SEC14 Phospholipid Transfer Protein Is Involved in Lipid Signaling-Mediated Plant Immune Responses in Nicotiana benthamiana

We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense–related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.


Introduction
Plants have evolved innate immune responses to detect and respond quickly to foreign infections [1]. Plants use transmembrane pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) at the cell surface. Plants perceive bacterial flagellin, EF-Tu, and fungal chitin oligomers through their cognate receptors FLS2, EFR, and CERK1, respectively [2,3]. Plants also recognize avirulent gene products as pathogen infections by polymorphic receptors typically containing nucleotide-binding leucine-rich repeat resistance (R) proteins [4].
After recognition events, intracellular signaling cascades, such as changes in ion fluxes, cytoplasmic Ca 2+ levels, oxidative burst, protein phosphorylation, and the production of stress-related hormonal substances, are required for the establishment of plant immune responses [5,6]. The increase in Ca 2+ concentration and activation of Ca 2+ -dependent protein kinases induces an oxidative burst in the potato after PAMPs recognition [7]. The generation of reactive oxygen species (ROS) and nitric oxide (NO) is also implicated in defense-related gene expression mediated by both PRRs and R proteins [8.9.10]. In Nicotiana plants, members of the mitogen activated protein kinase (MAPK) family, SIPK, WIPK, and NTF6, are involved in defense induction in response to PAMPs, INF1 and HWC [11,12]. Both WIPK and SIPK are also sufficient to induce N-gene mediated resistance to the tobacco mosaic virus [13]. In tomato, LeMKK2, LeMKK3, and LeMPK3 are required for Pto-mediated resistance against Pseudomonas syringae pv. tomato carrying AvrPto [14]. In Arabidposis plants, members of the MAPK family, MPK3 and MPK6, are implicated in PRRs and R protein-mediated defense responses [15,16]. Plant defense responses are also controlled by a complex, interconnected signaling network that includes the hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) with antagonistic interaction of the JA and SA signaling pathways [17]. In Arabidopsis thaliana, an SA-dependent cascade is critical for biotrophic immune responses against Pseudomonas syringae pv. tomato DC3000. In contrast, ET/JA pathways are required for necrotrophic resistance against Alternaria brassicicola [18].
Phospholipids-based signaling cascades are common signal transduction mechanisms during plant immune responses. Phospholipases are activated during defense signal transduction. For example, induction of phospholipase D genes (PLD) occurs after elicitor treatment of tomato cells [19]. Similarly, treatment of Nacetyl chitooligosaccharide elicitor could induce rapid activation of PLD, resulting in the accumulation of phosphatidic acid (PA) in rice cells [20]. An avirulent strain of Xanthomonas oryzae induced the expression of both phospholipase C (PLC) and PLD genes in rice [21]. Isoforms of tomato PLC are required for Cf-4-dependent immune responses, whereas SlPLC4 and SlPLC6 are required for general immune responses [22]. Among the phospholipids, PA has been shown as intracellular signaling molecule leading to plant immune responses. In tomato suspension-cultured cells, PA and diglycerol pyrophosphate accumulate in response to a xylanase elicitor [23]. PA also accumulates in tomato cells in response to a race-specific Avr4 elicitor in a Cf-4 dependent manner [24].
Phospholipid metabolism and signaling are important in plant immune responses, although the molecular regulatory mechanisms of phospholipid synthesizing enzymes have remained elusive. Previously, we identified a gene related to the SEC14-gene superfamily from Nicotiana benthamiana (NbSEC14). NbSEC14 rescued temperature-sensitive growth mutant of sec14 in yeast, and NbSEC14 protein showed phospholipid transfer activity. Moreover, acceleration of disease development of bacterial wilt and growth of R. solanacearum were observed in the NbSEC14silenced plants [25]. SEC14 protein belongs to the large yet undercharacterized Sec14-protein superfamily (1550 proteins) originally isolated from Saccharomyces cerevisiae [26,27,28]. SEC14 protein functions as phospholipid transfer protein and acts in the phospholipid metabolism and phosphoinositide signaling pathways involved in diverse cell functions [29,30,31]. However, there is no information about role of SEC14 phospholipid transfer protein in plant immune responses. In this study, we analyzed the role of NbSEC14 phospholipid transfer protein in plant immune responses in N. benthamiana. In addition, we also discuss a possible relationship between phospholipid turnovers and NbSEC14 protein that leads to plant immune responses.

Plant Materials
Nicotiana benthamiana was grown in a plant growth room as described before [32].

