Functional role of formate dehydrogenase 1 (FDH1) for host and nonhost disease resistance against bacterial pathogens.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.

Introduction

Nonhost resistance provides basic protection to plants and is also the most durable form of resistance to the majority of potential pathogens [1-3]. In general, both basal and nonhost resistance are controlled by quantitative trait loci (QTL). Disease resistance traits conferred by these QTLs have been widely used for developing new varieties for disease resistance [3-8]. In addition to QTLs, a number of studies have identified major plant genes involved in nonhost resistance against fungal and bacterial pathogens [2, 3, 6, 8–10]. However, the mechanism of nonhost resistance is not fully understood. Nonhost resistance against bacterial pathogens can be broadly classified as two types; type I (no visible hypersensitive response [HR] cell death) and type II (HR cell death) nonhost resistances [2]. The efficacy of nonhost disease resistance could be based on the recognition of pathogen-associated molecular patterns (PAMPs) and/or pathogen effectors. A number of studies have showed that genes associated with nonhost resistance is often involved in basal defense mechanism (pre- or post-invasive defense). For instance, stomatal innate immunity is an important mechanism of pre-invasive defense. Proteins involved in post-invasive responses could activate HR-type cell death or ROS accumulation to develop nonhost resistance. Organelles such as mitochondria and chloroplast have been well described for their important role for the ROS-mediated programmed cell death. PAMPs are mainly perceived at the plasma membrane where the PAMP-triggered immunity (PTI) could be induced as the first defense barrier against various pathogens [11, 12]. One known PTI response is stomatal closure which is circumvented by the phytotoxin coronatine (COR) produced by the host pathogen P. syringae pv. tomato DC3000 [13]. COR has structural and functional similarity to jasmonates and jasmonic acid-isoleucine (JA-Ile), and contributes to the virulence of P. syringae pv. tomato DC3000 [14-16]. COR disrupts the accumulation of the plant defense hormone salicylic acid (SA) for stomatal reopening and bacterial propagation in both local and systemic tissues of Arabidopsis [17]. COR is also involved in promoting the entry of nonhost bacterial pathogens via stomata and nonhost bacterial growth at the initial stage of infection [18]. In addition to PTI, a number of pathogen effectors secreted into host cells can also induce another type of defense response referred to as effector-triggered immunity (ETI) [19, 20]. ETI is typically associated with resistance proteins belonging to the nucleotide-binding domain (NBD) and leucine-rich repeat-containing (NLR) family. ETI triggers a type of cell death known as the HR [20]. Despite the plant immune systems, compatible host bacterial pathogens in susceptible plants suppress both basal and nonhost resistance responses thus causing disease.

Formate dehydrogenase 1 (FDH1) is a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that catalyzes the NAD-linked oxidation of formate to carbon dioxide. As a component of one-carbon metabolism in plants, most FDHs play an important role in response to various stresses in higher plants [21-26]. A previous report has shown that FDH1 regulates programmed cell death (PCD) in pepper against bacterial pathogens [23]. There is contradictory information regarding the localization of FDH1 in plant cell. According to the study by Choi (2014), FDH1 localizes to mitochondria and plays a role in hypersensitive cell death and the defense signaling pathway against bacterial pathogens in pepper. Several other reports also suggest there is mitochondrial localization of FDH1 in tobacco [27, 28]. Interestingly, several reports have described that FDH1 targets not only mitochondria, but also chloroplasts for its biological function [29, 30]. Chloroplast and mitochondria are the major targets of plant pathogen effectors, and the effectors targeting of these organelles inhibits the production of defense molecules including reactive oxygen species (ROS) [31-33]. Chloroplasts play a major role in generating ROS and nitric oxide to trigger defense responses such as PCD and HR against bacterial pathogens [34-36]. Mitochondria and chloroplasts also have been reported as the initial organelle to recognize bacterial effectors and trigger plant immunity against bacterial pathogens [37, 38]. In other studies, the co-localizations of mitochondria with chloroplasts has been well characterized [39-41]. The physical interactions between mitochondria and chloroplasts would provide the means to transfer genetic information directly to the organelle genome, as well as to mediate signaling transduction [42, 43]. However, how chloroplast and mitochondria are functionally integrated for bacterial disease resistance is not fully understood. Particularly, previous conflicting results regarding the cellular localizations of FDH1 may suggest possible roles of FDH1 in the chloroplast as well as mitochondria for bacterial disease resistance.

In the current study, we demonstrated the novel role of FDH1 in nonhost disease resistance in Nicotiana benthamiana and Arabidopsis. The cellular localization of FDH1 was confirmed to be mitochondria, but it was also found that the protein targets chloroplasts during the defense responses against host and nonhost bacterial pathogens. We speculate that FDH1 may coordinate mitochondria- and chloroplast-mediated defense responses against bacterial pathogens in plants.

Materials and methods

Plant materials

Nicotiana benthamiana plants were grown in 10-centimeter diameter round pots with BM7 soil (SUNGRO Horticulture Distribution, Inc., Bellevue WA) in the greenhouse using the conditions described in the previous study [44]. Plants grown four weeks were used for virus-induced gene silencing (VIGS) experiments as described below. The ecotype of Arabidopsis thaliana, Col-0, was used as wild-type. Arabidopsis T-DNA knockout mutants for AtFDH1 gene (At5g14780), SALK_118644 and SALK_118548, were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). To identify the homozygous knockout T-DNA mutant plants, seedlings grown from the SALK_118644 and SALK_118548 seeds and their progeny were harvested for PCR-based genotyping. Primers were designed from SALK T-DNA verification primer design (http://signal.salk.edu/tdnaprimers.2.html), and PCR was performed using REDExtract-N-Amp™ Tissue PCR Kit (Sigma-Aldrich, St. Louis, MO). All mutant plants were made homozygous for their respective T-DNA insertion, and seeds were harvested for further experiments. For seedling-flood inoculation (45), Arabidopsis plants were grown in ½ Murashige and Skoog (MS) agar medium plates at 25°C under short day condition (12 h light).

VIGS in Nicotiana benthamiana

VIGS in N. benthamiana was performed as described [2]. In brief, Agrobacterium tumefaciens GV2260 containing TRV1, TRV2::00, and TRV2::NbFDH1 was grown overnight on LB medium containing antibiotics (rifampicin, 25; kanamycin, 50) at 28°C. Bacterial cells were harvested and re-suspended in induction medium (10 mM MES, pH 5.5; 200 μM acetosyringone), and incubated at room temperature on an orbital shaker for 5 hrs. Bacterial cultures containing TRV1 and TRV2 were mixed in equal ratios (OD600 = 1) and infiltrated into N. benthamiana leaves using a 1 ml needleless syringe. The infiltrated plants were maintained in a greenhouse and used for studies 15 to 21 days post-infiltration.

Bacterial culture and inoculation

Bacterial pathogens, Pseudomonas syringae pv. tabaci (Pstab), P. syringae pv. tomato T1 (Pst T1), and P. syringae pv. maculicola (Psm) were grown in King’s B (KB) medium at 28°C overnight. The bacterial culture was centrifuged at 5,000 rpm for 10 min, and the cell pellet was re-suspended in 5 ml sterilized distilled water. For the inoculation assays in N. benthamiana, bacterial vacuum infiltration was performed using the concentration of 1×104 CFU/ml for both N. benthamiana host (Pstab) and nonhost (Pst T1) pathogens. For the inoculation assays in Arabidopsis, host (Psm) and nonhost (Pstab) pathogens were used for the inoculation followed by the seedling flood-inoculation method [45, 46].

