Proteomic analysis of exudate of Cercospora armoraciae from Armoracia rusticana

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Microbiology

Introduction

Armoracia rusticana Gaertn., Mey. & Scherb. (Brassicaceae), horseradish, is a perennial vegetable well known for its spicy roots (Kroener & Buettner, 2017). In Europe, the plant is also used locally for medical purposes, including treatment of edema, toothache, stomach, gout and rheumatism, and to promote perspiration (Bladh & Olsson, 2011). Armoracia rusticana is native to Eastern Europe but can now be found worldwide (Papp et al., 2018).

Cercosporoid fungi, one of the largest groups of hyphomycetes, are plant pathogens responsible for numerous economically devastating plant diseases. Although distributed worldwide, these fungi are especially abundant and diverse in tropical and subtropical areas (Guo, 2016). More than 5,000 names have been published (Groenewald et al., 2013). Cercospora armoraciae causes leaf spot disease on A. rusticana. The disease initially manifests as scattered, elliptical or irregular, taupe lesions surrounded by brown margins that eventually coalesce and rupture. As the disease progresses, almost 90% of leaves become infected, which threatens production.

Droplets from C. armoraciae, when grown on PDA, distinguishes this fungus from other members of the genus Cercospora. Many fungi produce exudates (Liang, Strelkov & Kav, 2010; Aliferis & Jabaji, 2010; Wang et al., 2017a), which contain diverse biomolecules (Cooke, 1969; Jones, 1970; Colotelo, 1971). These components relate to various functions of pathogens. For example, carbohydrates in the droplets may contribute to long-term survival of sclerotia (Daly, Knoche & Wiese, 1967; Willetts, 1971; Chet & Henis, 1975; Willetts & Bullock, 1992), while phenolic acids exuded by Rhizoctonia solani influence its antifungal, phytotoxic, and antioxidant activities (Aliferis & Jabaji, 2010). An understanding of exudate compositions should thus help elucidate pathogen functions and modes of action.

Transcriptome analysis is a rapid, accurate, cost-effective method for study non-model organisms (Qiao et al., 2013). To identify the genes expressed during exudation and the particular genes that correspond to proteins which found in the exudate, we constructed a library of transcripts. We then used the nano-HPLC-MS/MS analysis method to evaluate protein components in the exudate. A total of 14,937 unigenes were assembled and 576 proteins were identified from the exudate from C. armoraciae.

Materials and Methods

Fungal materials

Cercospora armoraciae strains (SCa-01, SCa-02 and SCa-03) used in this study were isolated from infected A. rusticana in ShenYang Botanical Garden, Liaoning Province, China, and maintained and subcultured on PDA plates at 25 ± 1 °C. To harvest pure mycelia of C. armoraciae, a piece of cellophane was placed under the mycelial plugs before transfer. The samples were stored at −80 °C in a 1.5 mL DNase/RNase free microcentrifuge tube (Sangon Biotech, Shanghai, China). After 20 days, mixed exudate was collected from hyphae using a capillary tube (20 µL), and the liquid was stored at −20 °C in a 1.5 mL microcentrifuge tubes (GEB, Torrance, CA, USA).

Pathogenicity assays

Pathogenicity assays were performed on strain SCa-01 and its exudate. The fleshy roots of A. rusticana were grown under greenhouse conditions (temperature, 25–28 °C; relative humidity 75%; and natural daylight) in August. After 1-month cultivation, the same growth vigour of second youngest leaf was selected for inoculation with colonized PDA blocks (diameter, 5 mm) and a cotton ball moistened with 100 μL exudates on the adaxial surface of wounded leaves. All treatments were covered with parafilm to maintain moisture. Five replicates were used. Both treatments were placed at high relative humidity (~95%) for 24 h and then transferred the greenhouse. After 5 d and 10 d, the size of the lesions within each leaf piece was observed.