Bacterial Isolates, Culture Conditions, and Inoculation
Bacterial strains used in this study are listed in Table S1. Ralstonia solanacearum strains 8107 (Rs8107), Pseudomonas cichorii SPC9018 were cultured in PY medium containing 20 mg/mL rifampicin. The density of bacterial suspension was adjusted to 1.0610 8 CFU/mL and inoculated by leaf infiltration as described in Maimbo et al., [32].

Primers and Plasmids
Primers and plasmids used in this study are listed in Tables S2 and S3, respectively.

RNA Isolation
Total RNA was prepared from N. benthamiana leaves with RNAiso (Takara Bio, Shiga Japan) according to the manufacturer's procedure. RNA samples were then treated with DNase I (RNase-free; Takara) to degrade contaminating genomic DNA as described previously [32].

Quantitative Real Time PCR
Quantitative real time PCR was performed by the method described in Maimbo et al., [32]. Reverse transcription was performed with 1 mg total RNA using PrimeScript RT reagent Kit (Takara), and qRT-PCR with 20 mL of a reaction mixture containing 1 mL of cDNA template, and 10 pM of the respective primers using the SYBR GreenER qPCR Reagent System (Invitrogen, Tokyo Japan) and an Applied Biosystems 7300 real time PCR instrument. Cycling parameters were the same for all primers: an initial 50uC for 2 min and 95uC for 10 min, followed by 40 cycles of 95uC for 10 s and 60uC for 1 min. Melting curve runs were performed at the end of each PCR reaction to verify the specificity of primers by the presence of a single product. The expected single DNA product and its molecular weight were verified by agarose gel electrophoresis. We also checked the sequence of amplified DNA products by direct sequencing with an upper primer. Relative quantification of gene expression was performed according to the instructions for the Applied Biosystems 7300 real-time PCR system using the comparative cycle threshold [Ct] method for the calculation of Qty value. All values were normalized to the expression values of the actin gene used as an internal standard in each cDNA stock. Expression analyses were performed with at least two biological replications to ensure that expression patterns were reproducible, and representative data are presented. Standard deviations and differences between expression ratios of non-treated controls and other samples were tested for statistical significance using the Student t-test.

Vector Constructs and Seedling Infection for Virusinduced Gene Silencing
A 389-bp cDNA fragment of the 39-terminal region of NbSEC14 was used for Virus-Induced Gene Silencing (VIGS) experiments as described previously [25]. Construct for NbCoi1-silencing was prepared as described previously [33]. Plasmid pPVX201 with no insert was used as a control. All binary plasmids were transformed into A. tumefaciens strain GV3101 [34] and inoculated into N. benthamiana leaves as described previously [32]. Specificity of NbSEC14-silencing was tried to confirm by NbSEC14-silencing with two different parts of NbSEC14 cDNAs ( Figure S1) and Southern blot analysis with cDNA fragment (sec14P1) as probe. According to results of Southern blot, we could observed single band, suggesting specific silencing of NbSEC14 [25]. However, we found out two different contigs (Nbs00015170g0012.1 and Nbs00058777g0001.1) in Sol Genomics database (http:// solgenomics.net/). Because they have more than 92% nucleotide identities with NbSEC14, we judged that silencing might affect all members of NbSEC14 family, and we therefore observed an overall effect of gene silencing of NbSEC14 family on plant immune responses.

Plasmid Construction for Agrobacterium-mediated Transient Expression
A full-length open reading frame (ORF) of NbSEC14 with a FLAG tag was amplified with secORF-S and secFlag-A primers using pGEMNbSEC14 [25] as a template, and the PCR product cloned into pGEMT-Easy vector (pGEMNbSEC14Flag). pGEMNbSEC14Flag was then digested with BamHI and SacI (Takara), and insert was cloned into the pBI121 vector (CLONTHEC, Tokyo, Japan) digested with the same enzymes. The final construct was designated pBI-NbSEC14. For agroinfiltration experiments, we also used the binary vector p35S-INF1 [35]. The binary vector p35S-GUS containing the GUS gene [36] was used as a control. These binary plasmids were transformed into A. tumefaciens strain GV3101, and inoculated into N. benthamiana leaves as described previously.

Phytohormone Analysis
Phytohormone contents were measured by the method described previously [37]. Extracted samples were subjected to measurement on a triple quadrupole LC-MS/MS 6410 (Agilent Technologies, USA) equipped with a Zorbax SB-C18 column [2.1 mm id650 mm, (1.8 mm), Agilent Technologies]. Hormone amounts were calculated from the ratio of endogenous hormone peak and known amount of internal standards spike, and related to actual fresh mass of the samples used for extraction.