Bacterial disease assay in N. benthamiana and Arabidopsis

For disease assays in N. benthamiana, bacterial suspensions of host and nonhost pathogens (1×105 CFU/ml) were vacuum-infiltrated in both silenced and control plants 2-week after TRV infection. The fully expanded leaves were used for disease assays, and the inoculated plants were kept in a growth chamber at 20–22°C. The number of bacterial cells in leaf apoplast were measured 1, 2, and 3 days after inoculation in N. benthamiana. The bacterial population at day 0 was estimated from leaves harvested 1 hr after inoculation. Two leaf discs (0.5 cm2) from each leaf were collected in 1.5 ml centrifuge tube containing 100 ul of sterilized distilled water. Samples were homogenized and plated on KB agar medium for measuring colony-forming units (CFU) per cm2 of leaf area. A total of three leaves were used for each experiment. To visualize bacterial colonization at infected sites in leaves, GFPuv-expressing P. syringae pv. tabaci and P. syringae pv. tomato T1 were vacuum infiltrated, and plants were examined under UV light 3 days after inoculation [47].

For disease assays in Arabidopsis, a flood inoculation method was used to infect Arabidopsis [45, 46]. Disease symptoms were observed 3 days after inoculation. For bacterial counting, leaves were surface-sterilized with 10% bleach for one min to eliminate epiphytic bacteria and then washed with sterile distilled water twice. The leaves were then homogenized in sterile distilled water, and serial dilutions were plated onto KB plates. Bacterial growth was evaluated in three independent experiments.

FDH1 protein localization in N. benthamiana and Arabidopsis

The full-length sequence of AtFDH1 with native promoter was cloned into pMDC107 for GFP expression (AtFDH1-GFP). Stable Arabidopsis transgenic lines for the expression of AtFDH1-GFP were developed by floral dip transformation [48]. The localization of AtFDH1-GFP in epidermal cells was determined under the confocal laser scanning microscope (NIKON, Japan).

To observe the localization of AtFDH1, Arabidopsis wild-type Col-0 and AtFDH1-GFP expressing (under the control of AtFDH1 promoter) transgenic plants in Col-0 were grown in ½ MS media for four weeks, and AtFDH1-GFP expression in epidermal cells of Arabidopsis was visualized using a confocal laser scanning microscope (NIKON, Japan). The leaf tissues were floated with the bacterial suspension of host pathogen P. syringae pv. maculicola (1×106 CFU/ml) and nonhost pathogen P. syringae pv. tabaci (1×106 CFU/ml). After one hour inoculation, the leaf tissues were washed with distilled water, and localization of FDH1-GFP was observed. For wounding stress, the adaxial epidermal peels from wild-type Col-0 and AtFDH1-GFP expressing transgenic plants were prepared in the MES buffer (10 mM, pH 6.5), and localization of AtFDH1 was imaged under the confocal laser scanning microscope (NIKON, Japan).

Isolation of chloroplast and mitochondria

Arabidopsis leaves (10 g) were homogenized in 100 ml of grinding buffer containing 50 mM HEPES (pH 8.0), 2 mM EDTA, 1 mM MgCl2, 0.33 M sorbitol, and 0.5 g/L BSA by using a motor-driven blender (WARING 51BL30, two 5 s bursts at maximum speed). The homogenate was filtered through 3 layers of miracloth (Sigma-Aldrich, St. Louis MO, USA). The cleared homogenate was centrifuged at 1,500 g for 10 min at 4°C. The supernatant was used for isolation of mitochondria, and the pellet was used for chloroplast extraction. For the isolation of chloroplast, the pellet was re-suspended in 3 ml of grinding buffer with a paint brush. The chloroplast suspension was then loaded on top of linear Percoll gradient (2 ml of 70% PBF-Percoll (v/v), 4 ml of 50% PBF-Percoll (v/v), and 4 ml of 40% PBF-Percoll (v/v)) and centrifuged at 16,000 g for 20 min at 4°C. The lower green bands were collected for intact chloroplasts with a glass pipette, washed twice with wash buffer (50 mM HEPES, pH 8.0, 2 mM EDTA, 1 mM MgCl2, 0.33 M sorbitol), and centrifuged at 1,500 g for 10 min at 4°C. The supernatant was discarded and the washed chloroplast pellet was collected for chloroplast protein extraction.

For the isolation of mitochondria, the supernatant was centrifuged at 3,000 g for 5 min at 4°C. The supernatant was transferred into a fresh centrifuge tube and centrifuged at 18,000 g for 20 min at 4°C. The greenish mitochondrial pellet was re-suspended carefully in 1 ml wash buffer with a fine paint brush and adjusted the final volume to 4.8 ml. 1.2 ml of 100% Percoll (Sigma-Aldrich, St. Louis, MO) was added and the total 6 ml of mitochondria homogenate was then loaded on top of linear Percoll gradient (5ml of 80% PBF-Percoll (v/v), 5ml of 33% PBF-Percoll). The mitochondria homogenate was centrifuged at 18,000 g for 1 hr and greenish upper band was collected. Mitochondria was rinsed twice with 15 ml wash buffer and centrifuged at 18,000 g for 20 min at 4°C. The supernatant was removed and the pellet was saved for mitochondria protein extraction.

Protein extraction from chloroplast and mitochondria

The mitochondrial and chloroplast proteins were isolated [49] in protein extraction buffer; 50 mM Tris-HCL, pH 7.5, 75 mM NaCl, 0.2% Triton X-100, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 100 uM MG132, 10 mM NaF, 2 mM Na2VO4, and 1% protease inhibitor cocktail (Sigma Aldrich, St. Louis, USA). The extracted proteins were quantified using Bradford method [50], and equal known concentrations were taken for the assay. Proteins were blotted on a polyvinylidene fluoride (PVDF) membrane and Cox II antibody (Agrisera, Sweden, cat no. AS04 053A) for mitochondria and Rubisco or RBCL (Abiocode, CA, USA, cat.no. R3352-2) for chloroplast was used as markers to confirm the proteins. GFP antisera (Miltenyl Biotec, San Diego, CA, USA cat. no. 130-091-833) was used to detect the FDH1 protein levels. The primary HRP-conjugated GFP antisera were diluted to 1:10,000 and visualized using ECL solution (GE Healthcare Bio-Sciences, Pittsburgh, USA) and protein gel blots were imaged. The raw image data for western blot analysis is show in (S6 Fig).

Quantitative reverse transcription PCR (RT-qPCR) analysis

Total RNA was extracted from Arabidopsis leaves infiltrated with water (mock control), host pathogen (P. syringae pv. maculicola) and nonhost pathogen (P. syringae pv. tabaci), sampled at 0, 12 and 24 hrs post-inoculation (hpi). RNA samples were treated with DNAseI (Ambion, Austin, TX) and used for cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen, Grand Island, NY, USA). The cDNA was diluted to 1:20 and used for RT-qPCR using Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) with an ABI Prism 7900 HT sequence detection system (Applied Biosystems, Foster City, CA, USA). Arabidopsis Ubiquitin 5 (UBQ5) and Elongation factor 1α (EF1α) were used as internal controls to ensure an equal amount of cDNA in individual reactions. Average Cycle Threshold (Ct) values calculated using Sequence Detection Systems (version 2.2.2; Applied Biosystems) from duplicate samples and were used to determine the fold expression relative to controls. Two biological replicates of each sample and three technical replicates of each biological replicate were analyzed for RT-qPCR analysis.

