http://www.psort.org/), confirmed that a mitochondrial targeting sequence exists in the motif (TRQLQA). The signal peptide of potato MFDH is proposed to be cleaved after entering mitochondria, indicating that the 43-kD MFDH protein may be cleaved to generate a 40-kD polypeptide (Colas des Francs-Small et al., 1993). These results suggest that pepper FDH1, which has a cleavage pattern similar to that of potato MFDH, may localize to mitochondria. Activation of FDH1 by Biotic and Abiotic Stresses

RNA and protein gel blot analyses were used to investigate FDH1 expression patterns in pepper plants exposed to biotic and abiotic stresses. In healthy plants, FDH1 was constitutively and strongly expressed in flowers but weakly expressed in stems, roots, and green fruits (Fig. 1A). No FDH1 transcripts were detectable in leaves and red fruits. Xcv infection greatly induced FDH1 in pepper leaves during compatible and incompatible interactions, and it was much faster and stronger

Figure 1. Patterns of FDH1 expression in pepper. A, RNA gel blot analysis of FDH1 expression in various organs. RNA gel blot (B) and immunoblot analyses (C) of FDH1 expression in leaves inoculated with virulent (compatible) Ds1 and avirulent (incompatible) Bv5-4a strains of Xcv. FDH1-specific antisera raised against an FDH1 peptide were used to detect CaFDH1 in leaf extracts. CBB, Coomassie Brilliant Blue; H, healthy; mock, infiltrated with 10 mM MgCl2; rRNA, ribosomal RNA. 1300

in the incompatible interaction that triggered rapid and localized HR-like cell death (Fig. 1B). FDH1specific antiserum raised against a specific FDH1 peptide was used for immunoblot analysis. Infection by avirulent Xcv Bv5-4a induced rapid and strong FDH1 protein expression compared with that for the virulent Xcv Ds1 infection (Fig. 1C). This result was supported by RNA gel blot analysis of the transcriptional induction of FDH1 by Xcv infection (Fig. 1B). In potato and Arabidopsis plants, FDH expression was induced by abiotic stresses, such as hypoxia, chilling, heating, drought, dark, wounding, and osmotic stress (Hourton-Cabassa et al., 1998; Li et al., 2002). RNA gel blot analyses showed that pepper FDH1 was also rapidly and specifically induced in pepper leaves in response to the phytohormones ethylene and SA and during abiotic stresses, such as drought and high salinity (Supplemental Fig. S3). Together, these results indicate that FDH1 is significantly induced in pepper leaves by biotic and abiotic stresses. FDH1 Is Required for Induction of HR-Like Cell Death

FDH1 is strongly up-regulated during the incompatible interaction between Xcv and pepper, which was accompanied by a rapid and localized cell death response (Fig. 1). We next investigated whether FDH1-OX triggers hypersensitive cell death in pepper leaves using the A. tumefaciens-mediated transient expression system (Fig. 2). Visible and UV light images showed typical cell death phenotypes in pepper leaves transiently expressing FDH1 3 d after agroinfiltration (Fig. 2A). Immunoblot analysis confirmed that the GFP-tagged FDH1 was expressed in pepper leaves (Fig. 2B). The levels of cell death response induced by transient expression of FDH1 were quantified by measuring electrolyte leakage from leaf discs infiltrated with A. tumefaciens carrying empty vector (GFP) or FDH1-GFP constructs (Fig. 2C). Transient expression of FDH1 in pepper leaves significantly enhanced electrolyte leakage because of cell death damage at 32 to 48 h after agroinfiltration compared with that of the empty vector controls. These results indicate that FDH1 is involved in the induction of hypersensitive cell death. Staining with 3,39-diaminobenzidine (DAB)-HCl showed the FDH1-mediated accumulation of ROS in the agroinfiltrated leaves (Fig. 2D). Induction of hydrogen peroxide (H2O2) accumulation occurred earlier than the cell death response in leaves transiently expressing FDH1. FDH1 Expression Induces Defense-Related Gene Expression and SA Production in Pepper

We next investigated whether transient expression of FDH1 induces pepper defense gene expression. Some of the tested pepper defense-related genes, including PATHOGENESIS-RELATED PROTEIN1 (CaPR1, Kim and Hwang, 2000; CaPR10, Choi et al., Plant Physiol. Vol. 166, 2014



downstream signaling, such as induction of defense response gene expression and SA accumulation. Subcellular Localization of FDH1

Most FDHs in higher plants are localized to mitochondria (Halliwell, 1974; Oliver, 1981; Colas des Francs-Small et al., 1993), but Arabidopsis FDH is targeted to both mitochondria (Jänsch et al., 1996) and chloroplasts (Olson et al., 2000). Consistent with these reports, pepper FDH1 has a mitochondrial targeting sequence (Supplemental Fig. S1). A potentially cleaved, 33-amino acid sequence (1-MAMRRVASTAARAFASPSSLVFTRQLQASPGPK-33)

Figure 2. Transient FDH1 expression induces cell death and promotes H2O2 production in pepper leaves. A, Induction of cell death by A. tumefaciens-mediated transient expression of FDH1. Photographs were taken at 72 h after agroinfiltration. Experiments were performed three times with similar results. B, Immunoblot analysis to detect FDH1 expression at 24 h after agroinfiltration. CBB, Coomassie Brilliant Blue. C, Electrolyte leakage analysis to assess cell death response in leaf discs from empty vector control and transient FDH1 expression plants after agroinfiltration (optical density at 600 nm [OD600] = 0.5). Data are means 6 SD from three independent experiments. Asterisks indicate significant differences in ion conductivity as determined by two-tailed t test (P , 0.05). D, Measurement of H2O2 using a ferrous ammonium sulfate/xylenol-orange assay. Data are means 6 SD (n = 3) from three independent experiments. Asterisks indicate significant differences in H2O2 production as determined by two-tailed t test (P , 0.05). [See online article for color version of this figure.]

2012) and DEFENSIN1 (CaDEF1; Do et al., 2004) were up-regulated 24 h after agroinfiltration (Fig. 3A). We investigated whether transient FDH1-OX triggers production of the defense-related plant hormone SA (Fig. 3B) and found that transient expression of FDH1 significantly induced greater SA levels in pepper leaves compared with that of empty vector control leaves. Collectively, these results indicate that FDH1 is essential for the induction of the hypersensitive cell death response in an SA-dependent manner. To examine whether FDH activity increases in response to transient FDH1 expression, FDH activity was measured using the fractionated supernatant of total protein extracts from leaves of empty vector control and transgenic FDH1-expressing pepper plants (Fig. 3C). FDH1 activity was significantly induced in leaves transiently expressing FDH1; however, weak FDH1 activity was detectable in empty vector control leaves. The increase in FDH1 activity in pepper leaves transiently expressing FDH supports the notion that FDH1 is required for HR-mediated cell death, which leads to

Figure 3. Transient expression of FDH1 in pepper leaves induces defense marker genes, SA production, and FDH activity. A, qRT-PCR analysis of pepper PR1, PR10, and DEF1 at the indicated time intervals after agroinfiltration. Data are means 6 SD. Pepper ACTIN was used to normalize the expression of defense marker genes. Asterisks indicate significant differences in gene expression as determined by the twotailed t test (P , 0.05). B, Levels of free and total SA (free SA plus glucoside-conjugated SA) at 48 h after agroinfiltration. Data are means 6 SD (n = 3) of three independent experiments. FW, Fresh weight. Asterisks indicate significant differences in SA contents as determined by the two-tailed t test (P , 0.05). C, Quantification of FDH activity at the indicated time intervals after agroinfiltration. Data are means 6 SD (n = 3) of three independent experiments. Asterisks indicate significant differences in FDH activity as determined by the two-tailed t test (P , 0.05).