Protein Analysis
Preparation of crude protein fractions and protein analysis were performed as described [38]. Crude protein fractions isolated from N. benthamiana leaves were separated by 12.5% SDS-PAGE and then electroblotted onto polyvinylidene difluoride membranes (Bio-Rad Labs., Hercules, CA). The blots were subjected to western blot analyses with a monoclonal antibody raised against the Flag-tag sequence (Sigma-Aldrich, Tokyo, Japan). Crossreacting proteins were visualized with a goat alkaline phosphatase secondary antibody (BioRad) conjugated with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium (Nacalai Tesque, Kyoto, Japan). Equal loading of protein fractions was estimated by Coomassie brilliant blue staining of Rubisco large subunit.

In vivo [ 32 P]Phospholipid Labeling
N. benthamiana leaves were detached and leaf petioles were dipped in water containing 0.59 Mbq carrier-free [ 32 P]orthophosphate (Muromachi Chemical, Tokyo, Japan) and incubated at 25uC for 12 h.

Phospholipid Extraction, Separation and Analysis
Total lipids were extracted in CHCl 3: MeOH:HCl (50:100:1, v/v/v) according to the method described in Munnik et al., [38]. Total lipid extracts were dried by vacuum centrifugation, dissolved in CHCl 3 , and separated by thin layer chromatography (TLC) with Silica [40]. Radiolabelled lipids were visualized and quantified by autoradiography, with densitometry scans performed on a GE Storm 860 with ImageQuant TL (GE Healthcare, Tokyo, Japan). Relative amount of phospholipid was calculated as relative to the none-treated sample.

DAG Quantification by DAG Kinase Reaction
Quantification of DAG levels was performed by measuring DAG 32 P-phosphorylation using Escherichia coli DAG kinase as described by Zien et al., [41], with slight modifications. First, leafextracted lipids were dried for a short time period under nitrogen. Second, micelles (20 mL) containing 7.5% octyl-L-D-glucopyranoside (Nacalai) and 20 mg mL 21 of 1,2-dioleoyl-sn-glycero-3phosphoglycerol (Funakochi, Tokyo, Japan) were added to the plant lipid mixture. Third, a reaction mixture (50 mL) containing 10 mM imidazole (pH 6.6), 10 mM LiCl, 25 mM MgCl 2 , 2 mM EDTA, and 19.4 mL of dilution buffer containing 10 mM imidazole (pH 6.6), 1 mM diethylenetriaminepentaacetic acid. This was followed by the addition of 10 mL of 2 mM ATP with 2.5 mCi of c-32 P-ATP to the previous mix. Fourth, the reaction of DAG 32 P-phosphorylation was started by the addition of 100 units of E. coli DAG kinase (Sigma) and incubated at 25uC for 1 h. Lipids were extracted with the procedure described by Bligh and Dyer [42]. The organic phase was dried down under nitrogen, resuspended in chloroform, spotted on TLC plates, and separated with an ethyl acetate solvent system. Radio-labeled PA were visualized by autoradiography, and densitometry scans of autoradiograms were performed using a GE Storm 860 and ImageQuant TL (GE Healthcare). DAG content was normalized to leaf fresh weight.

Assay for Phospholipase Enzymatic Activities
PLD activity was measured as the production of phosphatidylbutanol (PBut), as described previously [43,44]. Briefly, N. benthamiana leaves were prelabeled with 32 P for 12 h, and infiltrated with Rs8107 suspension with 0.25% n-butanol. Incubations were stopped and lipids extracted as described above. 32 P-labelled PBut was separated by an ethyl acetate TLC system, and its radioactivity visualized and quantified as described above. Relative phospholipase activity was calculated relative to the absolute PVX at time 0.
In vitro phosphoinositide-specific phospholipase C activity was measured by the hydrolysis of 3 H-PIP 2 as described previously [45,46]. Briefly, total protein fractions were incubated at 25uC for 30 min in Tris-Malate (pH 6.0) containing 10 mM CaCl 2 and 200 mM PIP 2 spiked with 0.86 KBq 3 H-PIP 2 . Reactions were stopped by addition of chloroform:methanol (2:1, v/v). 0.2 M HCI. Samples were centrifuged at 10,0006g, and radioactivity in the water-soluble upper phase counted with a liquid scintillation system.

Statistical Analysis
Statistical analysis was carried out using t-test.