Results

Formate dehydrogenase 1 is involved in nonhost disease resistance

Using virus-induced gene silencing (VIGS)-based forward genetics screening in Nicotiana benthamiana, we identified the clone 24E07 (NbME24E07) to be involved in nonhost disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato T1 [44, 51]. The cDNA insert in 24E07 clone was sequenced and BLAST results of the sequence showed that it was homologous to NbFDH1. Protein sequence analysis showed that NbFDH1 is 96% identical to SlFDH1 and 80% identical to AtFDH1 (S1 Fig). FDH1 is a single copy gene in both monocot and dicot plants.

Tobacco rattle virus (TRV)-based VIGS of NbFDH1 in N. benthamiana plants did not cause a visible phenotype regarding plant appearance. The downregulation of NbFDH1 was about 70% in TRV::NbFDH1 inoculated plants when compared to TRV:00 (non-silenced control) inoculated plants (S2 Fig). NbFDH1-silenced and non-silenced control plants were inoculated with host and nonhost pathogens. Upon vacuum infiltration with the nonhost pathogen P. syringae pv. tomato T1 containing pDSK-GFPuv [41] at 1×104 CFU/ml concentration, the bacteria multiplied more in NbFDH1-silenced plants than the non-silenced control as visualized by green fluorescence under UV light (Fig 1A). NbFDH1 silenced plants showed necrotic disease symptoms in infected leaf tissues, while no disease symptoms were observed in the non-silenced control (Fig 1A). Further, the bacterial titer of nonhost pathogen P. syringae pv. tomato T1 was measured for three consecutive days after inoculation in both the NbFDH1-silenced and non-silenced control plants. Consistent with the disease symptoms and green fluorescence observed, NbFDH1-silenced plants had more bacterial titer compared to the non-silenced control (Fig 1B). In contrast to the nonhost pathogen, multiplication of the host pathogen P. syringae pv. tabaci was not different in NbFDH1 silenced plants when compared to non-silenced control (Fig 1C).

Fig 1

Virus-induced gene silencing of NbFDH1 compromises nonhost resistance and elicitation of hypersensitive response in N. benthamiana.

(A) GFP fluorescence associated with bacterial multiplication of nonhost bacteria in NbFDH1 silenced N. benthamiana leaves. Two weeks old N. benthamiana seedlings were inoculated with TRV1 + TRV::00 (control) or TRV1 + TRV::NbFDH1. Three weeks after TRV inoculation, nonhost bacterial pathogen P. syringae pv. tomato T1 expressing pDSK-GFP was vacuum infiltrated at 1×104 CFU/ml concentration. The photograph was taken under UV light 2 days post infection (dpi) as show in the upper panel. Visual disease symptoms were photographed at 5 dpi (lower panel). An increase in GFP fluorescence and disease symptoms were observed in TRV::NbFDH1 inoculated but not in the TRV::00 inoculated plants. (B) and (C) Bacterial titer of host and nonhost pathogens in both NbFDH1-silenced and control plants. TRV inoculated plants (described above) were vacuum inoculated with host (P. syringae pv. tabaci) or nonhost (P. syringae pv. tomato T1) bacterial pathogens (1×104 CFU/ml), and bacteria were quantified by plating serial dilutions of leaf extracts. Asterisks indicate a significant difference from the control using Student’s t test (P < 0.01). Bars represent mean, and error bars represent the standard deviation of three biological replicates (three technical replicates were used for each biological replicate). Each experiment showed similar results. (D) HR-related cell death in NbFDH1-silenced and control plants. High concentration (1×106 CFU/ml) of nonhost pathogen P. syringae pv. tomato T1 was infiltrated using a needless syringe into fully expanded N. benthamiana leaves, three weeks after TRV inoculation. Cell death due to nonhost HR was observed and photographed 24 and 48 hpi.

To check if NbFDH1 has a role in nonhost HR, NbFDH1-silenced and non-silenced control plants were syringe-infiltrated with a high level of inoculum (1×106 CFU/ml) of the nonhost pathogen P. syringae pv. tomato T1. Non-silenced control showed a typical nonhost HR after 24 hpi whereas in NbFDH1-silenced lines, the HR was delayed until 48 hpi (Fig 1D). Together, these results suggest that NbFDH1 plays a role in nonhost disease resistance against P. syringae pv. tomato T1 in N. benthamiana.

Arabidopsis fdh1 mutants show increased susceptibility to host-pathogen and nonhost pathogens

Two Arabidopsis T-DNA insertion mutants (Col-0 background) for AtFDH1 gene (SALK118548: Atfdh1-1 and SALK118644: Atfdh1-3) were identified in the Arabidopsis T-DNA insertion lines and were obtained from the Arabidopsis Biological Resource Center. Homozygous T-DNA insertion lines were generated by selfing and confirmed by PCR. When wild-type (Col-0) and Atfdh1 mutants were flood inoculated [45, 46] with the nonhost pathogen P. syringae pv. tabaci, Atfdh1 mutants showed disease symptoms characterized by chlorosis at 5-day post inoculation (dpi), while Col-0 did not (Fig 2A). In addition, Atfdh1 mutants had higher bacterial titer (approximately 18-fold) when compared to Col-0 plants at 3 dpi (Fig 2B). In response to an infection with a host pathogen, P. syringae pv. maculicola, both Col-0 and the Atfdh1 mutants showed similar disease symptoms (Fig 2A). Interestingly, in contrast to the observation in NbFDH1-silenced N. benthamiana where the host pathogen titer did not differ between silenced and control plants, Arabidopsis host pathogen, P. syringae pv. maculicola, grew slightly more in the Atfdh1 mutants when compared to Col-0 (Fig 2B).

Fig 2

Arabidopsis Atfdh1 mutants are compromised in basal, nonhost, and gene-for-gene resistance.

(A) Disease symptoms of Atfdh1-1 mutant after inoculation with host or nonhost pathogens. Two-week-old Arabidopsis wild-type (Col-0) and Atfdh1-1 mutants grown in 1/2 strength MS medium under short-day conditions (8 hrs of daylight) were flood-inoculated with host (P. syringae pv. maculicola) or nonhost (P. syringae pv. tabaci) pathogens at 3×106 CFU/ml. Photographs were taken at four days post inoculation (dpi). (B) Bacterial titer of host and nonhost pathogens in Atfdh1 mutants. Two-week-old Arabidopsis Col-0 and two Atfdh1 mutant alleles (Atfdh1-1 and Atfdh1-3) were flood-inoculated with host (P. syringae pv. maculicola) or nonhost (P. syringae pv. tabaci) pathogens at 1×105 CFU/ml. Bacterial titers at 0 to 3 dpi were measured by taking leaf disks from four inoculated plants for each line. (C) Quantification of host bacterial multiplication during gene-for-gene resistance. Leaves from 6-week-old plants of Col-0 and Atfdh1 mutant alleles were syringe-infiltrated with avirulent (P. syringae pv. tomato DC3000 [AvrRps4]) bacterial strain at 2.8×105 CFU/ml concentration. Bacterial titer was measured at 0 and 3 dpi. Bars represent mean, and error bars represent standard deviation for four biological replicates with two independent experiments. The bacterial growth was similar between the experiments. Asterisks above bars represent statistically significant differences in comparison with wild-type plants using Student’s t-test (P < 0.05).