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of FDH1 was designated as the putative mitochondrial signal (MS) peptide. We generated FDH1 deletion mutants, including a truncated form lacking the MS peptide (FDH1DMS; Fig. 4A). Web-based programs, such as Predotar (http://urgi.versailles.inra.fr/predotar/ predotar.html), TargetP (http://www.cbs.dtu.dk/ services/TargetP), and MitoProt (http://ihg.gsf.de/ ihg/mitoprot.html), were used to predict the localization of full-length and truncated FDH1 sequences to mitochondria (Table I). All scores from three webbased programs predict mitochondrial import of the full-length FDH1. The truncated deletion mutant forms of the MS peptide, such as FDH1N and FDH1DMS, have high scores for mitochondrial targeting, similar to those of the full-length FDH1 sequence. By contrast, FDH1DMS has an extremely low score for mitochondrial targeting, and these prediction data suggest that the MS peptide is essential for mitochondrial targeting of FDH1. The catalytic domain of 2-hydroxyacid dehydrogenase sequence (CDHS; FDH1CDHS) and the N terminusdeleted sequence (FDH1DN) have relatively high targeting scores, indicating that these deletions may potentially localize to mitochondria. The CDHS may be required for mitochondrial targeting of FDH1.

To investigate the subcellular localization of FDH1 in plants, we used GFP fusion constructs to be transiently expressed in N. benthamiana leaves. Immunoblotting of the protein extract fractions from transformed N. benthamiana leaves shows that all FDH1 deletion mutants were expressed in N. benthamiana leaves (Fig. 4B). MitoTracker Red dye (Invitrogen) was used to stain mitochondria (Fig. 4C), and confocal microscopy analyses revealed that GFP-fused full-length FDH1 localized to mitochondria in N. benthamiana leaves (Fig. 4C). FDH1-GFP was present in the mitochondrial fraction but not the cytosolic fraction (Fig. 4B). For positive controls, heat shock complex70 and isocitrate dehydrogenase were detected in the cytosolic and mitochondrial fractions, respectively. However, no fluorescence with MitoTracker Red was detected 72 h after agroinfiltration. The empty vector control (GFP) did not affect red fluorescence (Supplemental Fig. S4), suggesting the involvement of FDH1 in the mitochondrial membrane dysfunction. FDH1N and FDH1DC, which contain the MS peptide, were targeted to mitochondria. By contrast, we detected green fluorescence primarily in the cytoplasm of N. benthamiana cells transiently expressing FDH1DMS, FDH1CDHS, FDH1C, or FDH1DN. Together, these data indicate

Figure 4. Subcellular localization of FDH1 in N. benthamiana leaves. A, Maps of FDH1 protein and various deletion mutants used in this study. B, Immunoblots of protein extract fractions from transgenic N. benthamiana transiently expressing FDH1 and various FDH1 deletion mutants. C, Confocal images of GFP fusion protein subcellular localization for full-length or deletion mutants of FDH1 transiently expressed in N. benthamiana leaves. MitoTracker red dye was used to stain mitochondria. Bars = 20 mm. 1302




Table I. Predotar, TargetP, and MitoProt predictions of mitochondrial targeting for FDH1 deletion mutants Deletion

Predotara

TargetPb

MitoProtc

FDH1 FDH1DMS FDH1N FDH1CDHS FDH1C FDH1DC FDH1DN

0.89 0.01 0.89 0.3 0.01 0.89 0.3

0.938 0.083 0.938 0.623 0.088 0.938 0.663

0.9987 0.0449 0.9987 0.8429 0.1149 0.9987 0.8663

FDH1 Silencing Attenuates R Gene-Mediated Resistance in Pepper Leaves

D-Isomer-Specific 2-Hydroxyacid Dehydrogenase Signature Domain of FDH1 Is Required for Triggering HR-Like Cell Death and FDH Activity

To define the role of FDH1 in defense response of pepper to Xcv infection, we generated FDH1-silenced (tobacco rattle virus [TRV] :FDH1) pepper plants using VIGS with TRV-based vectors (Liu et al., 2002a; Choi and Hwang, 2011). Growth of the virulent Xcv strain in leaves of FDH1-silenced plants was similar to that in empty vector control (TRV:00) plants (Fig. 6A). However, the avirulent Xcv Bv5-4a grew 10-fold more rapidly in FDH1-silenced leaves than in empty vector control leaves. We investigated in vivo FDH activity in empty vector control and FDH1-silenced leaves of pepper plants infected by Xcv (Fig. 6B). FDH activity drastically increased in response to Xcv challenge in empty vector control plants, especially during incompatible interactions. By contrast, FDH1 silencing significantly attenuated FDH activity in Xcv-infected leaves. Rapid hypersensitive cell death response induced by avirulent Xcv infection was distinctly attenuated in FDH1-silenced pepper leaves (Fig. 6, C and D). As shown in Figure 6C, trypan blue staining confirmed that cell death was significantly reduced in FDH1-silenced leaves. Electrolyte leakage from leaf discs of empty

FDH1 deletion mutants were used to investigate whether the D-isomer-specific 2-hydroxyaciddehydrogenase signature domain of FDH1 functions in the induction of HR-like cell death. There are three CDHSs in FDH1 (Supplemental Fig. S2). We first generated the catalytic domain-containing mutants, such as FDH1CDHS (Fig. 4A). Cell death response was induced in N. benthamiana leaves by transient expression of all FDH1 deletion mutants, except for the FDH1C mutant that lacked CDHS (Fig. 5, A and B). Transient expression of constructs containing the catalytic CDHS domain distinctly triggered the cell death response. We monitored electrolyte leakage from leaf tissues at specific time intervals after agroinfiltration. Significantly high electrolyte leakage at the cell death sites was induced by transient expression of FDH1, FDH1DMS, FDH1DN, or FDH1DC (Fig. 5C), indicating the pivotal role of CDHSs for the induction of cell death. We investigated in vivo FDH activity in N. benthamiana leaves transiently expressing various FDH1 deletion mutants (Fig. 5D). FDH activity was drastically induced by transient expression of FDH1, FDH1DMS, FDH1DN, or FDH1DC. This suggests that CDHSs are essential for the induction of FDH activity in plants. We investigated whether mitochondrial localization of FDH1 affects FDH1-triggered cell death. As shown in Figure 5, transient expression of cytosolic FDH1DMS lacking the putative MS peptide resulted in cell death and ion leakage levels as well as FDH activity levels similar to those for expression of intact FDH1 that is targeted to mitochondria. These data indicate that mitochondrial localization of FDH1 is not required for induction of HR-like cell death responses as well as FDH activity in plants.

