Comparative Membrane-Associated Proteomics of Three Different Immune Reactions in Potato

Plants have evolved different types of immune reactions but large-scale proteomics about these processes are lacking, especially in the case of agriculturally important crop pathosystems. We have established a system for investigating PAMP-triggered immunity (PTI) and two different effector-triggered immunity (ETI; triggered by Avr2 or IpiO) responses in potato. The ETI responses are triggered by molecules from the agriculturally important Phytophthora infestans interaction. To perform large-scale membrane protein-based comparison of these responses, we established a method to extract proteins from subcellular compartments in leaves. In the membrane fractions that were subjected to quantitative proteomics analysis, we found that most proteins regulated during PTI were also regulated in the same way in ETI. Proteins related to photosynthesis had lower abundance, while proteins related to oxidative and biotic stress, as well as those related to general antimicrobial defense and cell wall degradation, were found to be higher in abundance. On the other hand, we identified a few proteins—for instance, an ABC transporter-like protein—that were only found in the PTI reaction. Furthermore, we also identified proteins that were regulated only in ETI interactions. These included proteins related to GTP binding and heterotrimeric G-protein signaling, as well as those related to phospholipase signaling.


Introduction
Plants possess an intriguing and unique immune system that is different from many other living organisms. Despite these differences, the innate immune system of plants performs similar functions as that in animals [1]. Plant immune responses can be broadly categorized into PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) [2]. PAMPs (pathogen-associated molecular patterns) are surface exposed, pathogen-associated molecules that are generally conserved across microbial kingdoms. Plants detect PAMPs via membrane-bound PRRs (pattern recognition receptors) that often have a kinase domain. PAMP recognition leads to molecular responses such as reactive oxygen species (ROS) production, MAP kinase and transcription factor activation, followed by defense gene expression [3]. Through evolution, however, successful pathogens have evolved to produce effector molecules that interfere with PTI responses, enabling successful infection. This is known as effector-triggered susceptibility (ETS) [2]. In response to this, plants have evolved the ability to counter effector molecules via the production of specialized resistance proteins (R-proteins). R-proteins recognize the presence of specific effectors (mainly in the cytoplasm). This interaction and the associated molecular reactions constitute ETI [2]. Phenotypically, ETI defense responses

Phenotypes of PAMP-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) Responses
Disarmed Agrobacterium-infiltrated wildtype cv. Désirée potato plants were used to study PTI responses and were compared with samples from two different ETI responses. The first ETI model was Blb1-containing Désirée infiltrated with Agrobacterium transformed with the IpiO effector gene, and the second was R2-containing Désirée infiltrated with Agrobacterium transformed with Avr2 effector gene. The infiltrated samples were subjected to phenotypic analysis at 18 hpi (hours post infiltration) and 3 dpi (days post infiltration).
None of the infiltrated samples showed visible phenotypic symptoms at 18 hpi. Three days post infiltration, a strong reaction and even cell death coinciding with the infiltration area were found in both the ETI interactions ( Figure 1C,D). Both these interactions had similar degrees of cell death at the whole infiltrated area. In the PTI interaction, small areas of cell death were observed occasionally in the zone of infiltration ( Figure 1B). No response was identified in cv. Désirée leaflets infiltrated with the infiltration medium only ( Figure 1A). Disarmed Agrobacterium-infiltrated wildtype cv. Désirée potato plants were used to study PTI responses and were compared with samples from two different ETI responses. The first ETI model was Blb1-containing Désirée infiltrated with Agrobacterium transformed with the IpiO effector gene, and the second was R2-containing Désirée infiltrated with Agrobacterium transformed with Avr2 effector gene. The infiltrated samples were subjected to phenotypic analysis at 18 hpi (hours post infiltration) and 3 dpi (days post infiltration).
None of the infiltrated samples showed visible phenotypic symptoms at 18 hpi. Three days post infiltration, a strong reaction and even cell death coinciding with the infiltration area were found in both the ETI interactions ( Figure 1C,D). Both these interactions had similar degrees of cell death at the whole infiltrated area. In the PTI interaction, small areas of cell death were observed occasionally in the zone of infiltration ( Figure 1B). No response was identified in cv. Désirée leaflets infiltrated with the infiltration medium only ( Figure 1A).