To check if AtFDH1 plays a role in gene-for-gene resistance, we infected Arabidopsis Col-0 plants that carry multiple resistance (R) genes, including RPS4 with avirulent P. syringae pv. tomato DC3000 (AvrRPS4). After 3 dpi, P. syringae pv. tomato DC3000 (AvrRPS4) grew ~3 logs in wild-type Col-0, but a significantly higher growth of bacteria was observed in the Atfdh1 mutant lines (Fig 2C). The delayed HR-associated cell death was also found in NbFDH1-silenced N. benthamiana plants (Fig 1D). These results suggest that AtFDH1 confers plant defense associated with gene-for-gene resistance mechanisms.

AtFDH1 is induced in response to host and nonhost bacterial pathogens

In the publically available gene expression databases (TAIR), AtFDH1 is strongly expressed after 24h of inoculation with the virulent pathogen P. syringae pv. tomato DC3000 and the avirulent pathogen P. syringae pv. tomato (AvrRPM1) (https://www.arabidopsis.org/servlets/TairObject?id=136173&type=locus; S3A Fig). This agrees with the previous study of mitochondrial FDH1 in pepper [23]. We also found that AtFDH1 gene expression is induced after host or nonhost pathogen inoculation (S3B Fig). After inoculation with the virulent pathogen P. syringae pv. maculicola, FDH1 expression increased slightly (less than 0.5-fold) in comparison to mock-inoculated plants. Inoculation with the nonhost pathogen P. syringae pv. tabaci caused a higher induction of FDH1 and its level of expression was about 2-fold higher than in mock-inoculated plants (S3B Fig). These results suggest that FDH1 may play a greater role in nonhost disease resistance.

Mutation of AtFDH1 alters the SA-mediated defense hormonal pathway to bacterial pathogens

The gene expression of AtFDH1 was induced in response to both host and nonhost pathogens (S3A and S3B Fig). To examine if the resistance mechanism was related to a known common defense pathway, such as salicylic acid (SA) and Jasmonic acid (JA), we conducted quantitative RT-PCR (RT-qPCR) for the expression of plant defense related genes in wild-type Col-0 and the Atfdh1 mutant without any pathogen inoculation and at 24 hpi with the host pathogen P. syringae pv. maculicola or the nonhost pathogen P. syringae pv. tabaci. These genes were composed of three representative genes related to SA pathway (PAD4, EDS1, and NPR1) and one gene related to JA pathway (PDF1.2). After 24 hpi with either pathogen in Col-0, the SA marker genes, PAD4 and EDS1, and JA marker gene PDF1.2were strongly induced, but the level of induction of these genes was significantly lower in the Atfdh1 mutant against both host and nonhost pathogens, compared to Col-0 (Fig 3). NPR1 was significantly induced at 24 hpi with the host pathogen in wild-type Col-0 and decreased 5-fold in the Atfdh1 mutant. NPR1 was not significantly induced after inoculation with the nonhost pathogen in both mutant and wild-type lines. These results suggest that AtFDH1 plays a role in plant defense responses via SA and JA mediated defense pathways.

Fig 3

Patterns of gene expression associated with SA-mediated defense signaling pathways in wild type (Col-0) and Atfdh1 mutant (Atfdh1-1).

The expression of SA-mediated defense-related genes were examined after 24 hrs post inoculation (hpi) in response to host, P. syringae pv. maculicola, and nonhost pathogen, P. syringae pv. tabaci. Four weeks old seedlings were flood-inoculated with the concentration of 1×105 CFU/ml bacterial suspension. Each column is the fold change of gene expression as determined by RT-qPCR at 24 hpi in pathogen-inoculated samples. The relative gene expression values normalized by Ubiquitin5 (UBQ5) and Elongation factor 1 alpha (EF1α) are represented as n-fold compared to the mock-treated plants. Fold changes are over the non-treated Col-0 or mutants. Asterisks above bars represent statistically significant differences in comparison with wild-type using Student’s t-test (P < 0.05). The gene expression was examined with four biological samples (three technical repeats for each sample). Psm24: 24 hours after the inoculation of P. syringae pv. maculicola, Pst24: 24 hours after inoculation of P. syringae pv. tabaci.

AtFDH1 localizes predominantly in mitochondria and targets to chloroplasts for bacterial defense responses

Localization of FDH1 in mitochondria and/or chloroplast has been the subject of extensive debate [23, 27–30]. We cloned AtFDH1 to be expressed under its native promoter and fused it to the C-terminal of Green Fluorescent Protein (GFP) gene and transiently expressed it in N. benthamiana. The results showed that AtFDH1-GFP predominantly localizes to mitochondria (S4 Fig). We generated Arabidopsis stable lines expressing AtFDH1-GFP in Col-0, and the localization of AtFDH1-GFP in mitochondria was confirmed using the live cell mitochondrial stain MitoTracker (Fig 4A). Upon challenging the plant with host (P. syringae pv. tomato DC3000) and nonhost (P. syringae pv. tomato T1) bacterial pathogens, AtFDH1-GFP signal was also found at the outer envelope membrane of chloroplasts in addition to mitochondria (Fig 4B). Similar results were observed with another nonhost pathogen (P. syrinage pv. phaseolicola) (S5 Fig). The expression of AtFDH1-GFP (observed as green fluorescence) was remarkably higher after nonhost (P. syringae pv. tomato T1 and P. syringae pv. phaseolicoa) and host (P. syringae pv. tomato DC3000) pathogen treatments than the expression in the detached leaf sample without pathogen challenge (Figs 4B and S5).

Fig 4

Localization of AtFDH1 in Arabidopsis leaves in response to host and nonhost bacterial pathogens.

AtFDH1 is localized to mitochondria (A). The expression and localization of AtFDH1-GFP was observed in peeled adaxial epidermal cells from leaves of transgenic Arabidopsis lines expressing AtFDH1-GFP. The protein localization was also examined in detached leaf samples after the treatment of P. syringae pv. tomato DC3000 (1×105 CFU/ml), P. syringae pv. tomato T1 (1×105 CFU/ml), and P. syringae pv. tabaci (1×105 CFU/ml) under a confocal microscope (B). MitoTracker red dye was used to stain mitochondria. Bars = 10 μm. For MitoTracker Red, a 561 nm excitation, 570–620 nm emission filter was used. Red channel (680 nm emission filter) shows chlorophyll auto fluorescence in chloroplasts of mesophyll cells. Green channel shows the fluorescence signal of AtFDH1-GFP in mitochondria and outer membrane of chloroplast. In merged images, the fluorescence signal overlapped by MitoTracker and AtFDH1-GFP is shown in yellow. In chloroplast image, yellow represents a merged signal of FDH1-GFP localization in mitochondria and chloroplast.