Figure 5. FDH1-triggered cell death and FDH activity in N. benthamiana leaves. A, Cell death phenotypes 3 d after A. tumefaciens-mediated transient transformation of various FDH1 deletion mutants into N. benthamiana leaves. Red, yellow, and black circles indicate full, partial, and no cell death, respectively. B, Levels of cell death in leaves transiently expressing various FDH1 deletion mutants. Different letters indicate significant differences in percentage of cell death as determined by the LSD test (P , 0.05). C, Electrolyte leakage from leaf discs at the indicated time points after agroinfiltration (OD600 = 0.5). D, Levels of FDH activity in leaves transiently expressing various FDH1 deletion mutants. Data are means 6 SD from three independent experiments.

a

Predotar estimates the probability that a sequence contains a mitob TargetP probability score for mitochondrial targeting sequence. c chondrial targeting peptides. MitoProt calculates the N-terminal protein region that can support a mitochondrial targeting sequence and the cleavage site.

that mitochondrial targeting of FDH1 requires the MS peptide, and transient expression of FDH1 causes mitochondrial dysfunction.



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Figure 6. VIGS of FDH1 in pepper leaves. A, Bacterial growth in leaves of empty vector control (TRV:00) and FDH1-silenced (TRV: FDH1) plants infiltrated with virulent (compatible) strain Ds1 and avirulent (incompatible) strain Bv5-4a of Xcv (5 3 107 cfu mL21). Asterisk indicates significant difference in bacterial growth as determined by the two-tailed t test (P , 0.05). B, FDH1 activity in leaves of empty vector control (TRV:00) and FDH1-silenced (TRV:FDH1) pepper plants infiltrated by Xcv. Data are means 6 SD (n = 3) from three independent experiments. Trypan blue staining (C) and ion conductivity measurement (D) of leaf discs of empty vector control (TRV:00) or FDH1-silenced (TRV:FDH1) pepper plants infiltrated with Xcv (5 3 107 cfu mL21). Asterisks indicate significant differences in ion conductivity as determined by the two-tailed t test (P , 0.05). DAB staining (E) and H2O2 measurement (F) of leaves of empty vector control (TRV:00) or FDH1-silenced (TRV:FDH1) pepper plants infiltrated with Xcv (5 3 104 cfu mL21). Asterisks indicate significant differences in H2O2 levels as determined by the two-tailed t test (P , 0.05).

vector control and FDH1-silenced plants was measured to quantify cell damage caused by HR-like cell death (Fig. 6D). During infection with avirulent (incompatible) Xcv Bv5-4a, electrolyte leakage from FDH1-silenced leaves was significantly lower than that from empty vector control leaves. By contrast, virulent (compatible) Ds1 infection did not induce significantly different electrolyte leakage in empty vector control and FDH1-silenced leaves. These data support the cell 1304

death phenotypes shown in Supplemental Figure S5. Inoculation of pepper leaves with a high titer of avirulent Xcv suspension causes HR-like cell death, which is accompanied by ROS production (Choi and Hwang, 2011). We investigated HR-associated ROS burst in empty vector control and FDH1-silenced leaves by DAB-HCl staining and the xylenol-orange assay for measurement of H2O2. Consistent with the results of the cell death assay, ROS accumulation was significantly attenuated in FDH1-silenced plants during avirulent Xcv infection (Fig. 6, E and F), indicating that FDH1 plays a pivotal role in early defense response associated with cell death of pepper plants during Xcv infection. Quantitative real-time (qRT) -PCR analysis showed that FDH1 transcript levels were significantly reduced in FDH1-silenced pepper leaves during Xcv infection (Fig. 7A). We also used qRT-PCR to determine whether FDH1 silencing affects the expression of downstream defense-related genes in pepper (Fig. 7A). Expression of the marker defense genes CaPR1, CaPR10, and CaDEF1 was distinctly down-regulated in FDH1-silenced leaves compared with that in empty vector control leaves during virulent or avirulent Xcv infection. To determine whether FDH1 silencing affects SA signaling, we analyzed SA levels in leaves of empty vector control and FDH1-silenced pepper plants during Xcv infection (Fig. 7B). In general, Xcv infection induced SA accumulation in pepper leaves. However, FDH1 silencing significantly reduced SA accumulation in infected leaves, especially in incompatible interactions. These results suggest that the greater susceptibility of FDH1-silenced plants to pathogen infection may be largely caused by lower SA levels and low FDH activity but may also result from reduced expression of downstream defense-related genes.

FDH1 Expression Is Required for Enhanced Resistance to Pst Infection in Arabidopsis

To investigate FDH1 gain of function in heterologous plants, we generated transgenic Arabidopsis Col-0 plants constitutively overexpressing pepper FDH1 by using the floral dipping method (Clough and Bent, 1998). Three transgenic lines with a single insertion of the pepper FDH1 transgene were selected for this study. We confirmed constitutive expression of FDH1 in these transgenic lines using reverse transcription (RT)-PCR (Supplemental Fig. S6). The transgenic FDH1-expressing Arabidopsis lines were evaluated for defense responses against Pst DC3000 and DC3000 (avrRpm1), which trigger HR in an RPM1 plant (Mackey et al., 2003). As shown in Figure 8A, constitutive OX of FDH1 strongly inhibited the growth of both virulent Pst DC3000 and avirulent DC3000 (avrRpm1) in transgenic Arabidopsis leaves. The cell death response induced by Pst infection was more prominent in transgenic FDH1-OX Arabidopsis leaves than wild-type leaves (Fig. 8, B and C). Trypan blue staining showed that avirulent Pst DC3000 (avrRpm1) infection induced a significantly higher level Plant Physiol. Vol. 166, 2014



Figure 7. Defense gene expression and SA levels in VIGS plants. A, qRT-PCR analyses of FDH1 and defense-related marker gene expression in leaves of empty vector control (TRV:00) and FDH1expressing (TRV:FDH1) pepper plants 24 h after infiltration with virulent (compatible) strain Ds1 and avirulent (incompatible) strain Bv5-4a of Xcv (5 3 107 cfu mL21). Defense response gene expression was normalized using pepper ACTIN expression levels. Data are means 6 SD (n = 3) from three independent experiments. Mock, Infiltrated with 10 mM MgCl2. Asterisks indicate significant differences in relative gene expression levels as determined by the two-tailed t test (P , 0.05). B, Quantification of free and total SA (free SA plus glucoside-conjugated SA) levels in leaves of empty vector control (TRV:00) and FDH1silenced (TRV:FDH1) pepper plants infiltrated by Xcv. Data are means 6 SD from three independent experiments. FW, fresh weight. Asterisks indicate significant differences in SA levels as determined by the twotailed t test (P , 0.05).

of cell death in transgenic FDH1-OX Arabidopsis leaves than in wild-type leaves (Fig. 8B). Electrolyte leakages from leaf discs of wild-type and transgenic FDH1-expressing plants were measured to quantify cell damage caused by HR-like cell death (Fig. 8C). During virulent and avirulent Pst DC3000 (avrRpm1) infection, electrolyte leakage was significantly higher from transgenic FDH1-OX leaves than from wild-type control leaves. Notably, avirulent Pst DC3000 (avrRpm1) infection was more effective than virulent Pst DC3000 infection in triggering cell damage in Arabidopsis leaves. We investigated callose deposition in wild-type and transgenic FDH1-OX plants by aniline blue staining. ROS bursts were detected by measuring H2O2 using DAB staining and the xylenol-orange assay (Fig. 8,