Subcellular Protein Fractionation
The fractionation procedure was based on successive centrifugation steps, wherein the supernatant at each step was extracted in a different buffer leading to four different buffers containing four protein fractions named as follows: cytoplasmic (CEB), membrane (MEB), solublenuclear (NEB), and chromatin-bound (CNEB). In order to identify whether differences existed in the banding pattern between the four fractions, each fraction was analyzed on an SDS-PAGE gel (Figure

Subcellular Protein Fractionation
The fractionation procedure was based on successive centrifugation steps, wherein the supernatant at each step was extracted in a different buffer leading to four different buffers containing four protein fractions named as follows: cytoplasmic (CEB), membrane (MEB), soluble-nuclear (NEB), and chromatin-bound (CNEB). In order to identify whether differences existed in the banding pattern between the four fractions, each fraction was analyzed on an SDS-PAGE gel ( Figure 2). The banding patterns of the four fractions were clearly different, indicating that the subcellular fractionation procedure had resulted in the isolation of different protein fractions. The CNEB fraction contained a very prominent band corresponding to the large Rubisco subunit ( Figure 2). Rubisco is one of the most abundant proteins in plant tissues and is associated with the stromal component of chloroplasts [26]. The CNEB fraction contained a strong band corresponding to histones ( Figure 2); this indicates that proteins associated with chromatin are indeed extracted in the CNEB fraction. The total amount of protein obtained from the different fractions also differed. The different lanes on the gel contain approximately equal amounts of protein in order to better display the differences in banding patterns. On average, a total of 470 µg was obtained from the CEB fractions, 150 µg from the MEB fractions, and 5 µg from the CNEB fractions. Since the MEB fraction seemed to contain large amounts of potentially interesting proteins, and little is specifically known about this fraction from plants in relation to immunity, this fraction was chosen for further analysis. The protein abundances in our 18 h PTI model and the two ETI models were compared using potato leaves infiltrated with only infiltration medium as control (Figure 3, Supplementary Tables S1 and S2). 2). The banding patterns of the four fractions were clearly different, indicating that the subcellular fractionation procedure had resulted in the isolation of different protein fractions. The CNEB fraction contained a very prominent band corresponding to the large Rubisco subunit ( Figure 2). Rubisco is one of the most abundant proteins in plant tissues and is associated with the stromal component of chloroplasts [26]. The CNEB fraction contained a strong band corresponding to histones ( Figure 2); this indicates that proteins associated with chromatin are indeed extracted in the CNEB fraction. The total amount of protein obtained from the different fractions also differed. The different lanes on the gel contain approximately equal amounts of protein in order to better display the differences in banding patterns. On average, a total of 470 µg was obtained from the CEB fractions, 150 µg from the MEB fractions, and 5 µg from the CNEB fractions. Since the MEB fraction seemed to contain large amounts of potentially interesting proteins, and little is specifically known about this fraction from plants in relation to immunity, this fraction was chosen for further analysis. The protein abundances in our 18 h PTI model and the two ETI models were compared using potato leaves infiltrated with only infiltration medium as control ( Figure 3, supplementary table1 and 2).

Membrane-Associated Proteins in the PTI Response
In the quantitative analysis of the PTI interaction, 585 proteins were used. The fraction contained predominantly chloroplastic, ribosomal, and mitochondrial proteins. In the PTI condition, 47 proteins were downregulated and 47 proteins were upregulated ( Figure 3, Table 1). Among the downregulated proteins (Supplementary Tables S1 and S2), a large number were chloroplast proteins involved in photosynthetic processes, such as chlorophyll a/b binding proteins, photosystem proteins, NAD(P)H-quinone oxidoreductases, and cytochrome bf-6 complex components. In total, 26 of the downregulated proteins are involved in photosynthesis. This is consistent with the wellestablished observation that infection results in the downregulation of components of the photosynthetic machinery [27].