Using the light-sheet microscope, the movement and co-localization of mitochondria with chloroplasts were observed after the inoculation of nonhost pathogen, P. syringae pv. tomato T1 and P. syringae pv. tabaci. By performing a time lapse image of FDH1 localization in the transgenic Arabidopsis line expressing FDH1-GFP, we found that mitochondrial specific FDH1 localization was highly motile after inoculation with a nonhost pathogen. As shown in the Fig 5, the majority of mitochondria localized FDH1 was in the vicinity of chloroplasts in outer-membrane regions. The arrow in each image (every 1.5 min) shows the movement of mitochondria localized FDH1 around chloroplasts. In the time-lapse image, we observed that the mitochondria (FDH1::GFP) aggregate with chloroplasts and later goes apart from the chloroplast. After this event, other mitochondria localized FDH1 translocate again to chloroplasts, and we observed the event of co-localization continuously during the 15 min of time-lapse imaging (S1 Video). These results suggest that in response to nonhost pathogen, FDH1 co-localizes to both mitochondria and chloroplast and the localization of FDH1 in chloroplast is transient.

Fig 5

Time lapse imaging of co-localization of AtFDH1 in mitochondria and chloroplasts in response to nonhost pathogen.

Detached leaves from transgenic Arabidopsis plants expressing AtFDH1-GFP driven by AtFDH1 promoter were treated with P. syrinage pv. tomato T1 (1×105 CFU/ml). AtFDH1 expression was monitored 40 min after the pathogen infection by using a light-sheet fluorescence microscope (Carl Zeiss, Germany). The images were observed for 15 min by time lapse imaging at 30 second intervals in Z-stack mode. For every 30 sec, the AtFDH1-GFP and chlorophyll fluorescence of chloroplast was captured and both live streaming videos were merged to generate time lapse video simultaneously. Images were taken from detached leaf samples 60 min after treatment of P. syrinage pv. tomato T1. Times shown at bottom-right of each image are in minutes:seconds.

To further investigate the specific localization of AtFDH1 in mitochondria and chloroplast upon host and nonhost pathogens, the protein of mitochondria and chloroplast were isolated separately from AtFDH1-GFP expressing plants and examined for the presence of AtFDH1 protein. Immunoblot analysis revealed that in total protein extract, AtFDH1-GFP accumulates in response to host and nonhost pathogens at 2 and 4 hpi, which coincides with the result of RT-qPCR (Figs 6 and S3). To validate the localization of AtFDH1, we isolated mitochondria and chloroplasts from AtFDH1-GFP expressing plants upon inoculation with host or nonhost pathogen. Mitochondria and chloroplast proteins were individually extracted and subjected to immunoblot analyses. AtFDH1-GFP protein was detected in mitochondria prior to pathogen infection, and the protein amount increased significantly after host or nonhost pathogen infection (Fig 6). By contrast, AtFDH1-GFP protein was not detected in the chloroplast protein extract prior to pathogen infection. Consistent with the cell biology data, AtFDH1-GFP was detected in the chloroplast protein extract after infection with host or nonhost pathogen infection (Fig 6). However, the accumulation of AtFDH1 protein was only found in the chloroplast protein fraction at 2 hours after infection with nonhost pathogen, and the expression level was much higher at 4 hpi when compared to host pathogen (Fig 6).

Fig 6

Accumulation of AtFDH1 protein in response to host or nonhost pathogens in mitochondria and chloroplasts.

The 8-week-old Arabidopsis wild-type (Col-0) were flood-inoculated with the concentration of 1×105 CFU/ml bacterial suspension of P. syringae pv. maculicola (host) or P. syringae pv. tabaci (nonhost) pathogens. Leaf samples were collected at 0, 2, and 4 hpi for the protein extraction, and 3 μg protein from mitochondria or chloroplast was used for the immunoblot assay (A). Because no AtFDH1-GFP was visible in chloroplast samples with 3 μg total protein, a total of 28 μg was used. Rubisco: internal control for total protein (bromophenol blue stained gel), COXII: mitochondria marker protein detected using polyclonal COXII antisera (Agrisera), RBCL: chloroplast marker protein detected using polylonal Rbcl antisera (Abiocod). The band intensities on western blot membrane were measured using ImageJ software (https://imagej.nih.gov/ij/) for comparison (B). The bars show the mean values and standard deviations of the mean (* 0.05 > P and ** 0.01 > P). The experiments were replicated three times. The level of AtFDH1-GFP was determined under confocal microscope after infection of host (P. syrinage pv. maculicola) and nonhost pathogen (P. syringae pv. tabaci) (C).

Discussion

FDH enzyme is found in various organisms such as bacteria, yeast, and plants. This protein has been reported to function during various abiotic and biotic stress responses. Expression of FDH is strongly induced during various abiotic and biotic stress responses such as pathogen, hypoxia, chilling, drought, dark, wounding and iron deficiency [21, 23, 24]. There is only one study showing that FDH1 is involved in regulating plant cell death and defense responses against bacterial pathogens in pepper plants [23]. In this study, mitochondrial targeting of FDH1 played an important role in PCD- and SA-dependent defense response, and silencing of FDH1 attenuates resistance against X. campestris pv. vesicatoria pathogen in pepper plants. Our study demonstrates that FDH1 is involved in plant innate immunity against both host and nonhost bacterial pathogens. Nonhost disease resistance is the most common form of plant defense against various pathogens [2, 5, 52–54]. HR cell death are typical symptoms in response to ETI-triggered nonhost resistance in plants [20]. ROS produced in various cellular compartments, including chloroplasts, mitochondria, and peroxisomes have been proposed to act as signals for HR and PCD [55-57]. Chloroplasts are the main source of ROS during various environmental stresses, including plant-pathogen interactions [33, 36]. In addition, ROS generated in mitochondria (mtROS) has been described in several studies to be an important factor in inducing HR cell death against plant pathogens [38, 57]. Possibly both chloroplasts and mitochondria have a role in nonhost resistance against invading bacterial pathogens. In this study, we demonstrate that the protein encoded by a single FDH1 gene in the nuclear genome is targeted to both mitochondria and chloroplasts in response to wounding and bacterial pathogens. Chloroplast localization of FDH1 was more abundant after inoculation with nonhost pathogens (Figs 4 and 5), thus suggesting a probable role of chloroplasts in nonhost disease resistance. A previous study has shown that chloroplast generated ROS is required for nonhost disease resistance in Arabidopsis [58]. In addition to nonhost resistance, we also show that FDH1 plays a role in basal and gene-for-gene resistance in Arabidopsis. It is intriguing that the silencing of NbFDH1 did not compromise basal resistance in N. benthamiana. Since the silencing of NbFDH1 decreased NbFDH1 transcripts by ~70%, we speculate that this might not be sufficient to compromise basal resistance. By contrast, the complete knockout of AtFDH1 in Arabidopsis compromised basal resistance.