D–F). Consistent with the results of the cell death assay, callose deposition and ROS accumulation significantly increased in transgenic FDH1-OX plants during Pst infection and especially during avirulent Pst DC3000 (avrRpm1) infection. These results indicate that pepper FDH1 plays an important role in the early defense response associated with cell death during Pst infection. qRT-PCR was used to determine whether pepper FDH1-OX affects the expression of defense-related genes in Arabidopsis during infection (Fig. 9A). Pepper FDH1 was constitutively overexpressed in transgenic Arabidopsis leaves independent of Pst infection. Expression of the SA-dependent defense genes PR1 and PR4 was more strongly up-regulated in FDH1-OX leaves than in wild-type leaves during virulent or avirulent Pst DC3000 infection. By contrast, the induction of plant defensin1.2 (PDF1.2), a marker gene for jasmonic acid-dependent defense responses, by virulent or avirulent Pst DC3000 infection was similar in wild-type and transgenic FDH1-OX leaves. Levels of the plant defense hormone SA were monitored during infection (Fig. 9B). During Pst DC3000 and DC3000 (avrRpm1) infection, higher SA levels were induced in FDH1-OX Arabidopsis leaves compared with those in wild-type plants (Fig. 9B). These data show that pepper FDH1-OX confers enhanced resistance to Pst infection in an SA-dependent manner. To investigate whether AtFDH1 is required for resistance to Pst infection, two T-DNA insertion mutant lines (fdh1-1, SALK_118548; fdh1-4, SALK_108749) of Arabidopsis FDH1 were obtained from the Arabidopsis Biological Resource Center. Sequence analysis indicated that fdh1-1 and fdh1-4 have a T-DNA insertion in the first intron and the third exon, respectively (Fig. 10A). Under normal growth conditions, the growth phenotype of these mutants was the same as that of wild-type (Col-0) plants (Supplemental Fig. S7). We examined disease symptoms on the fdh1-1 and fdh1-4 mutant leaves 5 d after Pst DC3000 and Pst DC3000 (avrRpm1) infection. Both fdh1 mutants displayed more rapid and severe disease development than wild-type (Col-0) plants (Fig. 10B). The severe disease symptoms correlated with greater Pst proliferation in fdh1 leaves (Fig. 10C). Both Pst DC3000 and Pst DC3000 (avrRpm1) grew well in the fdh1-1 and fdh1-4 leaves compared with wild-type (Col-0) leaves. Collectively, these results suggest that AtFDH1 expression is required for basal defense responses and R gene-mediated resistance to Pst infection. RPM1, a coiled coil/nucleotide-binding site/Leurich repeat protein, recognizes AvrRpm1 and activates hypersensitive cell death response to inhibit pathogen proliferation (Gao et al., 2011). To investigate whether AtFDH1 is involved in the HR pathway, we stained dead cells with trypan blue, which revealed that the number of dying cells in leaves of fdh1 mutants was lower than those in wild-type (Col-0) leaves (Fig. 10D). To verify plasma membrane damage caused by cell death, we measured electrolyte leakage from the dead cells as ion conductivity. Consistent with the results



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Figure 8. Enhanced resistance of transgenic FDH1-OX Arabidopsis plants to Pst DC3000 and DC3000 (avrRpm1) infection. A, Bacterial growth in leaves of wild-type (WT; Col-0) and transgenic FDH1-OX plants infiltrated with Pst DC3000 and DC3000 (avrRpm1; 5 3 104 cfu mL21). Data are means 6 SD from three independent experiments. Different letters indicate significant differences in bacterial growth as determined by the LSD test (P , 0.05). dai, Days after inoculation. B, Trypan blue staining of leaves of WT and transgenic FDH1-OX plants infiltrated with Pst DC3000 and DC3000 (avrRpm1; 107 cfu mL21). C, Electrolyte leakage from leaf discs of WT and transgenic FDH1-OX plants infiltrated with Pst DC3000 and DC3000 (avrRpm1; 107 cfu mL21). Data are means 6 SD from three independent experiments. D, Aniline blue staining 24 h after infiltration with Pst DC3000 and DC3000 (avrRpm1; 107 cfu mL21). The numbers of callose per millimeter2 are represented as means 6 SD. Asterisks indicate significant differences in callose deposition as determined by the two-tailed t test (P , 0.05). Bars = 100 mm. DAB staining (E) and H2O2 measurements (F) in leaves of WT and transgenic FDH1-OX plants infiltrated with Pst DC3000 and DC3000 (avrRpm1; 107 cfu mL21). hai, Hours after inoculation. Asterisks indicate significant differences in H2O2 levels as determined by the two-tailed t test (P , 0.05).

of trypan blue staining, ion conductivity decreased much more in infected fdh1-4 leaves than in infected wild-type leaves, especially in fdh1-4 leaves infected with Pst DC3000 (avrRpm1; Fig. 10E). Similarly, bacterial infection caused lower callose deposition in fdh1-4 mutants than in wild-type plants (Fig. 10F). Together, these data indicate that AtFDH1 plays a key role in the HR-mediated defense response pathway during infection with the bacterial pathogen Pst. DISCUSSION

In this study, we show that pepper mitochondrial FDH1 is required for HR-like cell death and R genemediated bacterial disease resistance in pepper and/or transgenic Arabidopsis. Our results show that a putative signal peptide is responsible for FDH1 localization in mitochondria, which is not essential to induce HR-like cell death. Transient expression or constitutive OX of FDH1 in plants is closely associated with changes in FDH activity and SA levels. In planta assays with FDH1-silenced pepper and Arabidopsis ortholog fdh1 mutants support the involvement of FDHs in HR-mediated cell death and defense responses to bacterial diseases. 1306

Pepper FDH1 was expressed at low levels in stems, roots, and green fruits but constitutively and strongly expressed in flowers. FDH1 expression was not detected in pepper leaves and red fruits. These results suggest that FDHs may function in nonphotosynthetic tissues, such as fruits and tubers, rather than in photosynthetic green leaf tissues. In potato, the lowest FDH transcript level was found in leaves, whereas a high level of FDH mRNAs was present in developing tubers (Hourton-Cabassa et al., 1998). Potato tubers were proposed to require FDH activity for cell respiration in the incomplete tricarboxylic acid cycle processing (Hourton-Cabassa et al., 1998). The role of FDH during flowering in higher plants remains to be determined. There is convincing evidence that FDH is induced by abiotic stresses, such as hypoxia, chilling, drought, dark, and wounding (Hourton-Cabassa et al., 1998; Ambard-Bretteville et al., 2003). A few studies have revealed the role of FDH in host-pathogen interactions (David et al., 2010). We showed that FDH1 is induced by pathogens in pepper leaves. Xcv infection induced FDH1 expression in pepper leaves, particularly in response to the avirulent Xcv strain Bv5-4a. These results suggest that FDH1 is involved in HR induction during the incompatible interaction of pepper with Xcv. Plant Physiol. Vol. 166, 2014



Figure 9. Defense-related gene expression and SA levels in leaves of wild-type (WT) and transgenic FDH1-OX Arabidopsis plants infected with Pst DC3000 and DC3000 (avrRpm1). A, qRT-PCR analysis of FDH1 and defense-related gene expression in leaves of WT and transgenic FDH1-OX plants infiltrated with Pst (107 cfu mL21). Expression values were normalized by Arabidopsis UBQ5 expression levels. Data are means 6 SD from three independent experiments. Asterisks indicate significant differences in relative gene expression levels as determined by the two-tailed t test (P , 0.05). B, Quantification of free and total SA (free SA plus glucoside-conjugated SA) levels in leaves of WT and transgenic FDH1-OX Arabidopsis infiltrated by Pst. Data are means 6 SD from three independent experiments. FW, fresh weight; hai, days after inoculation. Asterisks indicate significant differences in SA levels as determined by the two-tailed t test (P , 0.05).

