Proteomic analysis of banana xylem sap provides insight into resistant mechanisms to Fusarium oxysporum f. sp. cubense Tropical Race 4

Background Fusarium wilt is a destructive soilborne disease of banana caused by Fusarium oxysporum f. sp. cubense ( Foc ), especially Tropical Race 4 (TR4), which is a xylem-invading fungus. It was evident that xylem sap contained macromolecules, such as proteins, involved in disease-resistance processes. However, there is no research to analyze changes in banana xylem sap proteins response to TR4 to date. Methods To gain an integrated understanding of differential protein expression in banana xylem sap during TR4 infection, we performed a comparative proteomic analysis of xylem sap in resistant ‘Pahang’ and susceptible ‘Brazilian’ bananas inoculated with TR4. Results A total of 1036 proteins were detected in xylem sap of both bananas, among which some proteins are involved in ‘signal transduction’, ‘environmental adaptation’, ‘biosynthesis of secondary metabolites’ and ‘lipid metabolism’, indicating that xylem sap contained defense-related proteins. A number of 129 differential expression proteins (DEPs) were identied in 4 possible pairs between resistant and susceptible tested combinations. Of these DEPs, hypersensitive-induced response protein 1 (HIR1), E3 ubiquitin ligase (E3) might play negative roles in ‘Pahang’ response to TR4 attack, whereas chalcone isomerase (CHI), glycine-rich RNA-binding protein (GRP), carboxylesterase (CXE) and GDSL lipase (GLIP) might play positive roles in ‘Pahang’ defense against TR4 infection. Conclusions Banana xylem sap contained defense-related proteins, among which HIRP1, E3, CHI, GRP, CXE and GLIP involved in banana defense against TR4. To our knowledge, this is rst report to analyze changes in banana xylem sap proteins response to TR4, which help us to explore molecular mechanisms of banana resistant to Fusarium wilt.

inoculated plants were placed in an artificial climate chamber at 30 ℃ , 8 h light/16 h dark with 80% humidity (Fig. 1A). At 14 days post inoculation (DPI), the plants were rinsed off with tap water and dried with filter papers, and the pseudostems were transected at 0.5 cm above the corms with a sterile blade ( Fig. 1B, C). After removed the exudate from the cut cells, the xylem sap exuded spontaneously from the remaining pseudostems was collected with a pipette in time ( Fig. 1D-E). To obtain sufficient amounts of protein for analysis, the xylem sap isolated from at least 30 plants was concentrated into one independent biological replicate, and three independent biological replicates were conducted. The xylem sap was frozen with liquid nitrogen prepared for protein extraction. The Pahang and Brazilian inoculated with TR4 named P_dpi and B_dpi, respectively. The Pahang and Brazilian inoculated with sterile water named P_mock and B_mock, respectively.

Xylem sap protein extraction
At least 3 mL xylem sap was resuspended with benzyltriphenylphosphonium chloride (BPP) solution (containing 1% Poly vinyl pyrrolidone pvp, PVPP) in the ratio of 1:3, and vortexed for 10 min at 4℃. Equal volume of Tris-saturated phenol was added, and shook for 10 min at 4 ℃ . The mixture was centrifuged with 12,000 g at 4 ℃ , and the bottom phenol phase was transferred to a new reaction tube and reextracted by adding an equal volume of BPP solution, and shook for 10 min at 4 ℃ . The mixture was centrifuged with 12,000 g at 4 ℃ , and the bottom phenol phase was collected. The proteins were precipitated from the phenol phase with ammonium carbinol acetate solution in the ratio of 1:5 at -20℃ overnight. Proteins were pelleted by centrifugation. Pellets were washed twice with acetone. Proteins were dissolved in 8 M urea and 1% sodium dodecyl sulfate (SDS). After centrifuging at 4 ℃ the supernatant was collected.
iTRAQ labeling Protein concentrations were determined by Bicinchoninic acid (BCA) method by BCA Protein Assay Kit (Pierce, Thermo, USA), and the total of protein should be no less than 100 μg. Protein digestion was performed according to the standard procedure and the resulting peptide mixture was labeled using the 8-plex iTRAQ reagent (Applied Biosystems, 4390812) according to the manufacturer's instructions.