Library construction and sequencing

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA samples were then purified using Dynabeads® Oligo (dT) 25 (Life, Carlsbad, CA, USA). Then the enriched fragments of mRNA were transformed into short fragments by fragmentation buffer, and the fragments were retranscribed into cDNAs by random primers (Grabherr et al., 2011). DNA polymerase I, RNase H, dNTP and buffer were used to synthesize the second strand of DNA. Then the fragment was purified with 1.8× Agencourt AMPure XP beads, repaired, poly(A) tail added, and connected to the Illumina sequencing connector (Illumina Hi Seq 4000 system) (Zhu et al., 2018).

Raw read filtering

Reads were further filtered through fastp (version 0.18.0). The parameters were as follows: (1) reads with linkers; (2) readings with more than 10% unknown (“N”) nucleotides; (3) low-quality reads with >40% bases and a Q value less than 20.

De novo assembly

Transcriptome de novo assembly was synthesized using the short reads assembly program-Trinity (Version 2.1.1) (Grabherr et al., 2011). The Trinity package contains three modules: Inchworm, Chrysalis and Butterfly. Inchworm uses a greedy k-aggregate-based approach to assemble readings to generate sets of linear overlapping groups. Chrysalis aggregates the corresponding overlapping groups, which correspond to the parts of alternatively spliced transcripts or other unique parts of the byline homologous genes, and then constructs the de Bruijn map of each related overlapping group. Butterfly analyses the paths between read and read pairs in the context of the corresponding de Bruijn graph, and outputs a linear sequence of homologous transcripts for each variable splicing and from the side-line homologous genes.

Protein extraction

Total protein was extracted from the exudate with the cold acetone method (Liang, Strelkov & Kav, 2010). The resulting powder was dissolved in 2 mL pyrolysis buffer (8 M urea, 2% SDS), 1 protease inhibitor mixture (Roche, Basel, Switzerland). The mixture was treated by ultrasound on ice for 30 min and centrifuged at 13,000×g for 30 min at 4 °C. The supernatant was transferred to a new tube. For each sample, the protein was precipitated overnight with cold acetone at −20 °C. The precipitate was washed three times with acetone and dissolved in 8 M urea by ultrasonic treatment on ice. Protein quality was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Protein digestion

The protein concentration in supernatant was determined by BCA protein assay kit (Pierce, Rockford, IL, USA). The 100 μg protein was transferred to the new tube, and the final volume was adjusted to 100 μL with 8 M urea. In the protein solution, 11 μL of 1 M DTT (DL-dithiothreotol) was added and kept at 37 °C for 1 h, then transferred to 10 K ultrafiltration tube (Merck Millipore, Billerica, MA, USA). To remove urea, the sample was centrifuged three times by adding 100 Mm TEAB. A total of 120 μL 55 mM iodoacetamide was added to the sample, kept in room temperature for 20 min and protected from light. Then the proteins were digested overnight with sequence-level modified tryptase (Promega, Madison, WI, USA).

nano-HPLC-MS/MS analysis

The sample was divided into three equal parts and resuspended with 30 μL solvent C (C: 0.1% formic acid water; D: ACN containing 0.1% formic acid). The sample solution was separated by nanoLC and analyzed by on-line electrospray tandem mass spectrometry. The experiment was carried out on an Easy-nLC 1000 system (Thermo Fisher Scientific, Waltham, MA, USA), which is connected to the Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an online nano-electrospray ion source. The peptide sample (10 μL) was loaded onto a trap column (Thermo Scientific Acclaim Pep Map C18,100 um × 2 cm) at a flow rate of 10 μL/min for 3 min, then followed by a linear gradient from 3% D to 32% D in 120 min to separate with the analytical column (Acclaim PepMap C18, 75 μm × 15 cm). The column was re-equilibrated for 10 min with a flow rate of 300 nL/min under initial conditions. The electrospray voltage of the inlet of the mass spectrometer was 2 kV. Mass spectrometer operated in data-related acquisition mode to automatically switch between MS and MS/MS acquisition. Full scan MS spectrum (m/z 350–1,550) with mass resolution of 35 K was obtained. Then continuous high energy collision dissociation MS/MS scanning was performed with resolution of 17.5 K. The dynamic exclusion time was set to 20 s. Data are available via ProteomeXchange with identifier PXD019663.