Screening for proteins involved in PCD in pepper plants infected with the avirulent strain of Xcv led to the identification and isolation of FDH1 as a candidate involved in HR. Early induction of FDH1 during the incompatible interaction between pepper plants and avirulent Xcv suggests the involvement of FDH1 in HR-like PCD. Transient expression of FDH1 in pepper leaves resulted in HR-like cell death, which was accompanied by accumulation of ROS and SA. We used N. benthamiana, a well-studied Solanaceous model plant, for transient expression experiments (Leister et al., 2005; Pfalz et al., 2011). A cell death response was distinctly induced in N. benthamiana leaves infiltrated by A. tumefaciens harboring 35S:FDH1 or 35S: FDH1 deletion mutants. Deletion mutant analysis revealed that the CDHSs in FDH1 are required for

induction of the cell death response and FDH activity. Three CDHSs are present in pepper FDH1. Notably, the transient expression of constructs containing the catalytic CDHS region induced a significantly stronger cell death response and higher FDH activity than the transient expression of FDH1 mutants lacking the CDHS. Strong FDH activity caused by transient OX of FDH1 in pepper was accompanied by high levels of SA. By contrast, FDH1 silencing significantly attenuated FDH activity and also significantly reduced SA accumulation in Xcv-infected pepper leaves. Previous microarray analysis showed that Arabidopsis FDH was strongly induced by SA (Schenk et al., 2000). SA treatment also caused significantly higher NADH levels, which function as an electron donor in key metabolic pathways that become NAD+ (Ishikawa et al., 2010). FDH1 is an NAD+-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide, which generates NADH from NAD+. Therefore, these results suggest that FDH1 may function in NADH metabolism and SA signaling during plant immune responses. Previous studies showed that several FDH sequences were well conserved in plants (Olson et al., 2000; Tishkov and Popov, 2004; David et al., 2010). Pepper FDH1 shares a high similarity with other plant FDHs and contains a putative mitochondrial targeting sequence. Signal peptides function to target proteins to specific subcellular compartments (Hartl et al., 1989). The targeting prediction programs Predotar, TargetP, and MitoProt indicated a high probability for localization of FDH1 in mitochondria. Deletion analysis of the putative mitochondrial targeting peptide predicted that the resulting proteins were not targeted to mitochondria. Their localization was confirmed by visualization of GFP fusion proteins using confocal microscopy and immunoblotting cytosolic and mitochondrial fractions using a GFP-specific antibody. It has been suggested that the first three amino acid residues (MAM) of FDH play a crucial role for mitochondrial localization (Ambard-Bretteville et al., 2003). Consistent with this suggestion, the pepper FDH1 sequence starts from the MAM motif, which supports FDH1 mitochondrial targeting. The catalytic domain fragment is predicted to be localized in mitochondria with an intermediate probability estimate. The Webbased program predictions may not consistently support all of the experimental results regarding in vivo targeting of GFP-FDH1 into mitochondria (AmbardBretteville et al., 2003). We investigated whether cytosolic FDH1 induces HR using the deletion mutations of the mitochondrial targeting peptide. Previous reports indicate that PCD is associated with mitochondria in plants (Petit et al., 1996; Lam et al., 2001; Yu et al., 2002). However, the results of this study suggest that mitochondrial localization of FDH1 is not required for induction of cell death and FDH activity in plants. It has been proposed that mitochondrial FDH regulates formate concentration under stress conditions (Ambard-Bretteville et al.,



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Figure 10. Arabidopsis fdh1 mutants exhibit enhanced susceptibility to Pst infection. A, Map of T-DNA insertion sites of fdh1 mutants. Exons and introns are indicated by black boxes and lines, respectively. fdh1-1 (SALK_118548), fdh1-2 (SALK_117670), and fdh1-3 (SALK_118644) have T-DNA insertions in the first intron. fdh1-4 (SALK_108749) and fdh1-5 (SALK_108751) have insertions in the third exon. All mutants are in the Col-0 background. B, Disease symptoms on leaves of wild-type (Col-0) and fdh1 mutant plants 5 d after inoculation with Pst DC3000 and Pst DC3000 (avrRpm1; 105 cfu mL21). C, Bacterial growth in leaves of wild-type (Col-0) and fdh1 mutant plants after inoculation with Pst DC3000 and Pst DC3000 (avrRpm1; 5 3 104 cfu mL21). Different letters indicate significant differences in bacterial growth or ion leakage as determined by the LSD test (P , 0.05). D, Trypan blue staining of leaves of wild-type (Col-0) and fdh1 mutant plants inoculated with Pst DC3000 and Pst DC3000 (avrRpm1; 107 cfu mL21). E, Electrolyte leakage from leaf discs of wild-type (Col-0) and fdh1 mutant plants inoculated with Pst DC3000 and Pst DC3000 (avrRpm1; 107 cfu mL21). Data are means 6 SD from three independent experiments. F, Aniline blue staining 24 h after inoculation with Pst DC3000 and Pst DC3000 (avrRpm1; 107 cfu mL21). The numbers of callose per millimeter2 are represented as means 6 SD. Bars = 100 mm. Asterisks indicate significant differences in callose deposition, as determined by the two-tailed t test (P , 0.05).

2003; David et al., 2010). OX of FDH1 may induce mitochondrial membrane dysfunction. Together, these results suggest that FDH1 functions in cell death induction may be independent of mitochondrial localization. There is convincing evidence that FDH may be involved in plant defense response to pathogen attack. All three P. vulgaris FDH genes were up-regulated by infection with the fungal pathogen C. lindemuthianum (David et al., 2010). Alternaria brassicicola infection induced Arabidopsis FDH expression (Schenk et al., 2000). However, the role of FDHs in defense response to pathogen attack is not fully understood. In our study, rapid and strong induction of the pepper FDH1 gene occurred in response to avirulent Xcv infection compared with that to virulent Xcv infection. FDH1silenced pepper plants displayed enhanced disease susceptibility and reduced cell death response. During pathogen infection, FDH1 silencing blocked the generation of ROS and SA and the expression of defenserelated genes. For FDH1 gain-of-function analysis, we generated transgenic Arabidopsis overexpressing FDH1 using the floral dipping method (Clough and Bent, 1998). By contrast to the results in FDH1-silenced pepper plants, OX of pepper FDH1 in transgenic 1308