High pH RPLC Separation
Samples were fractionated using high pH reverse phase separation techniques to increase the depth of the proteome. The peptides were resuspended with a loading buffer (2% acetonitrile in ammonium hydroxide solution, pH 10), and separated by high pH reversed-phase liquid chromatography (RPLC, Acquity Ultra Performance LC, Waters, USA). The gradient elution was carried out on a high pH RPLC column (ACQUITY UPLC BEH C18 Column 1.7 µm, 2.1 mm × 150 mm, Waters, USA) at 400 μL/min with a gradient increased for 66 min (Phase B: 5mM Ammonium hydroxide solution containing 80% acetonitrile, pH 10). Twenty fractions were collected from each sample and these fractions were pooled to form 10 total fractions per sample.

Mass spectrometry analysis
The experiment was conducted on a Q Extraction mass spectrometer in combination with Easy-nLC 1200. 4 μL of each fraction was injected into nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Peptide mixture (2 μg) was loaded into a C18-reversed phase column (75 μm × 25 cm, Thermo, USA) in buffer A (2% acetonitrile and 0.1% formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a 300 μL/min flow rate. An electrospray voltage of 1.8 kV was used at the inlet of the mass spectrometer. Q Exactive mass spectrometer was operated in the data-dependent mode and automatically switched between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350-1300) were measured with a mass resolution of 70,000, followed by 20 consecutive high-energy collisional dissociation (HCD) MS/MS scans with a resolution of 17,500. In all cases, a microscan was recorded with a dynamic exclusion of 18 sec, and the MS/MS normalized collision energy was set at 30.

Sequence Database Searching
MS/MS spectra were searched using ProteomeDiscoverer (Thermo Scientific, Version 2.2) against Musa acuminata database (http://www.uniprot.org/proteomes/UP000012960) and the decoy database as the following parameters. The highest score for a given peptide mass (best match to that predicted in the database) was used to identify parent proteins. The parameters for protein searching were set as follows: tryptic digestion with up to two missed cleavages, carbamidomethylation of cysteines as fixed modification, and oxidation of methionine and protein N-terminal acetylation as variable modifications.
False discovery rate (FDR) of peptide identification was set as FDR ≤ 0.01. A minimum of one unique peptide identification was used to support protein identification.

Quality control of raw data
The quality evaluation for each sample of the original MS data was conducted, including the matching error of peptides, the distribution statistics of peptide number, the length distribution of identified peptides and the distribution of protein molecular weight.
iTRAQ quantitative proteomics analysis The basic information analysis process for iTRAQ quantitative proteomics using the free online platform of Majorbio Cloud Platform (www.majorbio.com). First, the raw mass spectrums generated by the mass spectrometer were subjected to the peak identification. Secondly, the reference proteomic database of banana (http://www.uniprot.org/proteomes/UP000012960) was established to identify peptides and proteins. All identified proteins were functional annotated using Cluster of Orthologous Groups of

Results
Data and quality control evaluation Xylem saps of Pahang (resistant) and Brazilian (susceptible) inoculated with TR4 or mock at 14 dpi were collected for comparative proteomic analysis. A total of 261,038 spectra were acquired through iTRAQ quantitative proteomics analysis, among which 31,450 spectra were matched to 6,503 peptides, 3,291 proteins and 1509 protein groups ( Fig. 2A). Since this experiment was divided into two labeled groups, some proteins only appeared in one labeled group, and 1036 proteins that existed in both labeled groups were used for subsequent analysis. The distribution of peptide matching error analysis indicated error distribution between the true and theoretical values of the relative molecular weights of all matched peptides was acceptable (Fig. 2B). There were 126 proteins only with one peptide, and approximately 87.84% of the proteins contained at least two peptides. Protein number decreased with increasing number of peptides (Fig. 2C). The length of the peptide ranged from 6 to 34 amino acids, among which the peptide of 10 amino acids was most abundant (Fig. 2D). The molecular weight of almost all proteins (99.5%) ranged from 1 kDa to 121 kDa. 21-41 kDa proteins account for 40.54%, followed by proteins with 41-61 kDa and 1-21 kDa (Fig. 2E). In terms of protein's sequences coverage distribution, about 70.14% of proteins constituted more than 10% protein's sequences coverage, and 45.56% of proteins constituted more than 20% protein's sequences coverage (Fig. 2F). It indicated that the identified proteins have good peptide coverage, and the data had highly confidence.

