Formate dehydrogenase (FDH1) localizes to both mitochondria and chloroplast to play a role in host and nonhost disease resistance

1 Noble Research Institute, LLC, Admore, OK, United States 2 Gulf Coast Research and Education Center, Institute of Food and Agricultural Science, University of Florida, Wimauma, FL, United States. 3 Current Address: Laboratory of Plant Functional Genomics, Regional Center for Biotechnology, Faridabad, Haryana, India 4 Current Address: Department of Entomology and Plant Pathology, University of Arkansas, Fayetteville, AR, United States 5 Current Address: National Institute of Plant Genome Research, New Delhi, India 6 Current Address: Department of Plants, Soils and Climate, Utah State University, Logan, Utah, United States


INTRODUCTION
Nonhost resistance provide basic protection to plants and are also the most durable form of resistance to the majority of potential pathogens [1][2][3][4][5]. 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 [4,[6][7][8][9]. In addition to QTLs, a number of studies have identified major plant genes involved in nonhost resistance against fungal and bacterial pathogens [4,5,[9][10][11].
However, the mechanism of nonhost resistance is not well 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 [10]. The efficacy of nonhost disease resistance is based on the recognition of pathogen-associated molecular patterns (PAMPs) and/or pathogen effectors. PAMPs are mainly located at the plasma membrane where the PAMP-triggered immunity (PTI) could be induced as the first defense barrier against various pathogens [12,13]. One known PTI response is stomatal closure that is circumvented by the phytotoxin coronatine (COR) produced by the host pathogen P. syringae pv. tomato DC3000 [14].
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 [15,16].
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 [21]. Despite the plant immune systems, compatible host bacterial pathogens in susceptible plants suppress both basal and nonhost resistance responses to cause disease.
Formate dehydrogenase (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 [22][23][24][25]. 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 defense signaling pathway against the bacterial pathogens in pepper. Several other reports also suggest mitochondrial localization of FDH1 in tobacco [26,27]. Interestingly, few reports described that FDH1 targets not only mitochondria but also chloroplasts for its biological function [28,29]. Chloroplast and mitochondria are the major targets of plant pathogen effectors, and targeting of these organelles by effectors inhibits the production of defense molecules including reactive oxygen species (ROS) [30,31]. Chloroplasts play a major role in generating ROS and nitric oxide to trigger defense responses such as PCD and HR against bacterial pathogens [32,33]. Mitochondria and chloroplasts also have been reported as the initial organelle to recognize bacterial effectors and to trigger plant immunity against bacterial pathogens [34,35]. In other studies, the co-localizations of mitochondria with chloroplasts has been well characterized [36][37][38]. The physical interactions between mitochondria and chloroplasts would provide the means of transferring genetic information directly to the organelle genome, as well as to mediate signaling transduction [39][40][41][42]. However, how chloroplast and mitochondria are functionally integrated for bacterial disease resistance is not well 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 a 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 to chloroplasts for the defense responses against host and nonhost bacterial pathogens. We speculate that FDH1 may coordinate mitochondria-and chloroplast-mediated defense responses to bacterial pathogens in plants.