Arabidopsis inhibited Pst growth, which was accompanied by enhanced ROS burst, cell death, and defense response gene expression as well as SA accumulation. In support of these data, the Arabidopsis fdh1 T-DNA insertion mutant exhibited enhanced susceptibility to Pst infection, leading to a decline in R gene-mediated resistance. Together, these results suggest that FDH1 is a positive regulator of cell death and defense responses to bacterial pathogens. Combining the data presented here, we propose a working model for pepper FDH1 involvement in hypersensitive cell death and defense signaling responses to bacterial pathogens (Supplemental Fig. S8). Induction of pepper FDH1 by Xcv challenge triggers ROS burst, promotes SA accumulation, and induces expression of some PR genes, including PR1 (Kim and Hwang, 2000) and PR10 (Choi et al., 2012). This ultimately leads to HR cell death and defense responses. Mitochondrial targeting of FDH1 requires the MS peptide, and FDH1 expression causes mitochondrial dysfunction. However, mitochondrial localization of FDH1 is not required for HR induction by FDH1 expression in plants. FDH1 silencing and OX in pepper and Arabidopsis, respectively, show that FDH1-OX confers enhanced resistance to bacterial Plant Physiol. Vol. 166, 2014



infection in an SA-dependent manner. Taken together, these results suggest that FDH1 may function upstream of ROS- and SA-mediated cell death signaling during bacterial infection.

experimental approach (Jung and Hwang, 2000; Choi and Hwang, 2011). XbaI and SacI sites were inserted at the initiating ATG and the terminal TAA, respectively, of the FDH1 open reading frame to subclone FDH1 into the binary vector pTOPO (Invitrogen). The full-length FDH1 was excised using XbaI and SacI and ligated into pBIN35S-GFP to generate FDH1-GFP under control of the 35S promoter from Cauliflower mosaic virus.

MATERIALS AND METHODS

Construction and A. tumefaciens-Mediated Transient Expression of FDH1 Deletion Mutants

Plants, Pathogen Inoculation, and Abiotic Stress Treatment Pepper (Capsicum annuum ‘Knockwang’), Nicotiana benthamiana, and Arabidopsis (Arabidopsis thaliana) Col-0 were grown in a growth chamber (24°C 6 2°C, 50% relative humidity, 80 mmol photons m22 s21 fluorescent illumination) with a 14-/10-h day-night cycle. Xanthomonas campestris pv vesicatoria (Xcv) strains Ds1 (virulent) and Bv5-4a (avirulent) and Pseudomonas syringae pv tomato (Pst) strains DC3000 and DC3000 (avrRpm1) were grown in yeast nutrient broth (4 g L21 yeast extract and 8 g L21 nutrient extract) at 28°C. Bacterial suspensions of 5 3 104 (for bacterial growth assay) and 5 3 108 (for cell death assay) colony-forming units (cfu) mL21 were infiltrated into fully expanded leaves. Arabidopsis Col-0 seedlings were grown at 16°C and 80% relative humidity. Six leaf-stage pepper plants were placed in a sealed glass bottle, which was injected with a final concentration of 5 mL of ethylene per 1 L of SA (5 mM) and NaCl (200 mM) were sprayed onto pepper plants supplemented with 0.01% (v/v) Tween 20. For drought stress, plants were removed from soil and placed in a growth chamber, and water was withheld.

RNA Gel Blotting and qRT-PCR Total RNA was isolated from plant tissues using TRIzol (Invitrogen) according to the manufacturer’s recommendations. RT was conducted using Moloney murine leukemia virus RT (Enzynomics). qRT-PCR was performed in an iCycler iQ (Bio-Rad) using SYBR Green Reagent (Bio-Rad) as a dye. The primer sets used in this study were FDH1 (forward, 59-ATGGCGATGAGGCGTGTGGCATCT-39; reverse, 59-TTAGCGATACTGGGGTGCCAGTTCC-39), CaPR1 (forward, 59-CAGGATGCAACACTCTGGTGG-39; reverse, 59-ATCAAAGGCCGGTTGGTC-39), CaPR10 (forward, 59-ATGGGTGCTTATACCTTTACTGAC-39; reverse, 59-TTAAACATAGACAGAAGGATTGG-39), CaDEF1 (forward, 59-CAAGGGAGTATGTGCTAGTGAGAC-39; reverse, 59-TGCACAGCACTATCATTGCATAC-39), AtPR1 (forward, 59-AGTATGGCTTCTCGTTCACA-39; reverse, 59-GGAGCTACGCAGAACAACTA-39), AtPR4 (forward, 59-GCCATCTCATTGTTGACTACCAATTT-39; reverse, 59-ATCAATGGCCGAAACAAGCA-39), and AtPDF1.2 (forward, 59-TGTTCTCTTTGCTGCTTTCGACGC-39; reverse, 59-TGTGTGCTGGGAAGACATAGTTGC-39). Relative gene expression was normalized using pepper Actin (Kim et al., 2010) and Arabidopsis Ubiqutin5 (UBQ5; forward, 59-GTAGTGCTAAGAAGAGGAAGA-39; reverse, 59-TCAAGCTTCAACTCCTTCTT-39) as internal controls. Average expression was calculated from three experimental replicates.

VIGS in Pepper The pTRV vector and Agrobacterium tumefaciens were prepared as described previously (Senthil-Kumar et al., 2007) for VIGS analysis. A 216-bp genespecific 39-untranslated region of FDH1 was inserted into the pTRV2 vector to yield pTRV2:FDH1. The pTRV1 vector and the pTRV2 vector with or without FDH1 were transformed into A. tumefaciens strain GV3101. A 5-mL culture of each strain was grown overnight at 28°C in yeast extract and bactopeptone broth (10 mg mL21 yeast extract, 10 mg mL21 peptone, and 5 mg mL21 NaCl) with appropriate antibiotics (50 mg mL 21 kanamycin and 50 mg mL 21 rifampicin). The cells were resuspended in A. tumefaciens infiltration buffer [10 mM MgCl and 10 mM 2-(N-morpholino)-ethanesulfonic acid, pH 5.7] and adjusted to OD600 = 0.4. Cultures were then exposed to 150 mM acetosyringone at room temperature with shaking for 3 h. A. tumefaciens strains containing the pTRV1 vector and pTRV2:00 or pTRV2:FDH1 were mixed at a 1:1 ratio and coinfiltrated into the cotyledons of pepper seedlings. A. tumefaciens-infiltrated pepper plants were grown at 25°C with a 16-/8-h lightdark cycle.

Isolation of FDH1 cDNA A full-length FDH1 cDNA was isolated from a pepper cDNA library constructed from Xcv-infected pepper leaves using a differential hybridization

For subcellular localization and cell death analyses of FDH1 deletion mutants, partial coding regions for the mitochondrial targeting signal peptide deletions were as follows: full-signal peptide (DMS), N terminus, CDHSs, C terminus, and deletion mutants without N (DN) or C (DC) terminus. Deletion mutants were PCR amplified using full-length FDH1 as the template. Primer sets used in the constructions are listed in Supplemental Table S1. These FDH1 deletion constructs were introduced into A. tumefaciens strain GV3101 for transient expression experiments. A. tumefaciens strain GV3101 harboring various deletion mutants (5 3 108 cfu) was infiltrated into leaves of pepper and N. benthamiana.