Database search

Proteome Discoverer 1.2 (Thermo Fisher Science, Waltham, MA, USA) was used to convert mass spectrometry data into MGF files. The Mascot search engine (Version 2.3.2; Matrix Science, London, UK) was used to identify proteins by comparison to the reference transcriptome (accession number: SRR9163995). We searched the Mascot database for protein identification by using the C. armoraciae reference transcriptome in NCBI nr/Swiss Prot/Uniprot/IPI databases. The search parameters of Mascot were set to 0.050 Da for fragment ion mass tolerance and 10.0 ppm for parent ion tolerance.

Protein functional annotation and enrichment analysis

GO (http://www.geneontology.org/), KEGG (http://www.genome.jp/kegg/pathway.html), and COG/KOG (https://www.ncbi.nlm.nih.gov/COG/) databases were used to annotate the protein functions and pathway.

Results

Pathogenicity tests

Exudation from C. armariaciae appeared to damage horseradish plants in potted plant trials (Fig. 1). At 5 d-inoculation, yellowing and necrosis of the leaves was observed, and exudate infected site also showed pitchy edges, but there was no obvious size difference between pathogen and exudate inoculation site (Fig. 2A). At 10 d-inoculation, there were significant differences in necrotic spots caused by pathogens and their exduate. The pathogen inoculated lesions coalesced with a taupe brown center, which showed the similar symptom in the field. Exudate inoculated site of leaves did not expand significantly (Fig. 2B).

The formation of exudate droplets C. armoraciae accompanying with mycelial growth.

Figure 1: The formation of exudate droplets C. armoraciae accompanying with mycelial growth.

Pathogenicity tests results depicting different leaf spot lesions on A. rusticana with pathogen inoculated and exudate inoculated respectively, after 5 d (A) and 10 d (B).

Figure 2: Pathogenicity tests results depicting different leaf spot lesions on A. rusticana with pathogen inoculated and exudate inoculated respectively, after 5 d (A) and 10 d (B).

Left side of leaf: exudate inoculated site. Right side of leaf: pathogen inoculated site.

Illumina sequencing and de novo assembly of the C. armoraciae transcriptome

By Illumina sequencing, there were 51,659,554 raw reads obtained before filtering. Of them, 50,648,514 clean reads were combined and used for transcript assembly after filtering low-quality reads, resulting in 14,937 unigenes. The length of all transcripts ranged from 201 to 23,020 nucleotides, with a N50 length of 2,441 nucleotides. The raw data of RNA-seq were available at the national center for biotechnology information sequence read archive (SRA): accession number SRR9163995.

Protein identification

There were 576 proteins identified in the exudate including 1,538 peptides, of which 1,524 were unique (Table S1).

GO classification

In a GO analysis, 411 proteins (71%) were assigned to 27 GO functional groups. Among the 27 functional groups, the GO subcategories could be classified into 12 biological processes, 7 cellular components, and 8 molecular functions (Fig. 3). Metabolic process (301), cellular process (157) and single biological process (160) occupied the first three terms in the category of biological processes. While the cell (142) and cell part (142) were equally the most abundant terms in the cellular component category. Among the molecular function category, catalytic activity (263) and binding (153) accounted for an important proportion.

Gene ontology (GO) classification of exudate components from Cercospora armoraciae.

Figure 3: Gene ontology (GO) classification of exudate components from Cercospora armoraciae.

Biological process (green bars), cellular component (red bars), and molecular function (blue bars).