Formate dehydrogenase is involved in nonhost disease resistance
Using virus-induced gene silencing (VIGS)-based forward genetics screening in N.
benthamiana, we identified the clone 24E07 (NbME24E07) to be involved in nonhost disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato T1 [43,44]. The cDNA insert in 24E07 clone was sequenced. 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 (Supplementary Figure 1). FDH1 is a single copy gene in both monocot and dicot plants.  Figure S2). 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 (Wang et al., 2007) at 1×10 4 CFU/ml concentration, the bacteria multiplied more in NbFDH1-silenced plants when compared to non-silenced control as visualized by green fluorescence under UV light ( Figure 1A). In correlation with the increased nonhost bacterial multiplication, NbFDH1 silenced plants also showed disease symptoms characterized by necrosis and chlorosis. In contrast, no disease symptoms were observed in the non-silenced control ( Figure 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 non-silenced control ( Figure 1B). In contrary to nonhost pathogen, multiplication of the host pathogen P. syringae pv. tabaci was not different in NbFDH1 silenced plants when compared to non-silenced control ( Figure 1C).
To check if NbFDH1 has a role in nonhost HR, NbFDH1-silenced and nonsilenced control plants were syringe-infiltrated with a high level of inoculum (1×10 6 CFU/ml) of the nonhost pathogen P. syringae pv. tomato T1. Non-silenced control 7 showed a typical nonhost HR after 24 hours post inoculation (hpi) whereas in NbFDH1silenced lines, the HR was delayed until 48 hpi ( Figure 1D). Taken together, these results suggest that NbFDH1 plays a role in nonhost disease resistance against P. syringae pv. tomato T1 in N. benthamiana.
Arabidopsis Atfdh1 mutants show increased susceptibility to host-pathogen and nonhost pathogens.
To check if the role of FDH1 in nonhost resistance is conserved in more than one plant species, two Arabidopsis T-DNA insertion mutants 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, but not Col-0 showed disease symptoms characterized by chlorosis at 5-day post inoculation (dpi) (Figure 2A). In addition, Atfdh1 mutants had higher bacterial titer (approximately 18-fold) when compared to Col-0 plants at 3 dpi ( Figure 2B). In response to infection with a host pathogen, P. syringae pv. maculicola, both Col-0 and the Atfdh1 mutants showed similar disease symptoms ( Figure 2A). Interestingly, in contrast to the observation in NbFDH1silenced N. benthamiana where the host pathogen titer didn't 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 ( Figure 2B).
To check if AtFDH1 plays a role in gene-for-gene resistance, we infected Arabidopsis Col-0 plants that carry many 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 ( Figure 2C). This difference in growth was likely related to a deficiency in the production of ROS in the Atfdh1 mutant lines. It has been known that the mutation of AtFDH1 delays the production of ROS in response to P. syringae pv. tomato DC3000 (AvrRPM1) [23], and we also showed the delayed HRassociated cell death in NbFDH1-silenced N. benthamiana plants ( Figure 1D). These results suggest that AtFDH1 confers plant defense through ROS dependent 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; Supplementary Figure 3A). This agrees with the previous study of pepper mitochondrial FDH1 [23]. We also found that AtFDH1 gene expression is induced after host or nonhost pathogen inoculation (Supplementary Figure 3B). After inoculation with the virulent pathogen P. syringae pv. maculicola, FDH1 expression was increased slightly (less than 0.5-fold) in comparison with 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 (Supplementary Figure 3B). 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.
As shown above, Atfdh1 mutants are compromised in nonhost disease resistance, basal resistance, and gene-for-gene resistance. It was also found that the gene expression was induced in response to both host and nonhost pathogens (Supplementary Figure 3A). 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 gene expression of three representative genes related to SA pathway (PAD4, EDS1, and NPR1) and a gene related to JA pathway (PDF1.2) 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.
Without any pathogen infection, PAD4, EDS1, and NPR1, were not significantly different between Col-0 and Atfdh1-1, while the expression of JA marker gene PDF1.2 was remarkably increased in Atfdh1-1 (Supplementary Figure 3C). After 24 hpi with either pathogen in Col-0, the SA marker genes, PAD4 and EDS1, and JA marker gene, PDF1.2, were strongly induced, but the level of induction of these genes was significantly lower in the Atfdh1 mutant against both host and nonhost pathogens, comparing to Col-0 ( Figure   3). NPR1 was significantly induced at 24 hpi with the host pathogen in wild-type Col-0 and significantly reduced (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 plant defense pathways.

AtFDH1 localizes predominantly in mitochondria, but translocates to chloroplasts in response to abiotic and biotic stresses.
Localization of FDH1 in mitochondria and/or chloroplast has been the subject of extensive debate [23,[26][27][28][29]. We cloned AtFDH1 to be expressed under its native 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 ( Figure 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 ( Figure 6). More AtFDH1 protein was detected in the chloroplast protein fraction after infection with nonhost pathogen when compared to host pathogen ( Figure 6). These findings suggest that the localization of FDH1 in mitochondria may play a role for plant innate immunity against foliar bacterial pathogens, and FDH1 localization to chloroplasts may be important for nonhost disease resistance.