All proteins function annotation and expression analysis
All identified proteins (1,036) were conducted functional analysis, of which 938, 923 and 779 proteins were annotated with COG, GO and KEGG databases, respectively.
In terms of COG (

Signal transduction
There is no doubt that signal transduction pathways are responsible for induction of plant defense response [41,42]. Of these pathways, the mitogen-activated protein kinase (MAPK) cascades and plant hormone signals play pivotal roles in plant disease resistance [43]. Once plant perceives the invading pathogen, the activation of MAPKs is one of the earliest signaling events [44]. In the present studies, 10 proteins were related with MAPK signaling pathway-plant (Supplementary Table 1), such as nucleoside diphosphate kinase (NDPK, XP_009384691.1 and XP_009413273.1) inducing MPK3/6 expression through phosphorylation leading to hypersensitive response (HR) cell death in plant response to pathogen attack [45].
Among the plant hormone signals, salicylic acid (SA) and jasmonic acid (JA) are essential components of plant defense against pathogen [46]. SA and JA antagonize each other [47]. It is generally considered that SA enhances resistance to biotrophs, while JA is effectively against necrotrophs and insects [48,49]. However, there is exception that SA metabolism activation and signal transduction can improve banana resistance to TR4 [50]. In this study, 2 proteins was associated with SA and JA signals (Supplementary  table 1), including pathogenesis-related protein 1 (PR1, XP_009388942.1), a marker for systemic acquired resistance (SAR) from SA signaling pathway [51]; and coronatine-insensitive protein homolog (COI1, XP_009416210.1), a key regulator for JA-dependent induced systemic resistance (ISR) [48,52]. Further research is needed to determine whether SA-dependent SAR and JA-dependent ISR were simultaneously activated in banana.

Environmental adaptation
In the natural environment, plants were threatened by various abiotic and biotic stress. Over the evolutionary course during plant-pathogen interaction, plants have developed multi-layered innate immune system to defend against pathogen. The preliminary layer of immune is pathogen-associated molecular pattern (PAMP) perceived by pathogen recognition receptors (PRRs), and induces a series of physiological changes leading to PAMP-triggered immunity (PTI) [53]. These physiological changes include bursts of reactive oxygen species (ROS) and changes in calcium (Ca 2+ ) concentrations [54,55].

Lipid metabolism
Lipids and fatty acids involved in lipid metabolism were considered as signal transduction mediators of plant disease resistance [63,64]. In this study, long chain acyl-CoA synthetases (LACS, XP_009394139.1 and XP_009413949.1) involved in fatty acids metabolism that acting the synthesis of cutin conferred plant resistance to fungal pathogen [65,66], and phospholipase D α1 (PLDα1, XP_009407292.1, XP_009381115.1 and XP_009408984.1) involved in lipid metabolism which promote phosphatidic acid and ROS affecting plant immunity [67] were detected in xylem sap (Supplementary Table 1).