Membrane-Associated Proteins in the PTI Response
In the quantitative analysis of the PTI interaction, 585 proteins were used. The fraction contained predominantly chloroplastic, ribosomal, and mitochondrial proteins. In the PTI condition, 47 proteins were downregulated and 47 proteins were upregulated ( Figure 3, Table 1). Among the downregulated proteins (Supplementary Tables S1 and S2), a large number were chloroplast proteins involved in photosynthetic processes, such as chlorophyll a/b binding proteins, photosystem proteins, NAD(P)H-quinone oxidoreductases, and cytochrome bf-6 complex components. In total, 26 of the downregulated proteins are involved in photosynthesis. This is consistent with the well-established observation that infection results in the downregulation of components of the photosynthetic machinery [27]. Among the other downregulated proteins was a plasma membrane-associated temperature-induced lipocalin [28]. In Arabidopsis, temperature-induced lipocalins have been implicated in moderating tolerance to oxidative stress [29]. Another protein, a bacterioferritin homolog, was also downregulated. The closest Arabidopsis homolog is a peroxiredoxin Q. Similar to lipocalins, peroxiredoxins are also involved in protection against oxidative stress [30]. These results indicate that some components related to oxidative stress tolerance are downregulated during PTI.
The upregulated proteins during PTI were more varied in function than the downregulated proteins and are listed in Table 1. A number of ATP synthases were upregulated. This might reflect an increased need for energy for the activation of defenses [31]. Interestingly, an LRR-like receptor protein kinase (LRR-RK) was found to be upregulated. Expression of the orthologous Arabidopsis transcript has been hypothesized to correlate with auxin levels in Arabidopsis [32]. Another protein annotated as translationally-controlled tumor protein homolog was also upregulated. This protein has been shown to be upregulated in Arabidopsis in response to effectors produced by the Gram-negative bacterium Pseudomonas syringae pv. tomato [33]. The TAO1 (target of AvrB operation) protein that is necessary for Pseudomonas syringae AvrB-triggered resistance [34] was also upregulated.
Other proteins upregulated during PTI were glycolate oxidase and a peroxidase. Glycolate oxidase has been shown to generate hydrogen peroxide during stress [35]. Plant peroxidases belong to the PR9 family of PR proteins. They use hydrogen peroxide to catalyze the oxidation of a number of different substances [36]. The protein MAR binding filament protein (MFP1), which has been previously shown to be induced in tomato in response to the elicitor COS-OGA [37], was also upregulated. Treatment of Arabidopsis suspension cells and protoplasts with COS-OGA also generates hydrogen peroxide [38]. An ABC transporter-like protein that has been shown to be induced in response to oxidative stress [39] was also upregulated specifically in PTI. Therefore, the abovementioned proteins seem to be involved in reactive oxygen species (ROS) signaling.