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 [22][23][24]47]. 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 plays 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 required for plant innate immunity against both host and nonhost bacterial pathogens. Nonhost disease resistance is the most common form of plant defense against various pathogens [5,8,48,49]. HR cell death are typical symptoms in response to ETI-triggered nonhost resistance in plants [50,51]. ROS produced in various cellular compartments, including chloroplasts, mitochondria, and peroxisomes have been proposed to act as signals for HR and PCD [52]. Chloroplasts are the main source of ROS during various environmental stresses, including plant-pathogen interactions [53]. 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 [35]. 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 are 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 (Figure 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 [54]. 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 ~50%, we speculate that this is not 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 [55]. It has been reported that some proteins target to both mitochondria and chloroplast, and might be more common than thought but their functions are not well understood, especially for plant disease resistance [56][57][58][59][60]. FDH1 has a putative mitochondrial signal peptide, although AtFDH1 has been reported to localize to either mitochondria or chloroplasts [28,[61][62][63]. Therefore, FDH1 localization in plants remains controversial. There was one study showing that the dual localization of AtFDH1 in both chloroplasts and mitochondria when AtFDH1 is overexpressed in transgenic Arabidopsis and tobacco plants [64]. 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 [65]. This N-terminal sequence of AtFDH1 is quite different from potato, barley, and rice, suggesting AtFDH1 localizing in chloroplast could occur under certain conditions [63]. In our study, the localization of AtFDH1 in chloroplast was only detected under the conditions of wounding and pathogen stresses (Figure 4 and 5). As previously described, FDH1 is highly induced under various stress conditions [63]. We speculate that the localization of FDH1 in chloroplast is too low and transient to be detected under non-stress conditions, and this causes 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 mention above, FDH1 has been shown to play a role in disease resistance in pepper against a bacterial pathogen (Choi et al., 2014). 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 [66]. CRT3 is localized in the endoplasmic reticulum (ER) lumen, and has been known to associate with abiotic stress response and plant immunity [67][68][69]. FDH1 directly interacts with RING-type ubiquitin ligase Keep on Going (KEG), which is localized in trans-golgi and early endosomes [70]. 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 [71]. There are several reports describing the ROS-based signal transmission between mitochondria and chloroplasts [72][73][74][75]. Possibly, FDH1 protein could be transmitted to chloroplasts from mitochondria to interact with outer membrane proteins of chloroplasts and initiate a signal transduction pathway for the production of chloroplast-derived ROS.
In conclusion, we demonstrated a novel chloroplast-dependent pathway that regulates plant innate immunity, most likely through mitochondria-to-chloroplast integrated ROS signaling. Even though mitochondria is the main source of ROS, chloroplast also plays 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.

Plant materials
N. benthamiana plants were grown in 10-centimeter diameter round pots with BM7 soil (SUNGRO Horticulture Distribution, Inc., Bellevue WA) in the greenhouse using the condition described in the previous study [43]. Plants grown four weeks were used for virus-induced gene silencing (VIGS) experiments as described below. The ecotype of
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 × 10 4 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]. The bacterial population at 0 day was estimated from leaves harvested 1 hr after inoculation. Two leaf discs (0.5 cm 2 ) 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 cm 2 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 [76].

Bacterial disease assay in N. benthamiana and Arabidopsis
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.

Subcellular localization of FDH1 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 [77]. The subcellular location of AtFDH1-GFP in epidermal cells was determined under the confocal microscope.
To observe the localization of AtFDH1, Arabidopsis wild-type Col-0 and maculicola (1×10 6 CFU/ml) and nonhost pathogen P. syringae pv. tabaci (1×10 6 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 subcellular location of AtFDH1 was imaged under the confocal microscope.

Isolation of chloroplast and mitochondria
Arabidopsis leaves (10 g

Quantitative real-time 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),       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. 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 (BPB stained gel), COXII: mitochondria marker protein detected using polyclonal COXII antisera (Agrisera), RBCL: chloroplast marker protein detected using polylonal Rbcl antisera (Abiocod). 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. (C) Gene expression patterns of defense-related genes in wild-type and the Atfdh1 mutant without any biotic or abiotic stresses. Leaves of four weeks old Arabidopsis wild-type (Col-0) and Atfdh1 mutant (fdh1-1) plants were collected, total RNA was isolated, and subject to RT-qPCR to measure the transcripts of PAD4, EDS1, NPR1, and PDF1.2. 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).