Our study identified a dual-targeting role for AtFDH1 during plant defense responses against bacterial pathogens. Dual targeting of FDH1 to mitochondria and chloroplasts may be necessary for effective signaling during plant defense against bacterial pathogens. In the Arabidopsis nuclear genome, approximately 20–25% of the genes encode proteins that are targeted to either mitochondria or chloroplasts [59]. It has been reported that some proteins target to both mitochondria and chloroplast, and might be more common than thought. However, their functions are not well understood, especially for plant disease resistance [59-62]. FDH1 has a putative mitochondrial signal peptide, although AtFDH1 has been reported to localize to either mitochondria or chloroplasts [30, 63–65]. Therefore, FDH1 localization in plants remains controversial. One study showed the dual localization of AtFDH1 in both chloroplasts and mitochondria when AtFDH1 is overexpressed in transgenic Arabidopsis and tobacco plants [26, 66]. It is also reported that the N-terminal region of AtFDH1 is predicted to contain the signal peptide region that could target it to chloroplasts as well as mitochondria [67]. This N-terminal sequence of AtFDH1 is quite different from potato, barley, and rice, suggesting AtFDH1 localizing in chloroplast could occur under certain conditions [65]. In our study, the localization of AtFDH1 in chloroplast was mainly detected under the conditions of wounding and pathogen stresses (Figs 4 and 5). As previously described, FDH1 is highly induced under various stress conditions [65]. It is possible that the localization of FDH1 in chloroplast is low and transient to be detected under non-stress conditions, and this could cause controversy of the FDH1 localization in mitochondria or chloroplasts or both.

There are few reports that suggest FDH1 may have a role in biotic stress response in plants. As mentioned above, FDH1 has been shown to play a role in disease resistance in pepper against a bacterial pathogen [23]. FDH1 and Calreticulin-3 precursor (CRT3) directly interacts with the helicase domain of Cucumber mosaic virus (CMV) isolate-P1, suggesting that FDH1 has an important role in plant disease resistance [68]. CRT3 is localized in the endoplasmic reticulum (ER) lumen, and has been known to associate with abiotic stress response and plant immunity [69-71]. FDH1 directly interacts with RING-type ubiquitin ligase Keep on Going (KEG), which is localized in trans-golgi and early endosomes [25]. In Arabidopsis, the loss of function in KEG disrupts the secretion of the apoplastic defense proteins such as pathogenesis-related PR1, which indicates the involvement of KEG in plant immunity [72]. There are several reports describing the ROS-based signal transmission between mitochondria and chloroplasts [33, 73–75]. Possibly, FDH1 protein could be associated with a signal transduction pathway for the production of chloroplast-derived ROS.

Conclusions

In this study, we demonstrated the possible role of chloroplast-dependent pathway that regulates plant innate immunity, probably through mitochondria-to-chloroplast integrated ROS signaling. Even though mitochondria are the main source of ROS, chloroplasts also play a role in producing ROS during stress responses in plants. However, the signal transduction between these organelles for coordinated production of ROS is not well understood. Characterization of molecular functions of FDH1-interactors in both mitochondria and chloroplasts would provide insight into the role of FDH1 in cross-talk between these organelles during biotic and abiotic stress responses.

Sequence alignment of FDH1 protein from N. benthamiana (NbFDH1), tobacco (NtFDH1), tomato (SlFDH1), and Arabidopsis (AtFDH1).

Sequence information was obtained from the public database; TAIR, NCBI GenBank, and Sol Genomics Network. The software MEGA-X [76] was used for sequence alignment. The amino acid colors were in accordance with the default coloring schemes of ClustalX alignment, which depends on both residue type and the pattern of conservation within a column (http://www.clustal.org/clustal2/).

(PPTX)

Click here for additional data file.

The expression of the NbFDH1 gene is reduced in NbFDH1-silenced N. benthamiana plants.

Two weeks old N. benthamiana seedlings were inoculated with TRV1 + TRV::00 (control) or TRV1 + TRV::NbFDH1. Three weeks after TRV inoculation, leaf samples from three different biological replicates for each construct were collected, and gene expression was measured by RT-qPCR. NbActin was used as internal control for normalization. Bars represent mean, and error bars represent standard deviation for three biological replicates (four technical replicates for each biological sample). Asterisk represents statistical significance that was determined using Student’s t-test, (P < 0.01).

(PPTX)

Click here for additional data file.

AtFDH1 is upregulated upon inoculation with host and nonhost pathogens in wild-type Col-0, and some defense-related genes are differentially expressed in Atfdh1 mutant.

(A) Gene expression patterns of AtFDH1 against P. syringae bacterial pathogen in Arabidopsis. This data was obtained from Arabidopsis eFP Browser at bar.utoronto.ca [77]. (B) AtFDH1 is induced by host and nonhost pathogen inoculations. Four-weeks-old Arabidopsis wild-type (Col-0) were flood-inoculated with host (P. syringae pv. maculicola, Psm) or nonhost (P. syringae pv. tabaci, Pstab) pathogens. The 24 hours after inoculation, leaves were harvested, total RNA was extracted, and subject to RT-qPCR using AtFDH1 specific primers. AtActin was used as an internal control for normalization. Bars represent mean, and error bars represent standard deviation for three biological replicates (four technical replications for each biological replicate). Asterisks represent statistical significance as determined using Student’s t-test, (P < 0.01).

(PPTX)

Click here for additional data file.

Localization of AtFDH1 in N. benthamiana.

For Agrobacterium-mediated transient assay, a binary vector containing GFP gene fused to the C-terminal of AtFDH1 was transformed into the A. tumefaciens strain GV3101. The Agrobacterium suspension was (5×107 CFU/ml) was infiltrated using a needleless syringe into N. benthamiana leaves, and the green fluorescence representing AtFDH1 localization was observed 3 days after the agroinfiltration. Red channel (a 561 nm excitation, 570–620 nm emission filter) shows mitochondria stained with MitoTracker dye and green channel shows AtFDH1-GFP localization. Bars = 10 μm.

(PPTX)

Click here for additional data file.

Localization of AtFDH1 in Arabidopsis leaves.

The expression and localization of AtFDH1-GFP was observed in detached (no stress) and peeled adaxial epidermal cells (pathogen stress) from leaves of transgenic Arabidopsis lines expressing AtFDH1-GFP in Col-0. The protein localization was also examined in detached leaf samples 1-hr after the treatment of P. syringae pv. tomato DC3000 (1×105 CFU/ml) and P. syringae pv. phaseolicola (1×105 CFU/ml). Red channel (a 561 nm excitation, 570–620 nm emission filter), showing chloroplast; green channel showing AtFDH1-GFP. Bars = 10 μm.

(PPTX)

Click here for additional data file.

Western blot analysis for the expression of AtFDH1-GFP in mitochondria and chloroplast after infection of host (P. syringae pv. maculicola) and nonhost (P. syringae pv. tabaci) pathogens.

(A) AtFDH1-GFP expression in total protein, (B) Internal control for rubisco expression for total protein, (C) AtFDH1-GFP expression in mitochondrial protein, (D) COXII expression for the internal control of mitochondrial protein, (E) AtFDH1-GFP expression in chloroplast protein, (F) RBCL expression for the internal control of chloroplast.

(PPTX)

Click here for additional data file.

Live video for time lapse imaging of co-localization of AtFDH1 in mitochondria and chloroplasts in response to nonhost pathogen.

Experimental methods are described in Fig 5.