Proteins in ETI Responses
Seventy-four proteins were downregulated and 92 proteins were upregulated from one or both of the ETI interactions ( Figure 3). Proteins upregulated in the ETI interactions are mentioned in Table 2.
There was a substantial overlap with the proteins regulated in the PTI condition, particularly among the downregulated proteins ( Figure 3). Thus, out of the proteins downregulated in PTI, only 3 were uniquely downregulated, and all of the downregulated proteins discussed in the PTI section above were also downregulated in the ETI conditions. In addition, 30 more proteins were downregulated in ETI (Figure 3). Ten of these were chloroplast proteins involved in photosynthesis, as discussed in the PTI section.
The upregulated proteins in ETI overlapped with those upregulated in PTI, but less so than the downregulated proteins. Of the proteins upregulated in PTI, 10 were uniquely upregulated in that condition. Thirty-seven proteins were upregulated in both PTI and ETI and 59 were significantly upregulated in only ETI (Figure 3; Table 2); these latter included several proteins that showed the same tendency in all three sample types but did not reach the significance level in PTI. Among the proteins that were regulated significantly in both ETIs, a number were ribosomal proteins; possibly reflecting increased overall protein synthesis during this phase. Two superoxide dismutases were also upregulated. Superoxide dismutase catalyzes the dismutation of the superoxide radical. Their role is probably to protect the plant against the reactive oxygen species produced during the oxidative burst [40]. A further indication of active protective mechanisms to ROS is indicated by the upregulation of a chloroplastic lipocalin in ETI-IpiO, which has previously been shown to be involved in modulating tolerance to oxidative stress [29]. Interestingly, a lipocalin was downregulated in PTI (see above). A GTP-binding protein Era and an ethylene-responsive small GTP-binding protein were upregulated in the ETI interactions. A prominent role of regulation of plant immunity by GTP binding proteins has been suggested [41] and, based on our observations, it is tempting to speculate that GTP proteins might be specifically related to ETI plant immunity. Phospholipase A1 was upregulated only in ETI-Avr2. This protein belongs to a class of DAD (defective in anther dehiscence)-like proteins that is involved in jasmonic acid (JA) synthesis [42], possibly indicating a role for JA-mediated molecular signaling in Avr2-induced ETI. A heat shock protein 70-3 was also specifically upregulated in ETI-Avr2. This protein might connect oxidative stress induction and G-protein-dependent signaling in this ETI interaction as it has been shown to be involved in cGMP-dependent stress responses to hydrogen peroxide production [43] and again might underpin the involvement of GTP/GMP signaling in ETI. In comparison, a serine/threonine protein kinase was specifically upregulated in ETI-IpiO. The closest Arabidopsis homolog of this protein is annotated as an STN7 protein kinase, and it has been shown to link photosynthetic activity to ROS-induced molecular signaling during stress [44]. In combination with our observations with regards to chloroplastic lipocalin, upregulation of STN7 further supports the observation that ROS protection mechanisms might be necessary for ETI, specifically.

Plants and Agrobacterium Inoculation
Three sets of Solanum tuberosum (cv. Désirée) wildtype plants, AO1-22 (Désirée carrying Rpi-Blb1 resistance gene) [45,46] and T16 (Désirée plants carrying R2-type resistance gene) [13] were grown according to Abreha et al. [45]. Plants were initially grown in vitro on Murashige-Skoog (MS) media with vitamins in controlled growth conditions with 16 h light, day temperature of 23 • C and night temperature of 18 • C for 2 weeks. The plantlets were then transferred to soil and grown for 4 more weeks at approximately 22 • C with a cycle of 16 h of light and 8 h of darkness. The plants were supplemented with fertilizer (Rika S, SW Horto, Hammenhög, Sweden) once every second week. Agrobacterium strain AGL1 transformed with either an empty vector, IpiO effector gene, or Avr2 effector gene were grown according to Du et al. [47]. All antibiotics were used at a final concentration of 25 µg/mL except of spectinomycin that was used at a final concentration of 100 µg/mL. Agrobacterium strains were grown in 10 mL YEB medium supplemented with 1 µL acetosyringone (200 mM), 100 µL of 1 M MES buffer and appropriate antibiotics. The cultures were grown for 24 h at 28 • C, 200 rpm until OD 600 reached 1. The bacteria were harvested from the YEB medium by centrifuging at 3000× g for 10 min. The bacterial pellet was re-suspended in infiltration medium MMA medium (5 g/L MS salts, 1.95 g/L MES, 20 g/L sucrose, 200 µM acetosyringone, pH 5.6) to an OD 600 of 0.3. For infiltrations, the abaxial surface of a minimum of 5 leaflets on each plant was infiltrated using a 5 mL needleless syringe. A total of 4 plants belonging to each genotype (wildtype, AO1-22, and T16) were infiltrated. Three days post infiltration (dpi), a minimum of 1 leaflet from each plant (total 4 plants) was used to assess macroscopic cell death phenotype. The complete experiment was repeated twice.