KEGG pathway analysis

Exudate proteins from C. armoraciae were assigned to biochemical pathways labeled with the KO number of the homologous/similar protein. Among them, 314 proteins were categorized into 84 signaling/metabolic pathways (Table 1). The top five most highly represented pathways were protein processing in ribosome (72 members, path: ko03010), carbon metabolism (53 members, path: ko01200), biosynthesis of amino acids (34 members, path: ko01230), starch and sucrose metabolism (26 members, path: ko00500), and glycolysis/gluconeogenesis (19 members, path: ko00010). Sixteen pathways were associated with amino acid synthesis (path: ko01230, ko00290, ko00220, ko00400, ko00300), metabolism (path: ko00270, ko00330, ko00350, ko00380, ko00260, ko00460, ko00250, ko00360, ko00340, ko00410), and degradation (path: ko00310, ko00280). In addition, 15 pathways were related to carbohydrate metabolism: starch and sucrose metabolism; glycolysis/gluconeogenesis; pyruvate metabolism; glyoxylate and dicarboxylate metabolism; citrate cycle; galactose metabolism; amino sugar and nucleotide sugar metabolism; fructose and mannose metabolism; pentose phosphate pathway; pentose and glucuronate interconversions; inositol phosphate metabolism; ascorbate and aldarate metabolism; C5-branched dibasic acid metabolism; propanoate metabolism; and butanoate metabolism. Energy metabolism and lipid metabolism pathways also accounted for a large proportion of the annotated pathways.