Differential protein expression response to TR4 infection
To analyze differential protein expression response to TR4 infection, a number of 129 DEPs were identi ed in 4 possible pairs between resistant and susceptible tested combinations, but only 19 and 11 DEPs were identi ed in P_dpi vs P_mock and B_dpi vs B_mock, respectively (Supplementary Table 2). It suggested that TR4 did not induce highly dramatic changes in the overall xylem sap proteome. This result was similar to the proteomic analysis of melon phloem sap in response to viral infection [68]. However, these limited DEPs present in phloem sap might also play important roles in banana combatting with TR4.
Hypersensitive-induced response protein 1 (HIR1) may act as regulators of plant immunity by triggering hypersensitive cell death [69,70]. In the study, HIR1 (XP_018684918.1) decreased in abundance in Pahang but no signi cant changes occurred in Brazilian under TR4 infection, suggesting Pahang might decrease HIR1 expression to suppress the cell death, and enhanced resistant to TR4 due to Foc usual as hemibiotroph or necrotroph [71,72].
Ubiquitin involved in the ubiquitination system are key for plant immunity [73]. Ubiquitination is mediated by a three-step enzymatic cascades including activating (E1), conjugating (E2) and ligating (E3) enzymes [74]. E3 has received more attention in research. CaRING1, E3 ubiquitin ligase RING1 gene, played a positive role in pepper (Capsicum annuum) response to microbial pathogens [75]; whereas a homologous triplet of U-box type E3 ubiquitin ligases acted as negative regulators of PTI in Arabidopsis [76]. In the study, E3 (XP_009408396.1) with a RING zinc-nger domain decreased in abundance only in Pahang response to TR4, however, further studies are needed to prove whether this protein played a negative role in banana response to TR4.
Chalcone isomerase (CHI) is a key enzyme of avonoid pathway involved in the production of phytoalexin [77], which plays an important role in plant defense against pathogen. Overexpression of CHI enhanced resistance of soybean (Glycine max) against Phytophthora sojae [78]. In this study, chalconeavonone isomerase (also regard as CHI, XP_009384766.1) was increased in abundance in Pahang under TR4 infection, as well as in Pahang mocks compared with Brazilian mocks. It implied that this protein increased resistance against TR4 in banana.
Glycine-rich RNA-binding proteins (GRPs) function as regulators in diverse cellular processes, including response to stress in plants [79,80]. Over expressing TaRZ1, a wheat (Triticum aestivum) zinc ngercontaining GRP, in Arabidopsis thaliana increased resistance against necrotrophic bacteria Pseudomonas syringae [81]. In the present study, GRP (XP_009394303.1) containing an RNA recognition motif (RRM) domain was increased in abundance in Pahang response to TR4, but no signi cant changes in other pairwise comparisons. It indicated that this protein might play a positive role in banana response against TR4.

DEPs associated resistance in Pahang
To further explore the associated resistance mechanisms of Pahang, a number of 26 DEPs were identi ed by comparing TR4 inoculated Pahang with that of Brazilian, and the DEPs in mock inoculated Pahang versus that of Brazilian were excluded. Among which, 7 proteins whose function is unknown using the Uniprot annotation (based on version 1). Therefore, we added the RefSeq (NCBI) annotation locus codes and the V2 annotation names (column "C" and "D", respectively) for the 26 genes. Moreover, we added a more informative or alternative functional annotation which was available we added it in the "E" column. Finally, we checked if one or more paralogs or similar genes are present in Musa A genome (Column "F" and "G", respectively) (Supplementary Table 2). Of these 26 DEPs, two proteins are highly associated with resistance to pathogen in plant, including carboxylesterase and GDSL esterase/lipase Carboxylesterases (CXEs) have been implicated in plant defense. A conserved NbCXE inhibited accumulation of Tobacco mosaic virus (TMV) in Nicotiana benthamiana, which enhanced plant resistance [82]. Constitutive expression of PepEST, a fungus-inducible carboxylesterase in peper (Capsicum annuum) increased resistance against the hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides) [83]. In the present study, a CXE (XP_009406873.1), LOC103989673 (Ma06_t34160), was increased abundance in TR4 inoculated Pahang compared with that of Brazilian, but no changes in Pahang mocks compared with that of Brazilian.
GDSL esterase/lipases (GLIP) have been identi ed in many vascular plants and have been demonstrated that involved in plant defense against pathogens [84]. Overexpressing GLIP1 in Arabidopsis improved resistance against hemibiotrophic and necrotrophic pathogens [85,86]. In the study, a GLIP (XP_009382515.1), LOC103970461 (Ma11_t05100), was increased abundance in TR4 inoculated Pahang compared with that of Brazilian, but no changes in other pairs. It further validated our previous transcriptomic study that one GLIP gene was activated by TR4 attack [59].

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