Arabidopsis Transformation A. tumefaciens GV3101 harboring pBIN35S-FDH1 was used to transform FDH1 into Arabidopsis Col-0 by the floral dipping method (Clough and Bent, 1998). Transgenic Arabidopsis plants were selected using Murashige and Skoog media containing kanamycin (50 mg L21). Transcriptional expression of FDH1 in transgenic plants was confirmed by RT-PCR with Arabidopsis UBQ5 as an internal control (forward, 59-GTAGTGCTAAGAAGAGGAAGA-39; reverse, 59-TCAAGCTTCAACTCCTTCTT-39).

Isolation of T-DNA Insertion Arabidopsis Mutants The T-DNA insertion fdh1 mutants obtained from Arabidopsis Biological Resource Center were identified using the T-DNA left-border primer (59-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-39) and the AtFDH1-specific forward (59-AAGGGAGTGGAGATTAGGACG-39 for fdh1-1; 59-TTTGTTTTCTTGAACGTTGGG-39 for fdh1-4) and reverse (59-CACAGCTATTCTGGTCCGAAG-39 for fdh1-1; 59-TTGGCTCGGATCATATTGATC-39 for fdh1-4) primers.

Isolation of Mitochondria Mitochondria were isolated from pepper and N. benthamiana leaves as described previously (Teschner et al., 2010) with some modifications. Briefly, leaf samples (2 g) from 5-week-old plants were homogenized at 4°C in 5 mL of grinding buffer (0.3 M mannitol, 25 mM sodium pyrophosphate, 10 mM KH2PO4, 1 mM EDTA, 0.2% [w/v] bovine serum albumin, 0.5% [v/v] polyvinylpyrrolidone-40, 4 mM L-Cys, and 13 protease inhibitor cocktail, pH 7.6). The homogenate was centrifuged at 2,000g for 10 min at 4°C, and the supernatants containing cytosolic proteins were carefully removed. The pellet was resuspended in 3 mL of buffer (0.3 M mannitol, 10 mM KH2PO4, 2 mM Gly, 0.1% [w/v] bovine serum albumin, and 13 protease inhibitor cocktail, pH 7.2). The lysate was centrifuged at 1,000g for 10 min at 4°C, and the supernatant was transferred to a clean 1.5-mL tube. One more step of centrifugation was conducted at 6,000g for 10 min at 4°C. The final pellet was harvested and stored at 270°C until use.

Immunoblot Analysis Total proteins were extracted from leaf samples using extraction buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% [w/v] SDS, 10 mM dithiothreitol, and 13 protease inhibitor cocktail) as described previously (Choi and Hwang, 2011). The sample was centrifuged at 10,000g for 5 min, and the supernatant was used for immunoblot analysis. Anti-GFP (Santa Cruz Biotechnology) was used as a primary antibody. Primary FDH-specific antibody was obtained as antisera raised against an FDH1 peptide (Young in Frontier). Isocitrate dehydrogenase and heat shock complex70 antibodies (Agrisera) were used as mitochondrial and cytosolic markers, respectively. Immunodetection was performed using the appropriate secondary antibodies (anti-rabbit IgG or anti-mouse IgG) conjugated with horseradish peroxidase.



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Cell Death Assay Six leaf discs (12-mm diameter) were removed from leaves infiltrated with A. tumefaciens strain GV3101 harboring various deletion mutants, floated in 25 mL of distilled water, washed for 20 min, and transferred to 25 mL of fresh distilled water. Water conductance was measured using a sensION7 electrical conductivity meter (Hach) at indicated time points. To visualize cell death, bacteria-infiltrated leaves were stained with a solution containing lactophenoltrypan blue solution (2.5 mg mL21 trypan blue, 25% [v/v] lactic acid, 25% [v/v] phenol, and 25% [v/v] glycerol in water):100% (v/v) ethanol (1:1). Stained leaves were destained with chloral hydrate (2.5 g mL21 in water).

Hwang, 2011; Lee et al., 2011). MitoTracker Red CMXRos (Invitrogen) was used for mitochondrial staining. Agroinfiltrated leaves were soaked in MitoTracker Red solution (500 nM final concentration) for 20 min. Red fluorescence was collected using a band-pass filter (BP568) for excitation and a long-pass filter (LP590) for emission. Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: C. annuum FDH1 (JQ966303), C. annuum PR1 (AF053343), C. annuum PR10 (AF244121), C. annuum DEF1 (AF442388), C. annuum Actin (GQ339766), Arabidopsis PR1 (AT2G14610), Arabidopsis PDF1.2 (AT5G44420), Arabidopsis PR4 (AT3G04720), Arabidopsis FDH (AT5G14780), and Arabidopsis UBQ5 (AT3G62250).

Aniline Blue Staining Callose deposits in leaves were stained with an aniline blue solution as described previously (Dietrich et al., 1994; Choi and Hwang, 2011). Leaves were cleared with alcoholic lactophenol, stained in a solution containing 0.01% (w/v) aniline blue in 0.15 M K2HPO4 and observed under a UV epifluorescence microscope.

DAB Staining and H2O2 Measurement

The following materials are available in the online version of this article. Supplemental Figure S1. Nucleotide and deduced amino acid sequences of pepper FDH1 cDNA. Supplemental Figure S2. Alignment of pepper FDH1.

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Bacteria-infiltrated leaves were harvested and incubated in 1 mg mL of 3,39-DAB-HCl (pH 3.8; Sigma) for 6 h in the dark at room temperature. To quantify H2O2, a ferrous ammonium sulfate/xylenol-orange assay was performed as described previously (Galletti et al., 2008). Excised leaf discs were soaked in the assay reagent (500 mM ammonium ferrous sulfate, 50 mM H2SO4, and 200 mM sorbitol) and centrifuged at 3,000g for 20 min. The supernatant was added to 200 mM xylenol orange and incubated for 30 min. Absorbance of the Fe3+-xylenol-orange complex (A560) was detected. Data were normalized and expressed as micromolar H2O2 per centimeter squared of leaf discs.

Enzyme Activity Assay In vitro FDH enzyme activity assay was performed as described previously (Olson et al., 2000) with some modifications. Briefly, leaf tissues were homogenized in a chilled solution (50 mM Tris-Cl, pH 8.6, 10 mM NaCl, 1 mM MgSO4, 2% [w/v] polyvinylpyrrolidone, and 0.1% [v/v] 2-mercaptoethanol) and then filtered with Miracloth. After centrifugation for 30 min at 15,000g, the supernatant was fractionated using a Sephadex G-25 column. The column was equilibrated with 50 mM Tris-Cl (pH 8.6), 10 mM NaCl, and 1 mM MgSO4 before fractionation. FDH activity was monitored spectrophotometrically at 340 nm after incubation at 30°C in 100 mM potassium phosphate solution (pH 7.0) containing 50 mM sodium formate and 1 mM NAD+ as substrates.