NbSEC14 Protein Regulates the Expression of Defenserelated Genes
NbSEC14-silencing was carried out independently by NbSEC14silencing with two different parts of the NbSEC14 cDNA (P1, P2), showing very similar effect on immune responses in both cases (Figure 1, Figure S1). Therefore, we only used SEC14P1 fragment for further analysis. To determine the role of NbSEC14 in plant innate immunity, we focused on characteristic immune responses, including hypersensitive response (HR), salicylic acid (SA)-dependent and jasmonic acid (JA)-dependent signaling pathways. We could not observe any visible differences in HR induction in both control and NbSEC14-silenced plants ( Figure S2). In control leaves (empty vector VIGS) inoculated with Rs8107, expression of NbSEC14 and PR-4, a marker gene for JA signaling, showed peaks of expression at 12 and 24 hours after inoculation (HAI), respectively, but PR-4 transcript levels were greatly reduced in NbSEC14-silenced leaves. In contrast, expression of PR-1a, a marker gene for SA signaling, was rather enhanced in the silenced plants 48 HAI with Rs8107. In the case of hin1, the HR-related gene, the expression was much less affected by NbSEC14-silencing ( Figure 1). These results suggested a possible involvement of JA in NbSEC14-related immune responses. Then, we confirm JA and JA-Ile contents as well as SA content. As shown Figure 2, accumulation of JA and JA-Ile was observed in control plants challenged with Rs8107, whereas significant reduction of both hormone contents was observed in NbSEC14-silenced plants. In contrast, we could detect SA accumulation and hyper-accumulation of SA was observed in the silenced plants compared to control plants.
Because silencing of NbSEC14 reduced PR-4 expression, but not PR-1a and hin1, we tested the effect of the transient expression of NbSEC14 in N. benthamiana leaves on the expression of these genes (Figure 3a). qRT-PCR and western blot analysis confirmed a higher level of overexpressed NbSEC14, up to 8 times more relative to b-glucuronidase (GUS) gene-expressing control leaves at 48 hours (Figure 3b, c). Expression of PR-1a was significantly reduced, but hin1 expression was not affected. In contrast, PR-4 expression was significantly elevated in NbSEC14-overexpressing plants compared to control plants (Figure 3d). To strengthen our hypothesis of NbSEC14 interacting with JA signaling, we focused on NbCoi1 gene, which encodes F-box protein and have been well know as positive regulator of JA signaling [47]. Then, we examined the effect of NbCoi1-silencing on PR-4 induction by transient expression of NbSEC14. A strong reduction of PR-4 expression controlled by NbSEC14 in NbCoi1-silenced plants was observed ( Figure S3). Taken together, NbSEC14 protein may associate with the expression of defense-related genes related to JA-dependent pathway.

NbSEC14 Protein Regulates Phospholipid Signaling during Plant Immune Responses
NbSEC14 protein exhibits phospholipid transfer activities [25], and may be involved in plant immune response. Given these results, we evaluated the role of NbSEC14 protein in plant phospholipid metabolism in relation to its role in immunity. Among the phospholipids, we first focused on signaling phospholipids, diacylglycerol (DAG) and PA, and determined changes of DAG and PA in control and NbSEC14-silenced plants challenged with Rs8107. The formation of DAG and PA increased in Rs8107-inoculated control leaves, with peak DAG levels 12 HAI and peak PA levels at 24 HAI, whereas both phospholipid content was significantly reduced in NbSEC14-silenced plants (Figure 4a). In addition, NbSEC14-expressing plants showed increased DAG (24 HAI) and PA (48 HAI) compared to GUS-expressing controls (Figure 4b).

Regulation of Phospholipase Activities by NbSEC14 Protein
Diacylglycerol kinase (DGK)-PLC and PLD pathways are the two major metabolic pathways that produce DAG and PA [46]. A supporting pharmacological experiment showed that immune responses might be indeed related to PLC and PLD, since population of Rs8107 was stimulated in the concomitant presence of PLC and PLD inhibitors ( Figure S3). We therefore analyzed the relationship between NbSEC14 protein and these phospholipid metabolic enzymes during immune responses. DGK activity was enhanced 12-24 HAI, whereas activation of DGK activity was compromised in NbSEC14-silenced plants. NbSEC14-silencing blocked the increase in PLC activity at 6-24 HAI induced by Rs8107. NbSEC14 silencing also blocked the increase in PLD activity, as measured by 32 P-phosphatidylbutanol production, that occurred from 6-12 HAI (Figure 5a). In contrast, overexpression of NbSEC14 in N. benthamiana increased all DGK, PLC and PLD activities (Figure 5b).