Subcellular Protein Fractionation
Out of the four infiltrated plants belonging to each genotype, two plants were sampled for protein extraction at 18 hpi (hours post infiltration). Two leaflets from each plant were sampled for subcellular protein fractionation. From each infiltrated leaflet, two samples were taken, each containing two stabs (corresponding to 100 mg fresh weight) from the infiltrated area. In summary, four samples (containing two stabs each) were obtained from each genotype. Each sample was put in a 1.5 mL Eppendorf tube with sea sand on ice before further sample processing. The whole experiments were carried out twice and resulted in eight samples of each type. Subcellular protein fractionation into cytoplasmic (CEB), membrane (MEB), soluble-nuclear (NEB), and chromatin-bound (CNEB) fractions was performed using a Subcellular Protein Fractionation Kit for Tissues (ThermoFisher Scientific; Waltham, MA, USA, Catalog No. 87790) with minor modifications (see below). Phosphatase inhibitors (5 mM sodium phosphate, 50 µM sodium orthovanadate, and 10 nM calyculin A) were added to each buffer before use. Briefly, proteins were extracted in four different buffers consecutively and final supernatants were frozen at −80 • C until further use.
Each leaf sample was disrupted using pestle sticks in 1 mL ice-cold CEB. The sample was then passed through a tissue and centrifuged at 500× g for 5 min at 4 • C. The supernatant was cleared by re-centrifugation at 16,000× g for 10 min at 4 • C and saved as the cytoplasmic fraction. The 500× g CEB pellet was washed and centrifuged once with CEB and ice-cold MEB was added to the washed pellet. The pellet was then vortexed and incubated at 4 • C for 10 min with gentle mixing. After incubation, the solution was centrifuged at 3000× g for 5 min. The supernatant was cleared by re-centrifugation at 16,000× g for 10 min at 4 • C and the supernatant saved as the membrane fraction. The pellet obtained after the 3000× g centrifugation was washed once with MEB and centrifuged. To the resulting pellet, ice cold NEB was added, the sample was vortexed and incubated for 30 min at 4 • C with gentle mixing. After incubation, the sample was centrifuged at 5000× g for 5 min at 4 • C and the supernatant saved as the nuclear extract. The pellet obtained after the 5000× g centrifugation was washed and centrifuged once with NEB, and to the pellet room-temperature CNEB was added. The pellet was vortexed at maximum for 15 s and the sample was incubated at 37 • C for 15 min. After the room temperature incubation, the sample was centrifuged at 16,000× g for 5 min. The supernatant was defined as the chromatin sample.

Protein Concentration Determination
Protein concentration determination was performed using the bicinchoninic acid assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA; Catalog number: 23225) according the manufacturer's instructions. The buffer for each fraction (CEB, MEB and CNEB) was used to dilute the standard curves for each type of the three sample types.

Silver Staining
SDS-PAGE gels were stained with silver according to Blum et al. [48]. Briefly, gels were fixed in 40% ethanol, 10% acetic acid overnight. The gels were washed 3 times in water (20 min per wash). They were then incubated in 0.02% Na 2 S 2 O 3 for 1 min, washed 3 times in water (1 min per wash), and incubated for 20 min in 0.2% AgNO 3 , 0.02% formaldehyde. After this incubation, the gels were washed twice in water (1 min per wash) and developed in a solution of 3% Na 2 CO 3 , 0.05% formaldehyde, and 0.0005% Na 2 S 2 O 3 . The development process was stopped by washing once with water (1 min) and then incubated in 0.5% glycine solution.