Supplemental Figure S3. Patterns of FDH1 expression in pepper leaves after treatment with plant hormone and abiotic stress. Supplemental Figure S4. Transient FDH1 expression in N. benthamiana induces mitochondrial dysfunction. Supplemental Figure S5. Disease symptoms in empty vector control and FDH1-silenced pepper leaves infected by virulent and avirulent strains of Xcv. Supplemental Figure S6. RT-PCR analysis of FDH1 expression in wildtype and transgenic FDH1-overexpressing Arabidopsis plants. Supplemental Figure S7. Growth phenotypes of Arabidopsis wild-type and fdh1 mutants under normal growth conditions. Supplemental Figure S8. Proposed model for the role of the FDH1 in cell death-mediated defense signaling in plants. Supplemental Table S1. Oligonucleotides for plasmid constructs used in this study. Received July 13, 2014; accepted September 18, 2014; published September 18, 2014.

LITERATURE CITED

SA Measurement Leaf tissues were ground with liquid nitrogen to a fine powder in 90% (v/v) methanol (10 mL per 1 g of fresh weight of tissue) and sonicated for 15 min, and 3-hydroxybenzoic acid was added as an internal standard. The mixture was incubated for 1 h at 4°C with constant rotation and subsequently centrifuged at 3,000g for 20 min. The remaining pellet was reextracted with 100% methanol. All supernatants were pooled and subjected to vacuum evaporation. The residue was resuspended in 5 mL of 5% (v/v) trichloroacetic acid, and 5 mL of ethylacetate:cyclopentane:isopropanol (50:50:1) was added. The upper phase (organic extraction of free SA) was transferred to a new tube, whereas the aqueous phase was then reextracted as described above. The two organic phases were pooled. The resulting solution was dried with vacuum evaporation. The residue was suspended in 40% (v/v) methanol containing 0.1% (v/v) acetic acid and filtered through a 0.2 mM filter. For extraction of SA b-glucoside, the aqueous phase was acidified with HCl to pH 1 and boiled for 30 min. The released SA was then extracted as described above. SA and its glucoside were quantified using HPLC with a C18 analytical column (Xbridge C18; 5 mm, 4.6 3 250 mm; Waters). SA was detected spectrophotometrically using excitation and emission wavelengths of 305 and 405 nm, respectively.

Confocal Microscopy GFP expression in epidermal cells of N. benthamiana leaves was visualized using a Zeiss LSM 510 confocal microscope equipped for GFP fluorescence (excitation at 488 nm and emission between 505 and 530 nm; Hwang and 1310

Supplemental Data

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*

Supplemental Figure S1. Nucleotide and deduced amino acid sequences of pepper FORMATE DEHYDROGENASE1 (FDH1) cDNA. Deduced amino acid sequences are given below the nucleotide sequences. The transcriptional start site is shown in bold type and the termination codon is marked by an asterisk. The signal sequence that is cleaved on the entrance into the mitochondrion is underlined. The predicted amino acid sequence for targeting to mitochondria is shown in italic. The conserved catalytic domain of 2-hydroxyacid dehydrogenases is marked with the gray box. The box (aataaa) indicates the polyadenylation sequence.

Signal peptide

Mitochondria-targeting sequence

1

2

3

Supplemental Figure S2. Alignment of pepper (Capsicum annuum) FDH1. Comparison of the deduced amino acid sequence of pepper FDH1 with potato (Solanum tuberosum) mitochondrial formate dehydrogenase precursor (Accession no. CAA79702), tomato (Lycopersicon esculentum) formate dehydrogenase (Accession no. CAH60893), Arabidopsis thaliana formate dehydrogenase (Accession no. CAC01877), rice (Oryza sativa) putative formate dehydrogenase (Accession no. BAD38302), and barley (Hordeum vulgare subsp. vulgare) formate dehydrogenase (Accession no. BAA36181).

Ethylene H

1

5

10

15

SA 20

25 h

20

25 h

H

1

5

10

15

20

25 h

20

25 h

FDH1 rRNA NaCl

Drought H

1

5

10

15

H

1

5

10

15

FDH1 rRNA

Supplemental Figure S3. Patterns of FDH1 expression in pepper leaves after treatment with plant hormone and abiotic stress. RNA gel-blot analyses of FDH1 expression in leaves treated with ethylene (5 mL⋅L-1), salicylic acid (SA, 5 mM), drought, and NaCl (200 mM). RNA gel-blot membranes were hybridized with biotin probes specific for the FDH1 3′-UTR. Equal loading (10 μg) was verified by visualizing rRNA on gel stained with ethidium bromide.

MitoTracker

Merged

FDH1-GFP

00-GFP

GFP

Supplemental Figure S4. Transient FDH1 expression in Nicotiana benthamiana leaf cells three days after Agroinfiltration induces mitochondrial dysfunction. Confocal image of green fluorescence protein (GFP)-fused FDH1 in N. benthamiana leaves 72 h after agroinfiltration. MitoTracker Red dye was used to stain mitochondria. Bars = 50 μm.

TRV:FDH1

Incompatible

Compatible

TRV:00

Supplemental Figure S5. Disease symptoms in empty-vector control and FDH1-silenced pepper leaves infected by virulent and avirulent strains of Xcv (5 × 104 cfu⋅mL-1). Images were taken 7 days after inoculation.

WT

#1

#2

#3

FDH1 AtUBQ5

Supplemental Figure S6. RT-PCR analysis of FDH1 expression in wild-type and transgenic FDH1-overexpressing Arabidopsis plants. The AtUBQ5 transcript was used as an internal control.

Col-0

fdh1-1

fdh1-4

Supplemental Figure S7. Growth phenotypes of Arabidopsis wild-type (Col-0) and fdh1 mutants under normal growth conditions.

Bacterial pathogens

FDH1

SA

ROS burst

PR genes (PR1, PR10)

Cell death & defense

Supplemental Figure S8. Proposed model for the role of the FDH1 in cell death-mediated defense signaling in plants.

Pepper mildew resistance locus O interacts with pepper calmodulin and suppresses Xanthomonas AvrBsT-triggered cell death and defense responses.

Foliar application of the leaf-colonizing yeast Pseudozyma churashimaensis elicits systemic defense of pepper against bacterial and viral pathogens.

Innate Defense against Fungal Pathogens.

Pyroptotic cell death defends against intracellular pathogens.

Defense-Related Responses in Fruit of the Nonhost Chili Pepper against Xanthomonas axonopodis pv. glycines Infection.

Defense against bacterial drug resistance.

Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens.

SlBIR3 Negatively Regulates PAMP Responses and Cell Death in Tomato.

Bacterial gasotransmitters: an innate defense against antibiotics.

GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

Plant innate immunity against human bacterial pathogens.

Age-related defense against infection with intracellular pathogens.

Leukocytes--second line of defense against invading mastitis pathogens.

Intricate Roles of Mammalian Sirtuins in Defense against Viral Pathogens.

Mitochondrial Protein PGAM5 Regulates Mitophagic Protection against Cell Necroptosis.

Bacterial formate hydrogenlyase complex.

An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens.

Cloning and characterization of the pepper CaPAO gene for defense responses to salt-induced leaf senescence.

Arabidopsis HSP90 protein modulates RPP4-mediated temperature-dependent cell death and defense responses.

Pregnane X Receptor Regulates Pathogen-Induced Inflammation and Host Defense against an Intracellular Bacterial Infection through Toll-like Receptor 4.

The Three Bacterial Lines of Defense against Antimicrobial Agents.

Defense system in the biliary tract against bacterial infection.

Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens.

In vitro activity of tosufloxacin against bacterial enteric pathogens.