Table 1:
Pathway enrichment of exudates from Cercospora armoraciae.
No. Pathway All genes with pathway annotation (314) Pathway ID
1 Ribosome 72 (22.93%) ko03010
2 Carbon metabolism 53 (16.88%) ko01200
3 Biosynthesis of amino acids 34 (10.83%) ko01230
4 Starch and sucrose metabolism 26 (8.28%) ko00500
5 Glycolysis/Gluconeogenesis 19 (6.05%) ko00010
6 Pyruvate metabolism 17 (5.41%) ko00620
7 Glyoxylate and dicarboxylate metabolism 16 (5.1%) ko00630
8 2-Oxocarboxylic acid metabolism 16 (5.1%) ko01210
9 Citrate cycle (TCA cycle) 14 (4.46%) ko00020
10 Cysteine and methionine metabolism 14 (4.46%) ko00270
11 Arginine and proline metabolism 14 (4.46%) ko00330
12 Tyrosine metabolism 14 (4.46%) ko00350
13 Methane metabolism 14 (4.46%) ko00680
14 Galactose metabolism 13 (4.14%) ko00052
15 Tryptophan metabolism 12 (3.82%) ko00380
16 Glycine, serine and threonine metabolism 11 (3.5%) ko00260
17 Cyanoamino acid metabolism 11 (3.5%) ko00460
18 Amino sugar and nucleotide sugar metabolism 11 (3.5%) ko00520
19 Protein processing in endoplasmic reticulum 11 (3.5%) ko04141
20 Fructose and mannose metabolism 10 (3.18%) ko00051
21 Alanine, aspartate and glutamate metabolism 10 (3.18%) ko00250
22 Phenylalanine metabolism 10 (3.18%) ko00360
23 Peroxisome 10 (3.18%) ko04146
24 Valine, leucine and isoleucine biosynthesis 9 (2.87%) ko00290
25 Proteasome 9 (2.87%) ko03050
26 Pentose phosphate pathway 8 (2.55%) ko00030
27 Oxidative phosphorylation 8 (2.55%) ko00190
28 Arginine biosynthesis 8 (2.55%) ko00220
29 Purine metabolism 8 (2.55%) ko00230
30 Lysine degradation 7 (2.23%) ko00310
31 Glutathione metabolism 7 (2.23%) ko00480
32 Glycerolipid metabolism 7 (2.23%) ko00561
33 Phagosome 7 (2.23%) ko04145
34 Pentose and glucuronate interconversions 6 (1.91%) ko00040
35 Histidine metabolism 6 (1.91%) ko00340
36 Glycerophospholipid metabolism 6 (1.91%) ko00564
37 Nitrogen metabolism 6 (1.91%) ko00910
38 Fatty acid degradation 5 (1.59%) ko00071
39 Valine, leucine and isoleucine degradation 5 (1.59%) ko00280
40 Beta-Alanine metabolism 5 (1.59%) ko00410
41 Other glycan degradation 5 (1.59%) ko00511
42 Thiamine metabolism 5 (1.59%) ko00730
43 Riboflavin metabolism 5 (1.59%) ko00740
44 RNA transport 5 (1.59%) ko03013
45 Endocytosis 5 (1.59%) ko04144
46 Inositol phosphate metabolism 4 (1.27%) ko00562
47 Glycosphingolipid biosynthesis—globo series 4 (1.27%) ko00603
48 Pantothenate and CoA biosynthesis 4 (1.27%) ko00770
49 Sulfur metabolism 4 (1.27%) ko00920
50 Phosphatidylinositol signaling system 4 (1.27%) ko04070
51 Ubiquitin mediated proteolysis 4 (1.27%) ko04120
52 Ascorbate and aldarate metabolism 3 (0.96%) ko00053
53 Pyrimidine metabolism 3 (0.96%) ko00240
54 Phenylalanine, tyrosine and tryptophan biosynthesis 3 (0.96%) ko00400
55 N-Glycan biosynthesis 3 (0.96%) ko00510
56 C5-Branched dibasic acid metabolism 3 (0.96%) ko00660
57 Nicotinate and nicotinamide metabolism 3 (0.96%) ko00760
58 Aminoacyl-tRNA biosynthesis 3 (0.96%) ko00970
59 RNA degradation 3 (0.96%) ko03018
60 Taurine and hypotaurine metabolism 2 (0.64%) ko00430
61 Selenocompound metabolism 2 (0.64%) ko00450
62 Glycosaminoglycan degradation 2 (0.64%) ko00531
63 Sphingolipid metabolism 2 (0.64%) ko00600
64 Propanoate metabolism 2 (0.64%) ko00640
65 Terpenoid backbone biosynthesis 2 (0.64%) ko00900
66 Degradation of aromatic compounds 2 (0.64%) ko01220
67 mRNA surveillance pathway 2 (0.64%) ko03015
68 Spliceosome 2 (0.64%) ko03040
69 Protein export 2 (0.64%) ko03060
70 Cell cycle—yeast 2 (0.64%) ko04111
71 SNARE interactions in vesicular transport 2 (0.64%) ko04130
72 Regulation of mitophagy—yeast 2 (0.64%) ko04139
73 Fatty acid biosynthesis 1 (0.32%) ko00061
74 Aflatoxin biosynthesis 1 (0.32%) ko00254
75 Monobactam biosynthesis 1 (0.32%) ko00261
76 Lysine biosynthesis 1 (0.32%) ko00300
77 Various types of N-glycan biosynthesis 1 (0.32%) ko00513
78 Arachidonic acid metabolism 1 (0.32%) ko00590
79 Butanoate metabolism 1 (0.32%) ko00650
80 One carbon pool by folate 1 (0.32%) ko00670
81 Porphyrin and chlorophyll metabolism 1 (0.32%) ko00860
82 Fatty acid metabolism 1 (0.32%) ko01212
83 Ribosome biogenesis in eukaryotes 1 (0.32%) ko03008
84 Sulfur relay system 1 (0.32%) ko04122
DOI: 10.7717/peerj.9592/table-1

COG/KOG functional annotation

There were 450 proteins annotated against the COG/KOG database (Fig. 4). In all functional ontologies, O group (posttranslational modification, protein turnover, chaperones) had the most protein. J (translation, ribosomal structure, and biogenesis) and R (general function prediction only) groups also contained relatively more proteins. Y (nuclear structure) and N (cell motility) were two functional groups with less protein. In addition, 24 proteins were categorized as functionally unknown proteins.