(AVI)

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(PDF)

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(XLSX)

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(XLSX)

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(XLSX)

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23 Feb 2021

PONE-D-20-40925

Formate Dehydrogenase (FDH1) Localizes to Both Mitochondria and Chloroplast to Play a Role in Host and Nonhost Disease Resistance

PLOS ONE

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

This was an interesting topic that was generally well-received by the reviewers. However, in addition to several minor concerns that both reviewers raised, and that can likely be easily addressed, Reviewer 2 indicated that the localization studies were not yet compelling. I am in agreement that the use of a co-reporter will be useful here. If a suitable reporter is unavailable, other means of improving understanding of FDH1 localization would also be acceptable. In addition, comments made regarding the western blot analysis that should be addressed.

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Reviewer #1: The manuscript is overall well described the goals, importance and results; properly designed and soundly carried out, emphasizing the important of FDH1 in plant defense responses.

Minor points are;

In abstract, authors argued that SA signaling is involved in FDH1-mediated defense response. However, Fig. 3 and a corresponding chapter showed the role of both SA and JA in FDH1 activity. Authors should revise the abstract according to the Fig. 3 results, and may discuss potential implications on SA and JA coordination (?) or common understanding of SA/JA antagonisms (if possible, additional JA-marker genes could be added, and/or timely compared with SA-marker genes).

Line 245 and 398; the efficacy of NbFDH1-silencing was differently described between line 245 (70%) and line 398 (50%). Those ~20% may have somewhat critical effects on physiology, e.g., may explain different results observed between tomato-silencing and Arabidopsis-KO lines on the infection of host pathogens (Fig. 1C vs. 2B).

Line 292: chapter “AtFDH1 is … pathogens” compared the upregulation of FDH1 expressions in response to host or nonhost bacteria, and proposed a greater role of FDH1 in nonhost disease resistance. Is it possible that induction/response of FDH1 upregulation by nonhost bacteria is slower (or faster) than by host pathogens? RT-PCR on additional time points could confirm/strengthen the authors’ conclusion.

Reviewer #2: The manuscript entitled “Formate Dehydrogenase (FDH1) Localizes to Both Mitochondria and Chloroplast to Play a Role in Host and Nonhost Disease Resistance” by Lee et al. described isolation of a FDH1 gene encoding a formate dehydrogenase from two plant species, Nicotiana benthamiana and Arabidopsis thaliana. The authors concluded that the gene provides nonhost, basal and effector triggered immunity. Furthermore, they concluded that FDH1 in Arabidopsis is localized to mitochondria and following bacterial infection it moves to chloroplast.

It is an interesting study reporting new knowledge in the area of plant immunity.

Major Comments:

1. Localization section requires additional studies. It is hard to follow the photos relevant to mitochondrial and chloroplastic localization of FDH1. It will be best to co-localize FDH1 with a reporter protein; i. e, an RFP fused protein that has been already established to be a mitochondrial or chloroplast protein. Close-up photos will also help the readers to follow the changes in accumulation patterns following infection.

2. The western-blot looks good; but quantification has not made to claim that there is an enhanced accumulation of the fusion protein with time following infection (Figure 6).

This is a very interesting preliminary observation that FDH1 starts to accumulate in chloroplast following infection. Is not it possible that the protein is directly targeted to chloroplast from cytoplasm following infection? Note that in lane 3, the total protein is much more than that in mitochondria suggesting an increased synthesis of FDH1 following infection.

Minor Comments:

1. It is not evident in this study if FDH1 localization to mitochondria or chloroplast is involved in host and nonhost disease resistance; therefore, title may be revised.

2. The basal resistance section is less developed. How was it measured?

3. Lines 60 - 61: “The efficacy of nonhost disease resistance is based on the recognition of pathogen-associated molecular patterns (PAMPs) and/or pathogen effectors.” This statement should be revised as not all nonhost resistance mechanisms are controlled by receptors for PAMPs. A review of the nonhost resistance mechanisms/genes should be included in “Introduction.”

4. Line 252: The sentence starting with “In correlation with ….” should be revised.

5. Line 293: May delete the first section of the sentence up to the comma.

6. Lines 307-308: The sentence should be deleted. Basal resistance is not even referred in the previous paragraph.

7. Lines 324 -325: “AtFDH1 localizes predominantly in mitochondria, but translocates to chloroplasts in response to abiotic and biotic stresses” – No evidence is provided in support of the translocation of the protein from mitochondria to chloroplast.

8. The manuscript would be benefited from editing for English.

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Reviewer #1: No

Reviewer #2: No

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14 Dec 2021

Dear Editor,

Thank you for the valuable comments on our manuscript. We have followed the reviewers’ comments and thoughtfully revised the manuscript.

Please see below our responses and let us know for any questions.

Best Regards,

Seonghee Lee

-----------------------------------------------------------------------------------------------------------------------------------------

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

This was an interesting topic that was generally well-received by the reviewers. However, in addition to several minor concerns that both reviewers raised, and that can likely be easily addressed, Reviewer 2 indicated that the localization studies were not yet compelling. I am in agreement that the use of a co-reporter will be useful here. If a suitable reporter is unavailable, other means of improving understanding of FDH1 localization would also be acceptable. In addition, comments made regarding the western blot analysis that should be addressed.

Please submit your revised manuscript by Apr 02 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

1. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

2. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

3. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Reviewer #1: The manuscript is overall well described the goals, importance and results; properly designed and soundly carried out, emphasizing the important of FDH1 in plant defense responses.

Minor points are;

In abstract, authors argued that SA signaling is involved in FDH1-mediated defense response. However, Fig. 3 and a corresponding chapter showed the role of both SA and JA in FDH1 activity. Authors should revise the abstract according to the Fig. 3 results, and may discuss potential implications on SA and JA coordination (?) or common understanding of SA/JA antagonisms (if possible, additional JA-marker genes could be added, and/or timely compared with SA-marker genes).

-> That is correct. We found the important role of both SA and JA for host and nonhost resistance against bacterial pathogens. The statement was corrected.

Line 245 and 398; the efficacy of NbFDH1-silencing was differently described between line 245 (70%) and line 398 (50%). Those ~20% may have somewhat critical effects on physiology, e.g., may explain different results observed between tomato-silencing and Arabidopsis-KO lines on the infection of host pathogens (Fig. 1C vs. 2B).

-> As shown in the supplementary Figure 2, the silencing of NbFDH1 is approximately 70%. We corrected the statement in line 402.

Line 292: chapter “AtFDH1 is … pathogens” compared the upregulation of FDH1 expressions in response to host or nonhost bacteria, and proposed a greater role of FDH1 in nonhost disease resistance. Is it possible that induction/response of FDH1 upregulation by nonhost bacteria is slower (or faster) than by host pathogens? RT-PCR on additional time points could confirm/strengthen the authors’ conclusion.

-> As shown in supplementary Figure 3 and 5, we found that expression level of FDH1 was much higher in response to nonhost pathogen than by host pathogen. In qRT-PCR assay, we only determined the FDH1 expression at 24hpi; thus, it would be difficult to explain if the gene expression would be faster or slower in response host vs. nonhost pathogens. However, in Figure 6, FDH1 accumulation was elevated faster in response to nonhost pathogen (Pst, 2hpi) at chloroplast sample. The statement was revised in line 370.