Discussion
In this experiment, we could observe that NbSEC14-silencing caused dramatic changes of signaling phospholipids after plants were challenged with Rs8107 ( Figure 4). Among the phospholipids, DAG is well known as signaling phospholipid and is reportedly shown to play a crucial role in the response of tobacco cells to aluminum ions [48]. DAG is likely to act as a signaling molecule in tobacco pollen tubes [49]. PA is also recognized as signaling phospholipid [45]. PA is produced in response to xylanase treatments, and the accumulation of PA induces ROS production and cell death in tomato cells and Arabidopsis [50,51,52,53]. Wound-induced PA accumulation causes JA accumulation in Arabidopsis plants [54]. Here, we showed that NbSEC14-silencing blocked increases in JA contents and JA-dependent PR-4 genes, but did not affect hin1 gene and subsequent HR (Figure 1, 2 and Figure S2). Expression of SA-dependent PR-1a was rather enhanced in the silenced plants. Transiently increasing NbSEC14 protein enhanced the expression of defense-related PR-4, whereas PR-1a expression was significantly reduced (Figure 3). In addition, up-regulation of PR-4 gene expression was reduced in NbCoi1-   Figure S3). Therefore, these results suggested the direct or indirect relation of NbSEC14 on JA-dependent signaling pathway. NbSEC14 silencing caused faster growth of avirulent and virulent bacteria, and acceleration of disease development by virulent bacteria, and reduction PR-4 expression was also observed in the silenced plants in response to virulent bacteria [25]. The competitive interactions between SA and JA, and negative effects of JA on SA have been already described by Pieterse et al. [55] and others. Therefore, NbSEC14 protein may be closely associated with plant immunity related to the JA pathway, with interference of SA signaling by competitive interaction of the JA and SA signaling pathways.
Several plant SEC14-like proteins were reportedly shown to play a key role in the lipid-mediated signaling, and PA, DAG, and phosphoinositides (PIPs) regulate important cellular functions. AtSfh1p transfers phosphatidylinositol (PI) and phosphatidylcholine (PC) in vitro, in addition to stimulating intracellular and plasma membrane PIP 2 in a polarity landmark pattern that focuses membrane trafficking, Ca 2+ signaling, and cytoskeleton functions at the growing root hair apex [56]. AtPATL1, a novel cell-plateassociated protein, regulates membrane lipid composition (PI and PC) to activate PLD [57]. Ssh1p directly activates PI-3-kinase and PI-4-kinase in response to hyperosmotic stress [58]. Schaaf et al., [59] have shown that Sec14 protein is capable of stimulating the production of PIPs by presenting PI to PtdIns 4-kinase. PIP can then serve as a substrate for a PIP kinase to make another class of lipid signaling molecules, PtdIns-4,5-P 2 (PIP 2 ), and PIP 2 can then be a substrate for PLC and generate DAG and PA. PC can be hydrolyzed by either PLD to generate PA [60] or by PC-PLC to generate phosphocholine and DAG in plants [61]. In animals, PLC produces DAG as a second messenger [60]. NbSEC14silencing reduced PLC and PLD activity in response to Rs8107 inoculation, whereas transient expression of NbSEC14 activated both enzyme activities ( Figure 5). We could observe drastic changes of signaling phospholipids in NbSEC14-silenced plant as well as NbSEC14-expressing plants (Figure 4). NbSEC14 protein transfers PC and PI in vitro [25]. Unfortunately, although we could not determine actual substrate(s) for NbSEC14 protein in planta, we speculated that NbSEC14 protein affected lipid signaling-mediated plant immune systems in Nicotiana through PLC and PLD activities.
The PLC and PLD pathways are crucial in plant defense. Indeed, treatment of an N-acetyl chitooligosaccharide elicitor could induce rapid activation of PLD and the accumulation of PA, increasing elicitor-responsive genes as well as phytoalexin biosynthesis in rice cells [20]. Phytoalexin production induced by treatment with the glycopeptide elicitor from Mycosphaerella pinodes is mediated by a PIP 2 -PLC pathway [61,62]. Pharmacological experiments suggested that PLC and PLD might have an important role in the plant immune response against R. solanacearum ( Figure S3). Inversely, NbSEC14-silencing did not affect HR induction, but did affect resistance to both virulent and avirulent bacteria ( Figure S2) [25]. These results further imply that NbSEC14 protein may influence HR-independent defense via phospholipase-mediated phospholipid metabolism.
In conclusion, we have speculated that NbSEC14 protein may influence on PLC and PLD activities, as well as downstream PA and DAG production, associated with innate immune responses during bacterial infections ( Figure S5). With the capacity of NbSEC14 protein to change the expression of defense-related genes via JA signaling, further studies will be required to clarify the complex mechanism by which NbSEC14 protein is engaged in plant immunity, and to characterize the phospholipases and/or kinases involved in the signaling cascades.