Tryptic Digestion and Mass Spectrometry
Proteins from the analyzed fractions were separated on a 14% SDS-PAGE gel. The entire lane was excised, washed, and the proteins digested with trypsin (Promega Trypsin Gold, Madison, WI, USA, Mass Spectrometry Grade Trypsin Gold, Catalog number: V5280). The tryptic digests were desalted using C18-based spin columns (The Nest Group, Inc., Southborough, MA, USA) as described in Chawade et al. [49]. Tryptic digests were subjected to HPLC-MS/MS analysis using an Eksigent nanoLC2D HPLC system connected online-with an LTQ Orbitrap XL ETD. The peptide samples were loaded onto an Agilent Zorbax 300SB C18 (0.3 mm ID, 5 mm, 5 µm particle size) pre-column and separated on an in-house packed PicoFrit column (Santa Clara, CA, USA; Agilent Zorbax 300SB C18, 75 µm ID, 150 mm, 3.5 µm particle size). The analytical column was pre-equilibrated with a buffer consisting of 0.1% formic acid (FA) in 5% ACN for 10 min at a flow rate of 10 µL/min, and peptide separation was conducted in 0.1% FA buffer using a 55 min linear gradient from 5% to 40% ACN, followed by a 5 min linear gradient from 40% to 80% ACN, at a flow rate of 350 nL/min. The eluted peptides were analyzed using an LTQ Orbitrap XL ETD. The Orbitrap was operated in data-dependent mode with survey scan spectra 400-2000 Da in the Orbitrap mass analyzer at target resolution 60,000, followed by selection of the seven most intense ions for fragmentation in the LTQ, using a mass window of 2 Da for precursor ion selection. The precursor ions were fragmented with normalized collision energy of 35 (with activation Q set to 0.25 and an activation time of 30 ms). Dynamic exclusion with a repeat count of 2 and a repeat duration of 20 s and exclusion duration of 120 s were used, with an exclusion list size of 499 and a 10 ppm relative exclusion mass width.

Data Analysis
The raw data from the Orbitrap was converted to Mascot generic files (mgf) using ProteoWizard [50]. The Proteios software environment [51] was used to search the mgf files in Mascot version 2.3.01. The mgf files were searched against a database consisting of Solanum proteins from UniProt (www.uniprot.org), downloaded 24 August 2011; protein sequences from the Potato Genome Project [52] and the Agrobacterium proteins from UniProt, downloaded 10 March 2015, concatenated with an equal size decoy database (random protein sequences with conserved protein length and amino acid distribution, in total 36,512 target and decoy protein entries) generated using a modified version of the decoy.pl script from MatrixScience (http://www.matrixscience.com/help/decoy_help.html) [53]. Since the Potato Genome Project are from the diploid Solanum phureja and we used a tetraploid potato, the UniProt Solanum sequences were included to increase the number of identifications. Search tolerances were 7 ppm for precursors and 0.5 Da for MS/MS fragments. One missed cleavage was allowed and carbamidomethylation of cysteine residues was used as fixed modification and oxidation of methionines as variable modification. Search results were exported from Mascot as XML, including query level results, with a modification to the export script to include protein accession numbers also for the query (spectrum) level results. All search results, including the top-ranked peptide for each spectrum, were imported to Proteios where q values were calculated using the target-decoy method described by Käll et al. [54]. The search results were then filtered at a peptide-spectrum match q-value of 0.01 to obtain a false discovery rate of 1% in the filtered list. For quantitative analysis, a label-free approach based on precursor ion intensities was used [55] with all data processing steps performed within Proteios. MS1 peptide feature detection was performed using Dinosaur [56], while the other data processing steps were performed in Proteios, and subsequent feature matching and alignment between LC-MS/MS runs with a previously described workflow [57]. The resulting peptide data was normalized using Loess-G normalization [58] in the Normalyzer software [59]. The normalized data was analyzed using DanteR [60].

Plant Material
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Conclusions
Comparative quantitative proteomic analysis of PTI and ETI interactions revealed that in the PTI interaction proteins generally related to oxidative and biotic stress were upregulated, while proteins related to photosynthesis were downregulated. Furthermore, proteins related to antimicrobial defense and cell wall degradation were also upregulated. Analysis of the ETI interaction showed upregulation of several proteins that were also identified in the PTI interaction. However, we identified distinct upregulation in proteins related to oxidative stress tolerance and GTP binding proteins associated with heterotrimeric G-protein signaling only in the ETI interactions. In addition, proteins related to phospholipase and oxidative stress tolerance were significantly upregulated in only the ETI interactions, such as a chloroplastic lipocalin and a HSP-70 isoform. This study provides a basis for new mechanistic studies and breeding of sustainable resistance in potato.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/xxx/s1.