Reviewer #2: The manuscript entitled “Formate Dehydrogenase (FDH1) Localizes to Both Mitochondria and Chloroplast to Play a Role in Host and Nonhost Disease Resistance” by Lee et al. described isolation of a FDH1 gene encoding a formate dehydrogenase from two plant species, Nicotiana benthamiana and Arabidopsis thaliana. The authors concluded that the gene provides nonhost, basal and effector triggered immunity. Furthermore, they concluded that FDH1 in Arabidopsis is localized to mitochondria and following bacterial infection it moves to chloroplast.

It is an interesting study reporting new knowledge in the area of plant immunity.

Major Comments:

1. Localization section requires additional studies. It is hard to follow the photos relevant to mitochondrial and chloroplastic localization of FDH1. It will be best to co-localize FDH1 with a reporter protein; i. e, an RFP fused protein that has been already established to be a mitochondrial or chloroplast protein. Close-up photos will also help the readers to follow the changes in accumulation patterns following infection.

-> We have used MitoTracker red dye to determine the subcellular localization of FDH1, because previous studies suggested that FDH1 localized in mitochondria and possibly cloroplast. MitoTracker was also used as mitochondrial staining dye and validated the localization of FDH1 in pepper (Choi et al., 2014, Plant Physiology). In addition, there are a number of studies published using MitoTracker as the dye of mitochondria straining (few examples - labeling mitochondria with MitoTracker dyes, Cold Spring Harbor Protocols, 2010; Sorvina et al., 2018, “Mitochondrial imaging…. “, Scientific reports; Jeena et al., 2017, “Mitochondrial localization….”, Nature communication). We agree our localization image was not clearly zoomed in, so another high quality of close-up image (Figure 4) was included to show the localization of FDH1 in mitochondria.

Also, in Figure 5, we did not use RFP fused protein for examining the chloroplast localization. Instead of using RFP fusing protein, we confirmed chloroplast with red autofluorescence of chlorophyll in mesophyll cells. Because it is well known that chloroplasts exhibit very strong autofluorescence in red, with a peak of approximately 680 nm. Thus, we used red channel to show chlorophyll auto fluorescence in chloroplasts of the epidermal cell region (this information was added in line 516). There are numerous studies to use the red autofluorescence of chlorophyll to determine chloroplast. We agree that the image resolution of Figure 5 was not in good quality to understand colocalization of FDH1 in mitochondria and chloroplast. This Figure has been revised for better explanation of the distributional change of FDH1-GFP in response to bacterial pathogens in mitochondria and chloroplast. Please see the revised Figure 4.

2. The western-blot looks good; but quantification has not made to claim that there is an enhanced accumulation of the fusion protein with time following infection (Figure 6).

-> That is correct, so we measured western blot band intensities. Figure 6 was revised.

This is a very interesting preliminary observation that FDH1 starts to accumulate in chloroplast following infection. Is not it possible that the protein is directly targeted to chloroplast from cytoplasm following infection? Note that in lane 3, the total protein is much more than that in mitochondria suggesting an increased synthesis of FDH1 following infection.

-> There would be a possibility that FDH1 could be targeted to chloroplast from cytoplasm. Based on our confocal microscope observation in many different experiments, FDH1 is mainly localized in mitochondria, and the expression is drastically increased after nonhost bacterial pathogens. We also see the movement of FDH1 expressing mitochondria is much faster and colocalized to chloroplast after nonhost pathogen infection than host pathogen and water control. Figure 6 is updated and showed the increased synthesis of FDH1 in response to nonhost pathogen (also please see the supplementary Figure 5).

Minor Comments:

1. It is not evident in this study if FDH1 localization to mitochondria or chloroplast is involved in host and nonhost disease resistance; therefore, title may be revised.

-> We agrees the comment, and title has been revised.

2. The basal resistance section is less developed. How was it measured?

-> In this study, we found that FDH1 is upregulated against both host and nonhost pathogens. Also as shown in Figure 2, fdh1 mutant plants were more susceptible to host pathogen, and the bacterial population in infected leaf tissues were much higher than the bacterial population in wide type. Also similar result showed in another group study in pepper (Choi et al., 2014).

3. Lines 60 - 61: “The efficacy of nonhost disease resistance is based on the recognition of pathogen-associated molecular patterns (PAMPs) and/or pathogen effectors.” This statement should be revised as not all nonhost resistance mechanisms are controlled by receptors for PAMPs. A review of the nonhost resistance mechanisms/genes should be included in “Introduction.”

-> This sentence was revised as shown in line 63-70.

4. Line 252: The sentence starting with “In correlation with ….” should be revised.

-> This sentence was correct.

5. Line 293: May delete the first section of the sentence up to the comma.

-> Corrected as shown in line 280.

6. Lines 307-308: The sentence should be deleted. Basal resistance is not even referred in the previous paragraph.

-> corrected (line 317).

7. Lines 324 -325: “AtFDH1 localizes predominantly in mitochondria, but translocates to chloroplasts in response to abiotic and biotic stresses” – No evidence is provided in support of the translocation of the protein from mitochondria to chloroplast.

-> Yes we agree that, and the sentence was changed.

8. The manuscript would be benefited from editing for English.

- > The manuscript has been thoughtfully read and revised for any potential grammatical errors in English.

Submitted filename: Response to Reviewers.docx

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4 Jan 2022

17 Feb 2022

Dear Editor,

Thank you for the valuable comments on our manuscript. We have followed the reviewers’ comments and thoughtfully revised the manuscript.

Please see below our responses and let us know for any questions.

Best Regards,

Seonghee Lee

-----------------------------------------------------------------------------------------------------------------------------------------

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

--->> Response: The list of reference was updated and highlighted for the changes. Following the reviewer’s comments, some old references were excluded because there were new publications available. Also, we found that there were too many references cited in the manuscript, and retracted some references that describe duplicated information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer #1: (No Response)

Reviewer #2: The revised version is much-improved. I have however a few concerns, which should be addressed.

1. If feasible a video may complement the Figure 5. I could not follow the concept that mitochondria remain attach to the chloroplast for a minute …... see sentence below from the manuscript.

"The arrow in each image (every 1min:30sec) indicates that the mitochondria localized FDH1 moves to chloroplasts and attach there approximately for 1 min, and later goes apart from the chloroplast."

---> Response: This sentence was changed and also we included a video (supplementary figure) to describe the movement of mitochondria to chloroplast.

2. Figure 6B is an improvement but it lacks statistical analyses.

Corrected.

3. The manuscript does not have any references to any of the publications of 2020 and 2021. There has been a lot known in the last two years. This can easily be fixed. Certain places authors may consider to cite original references rather than reviews, which are old and do not accommodate new work. Instead of using multiple reviews, authors are encouraged to cite the original work so that readers will be able to access those easily.

---> Response: Reference list was updated.

________________________________________

Submitted filename: Response to Reviewers.docx

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22 Feb 2022

Functional Role of Formate Dehydrogenase (FDH1) for Host and Nonhost Disease Resistance Against Bacterial Pathogens

PONE-D-20-40925R2

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Reviewers' comments:

20 Apr 2022

PONE-D-20-40925R2

Functional Role of Formate Dehydrogenase (FDH1) for Host and Nonhost Disease Resistance against Bacterial Pathogens

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