Distribution of COG/KOG functional classes of exudate proteins from Cercospora armoraciae.

Figure 4: Distribution of COG/KOG functional classes of exudate proteins from Cercospora armoraciae.

Discussion

We performed de novo sequencing to profile the transcriptome and nano-HPLC-MS/MS analysis to study the proteomics of C. armoraciae exudate. This approach provided preliminary information needed for molecular profiling and functional studies of this, and related, plant pathogens that traditional methods of protein research cannot provide (Zhang et al., 2013; Lin et al., 2017; Wang et al., 2017b). Searching and annotating exudate proteins in the transcript database identified 576 proteins; however, lack of a reference genome limits the effective information obtained from only a single identification of a protein of its exudate.

Although many hyphomycetes exudate droplets under artificial culture conditions (Sprecher, 1959; Colotelo, 1978; Stefan et al., 2010), the role of exudates in the ecosystem is unclear. We speculate that exudation of droplets accompanies the entire process of colony growth and development and plays a vital role in plant–fungal interactions and maintenance of the physiological balance of fungi (Cooke, 1969). Guttation droplets of Metarhizium anisopliae serve as a water reservoir for maintaining the constant growth of aerial hyphae (Stefan et al., 2010). Exudate decreases during development of Sclerotinia (Liang, Strelkov & Kav, 2010; Wang et al., 2017a), which may reflect partial re-absorption of exudate into the fungus (Colotelo, 1978). In the case of Sclerotinia and Sclerotiorum species, excess nutrients, water, and metabolites of exudates translocate to mature sclerotia to sustain increased metabolic activity (Georgiou et al., 2006; Cooke, 1971; Punja, 1985).

As inferred from the GO analysis, some proteins were related to growth, development, and reproduction, which suggests exudates participate in the whole life history of the pathogen. Cell and cell part are the two most important cell component GO terms, and many proteins are involved in the formation of organelles and membranes. KEGG analysis indicated that 16 of 84 identified pathways were related to amino acid synthesis, metabolism, and decomposition; among these pathways, phenylalanine metabolism is associated with the shikimic acid pathway and enhances pathogenicity (Southerton & Deverall, 1990). KEGG analysis also identified 15 pathways related to carbohydrate metabolism, including 3 (glycolysis/gluconeogenesis, citrate cycle, and pentose phosphate) that cooperate to increase the adaptability of organisms (Kruger & Schaewen, 2003). Two identified pathways, the peroxisome metabolic pathway and the fatty acid biosynthesis pathway, may confer antifungal, antimicrobial, and antioxidant activities on exudates and protect the pathogens. Exudates of R. solani significantly reduce the spore germination of Stachybotrys elegans, Heterosporium solani, Fusarium sporotrichioides, and Trichoderma virens relative to the control (Aliferis & Jabaji, 2010), and exudates from Sclerotium rolfsii and R. solani also contribute to antioxidant activities (Pandey et al., 2007; Aliferis & Jabaji, 2010). COG/KOG analysis uncovered 24 proteins of unknown function, such proteins also account for 32% and 23% of exudate proteins of Sclerotinia sclerotiorum and S. ginseng, respectively (Liang, Strelkov & Kav, 2010; Wang et al., 2017a).

Conclusions

This first-ever detailed classification and annotation of proteins of exudate from C. armoraciae supports the hypothesis that exudates enhance virulence.

Supplemental Information

De novo assembly sequences of Cercospora armoraciae.

DOI: 10.7717/peerj.9592/supp-1

List of proteins identified with LC-MS/MS in the exudates of Cercospora armoraciae.

DOI: 10.7717/peerj.9592/supp-2
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