Open Access

Fungal and host transcriptome analysis of pH-regulated genes during colonization of apple fruits by Penicillium expansum

  • Shiri Barad1, 2,
  • Noa Sela3,
  • Dilip Kumar1,
  • Amit Kumar-Dubey1,
  • Nofar Glam-Matana1, 2,
  • Amir Sherman4 and
  • Dov Prusky1Email author
Contributed equally
BMC Genomics201617:330

https://doi.org/10.1186/s12864-016-2665-7

Received: 29 March 2016

Accepted: 26 April 2016

Published: 4 May 2016

Abstract

Background

Penicillium expansum is a destructive phytopathogen that causes decay in deciduous fruits during postharvest handling and storage. During colonization the fungus secretes D-gluconic acid (GLA), which modulates environmental pH and regulates mycotoxin accumulation in colonized tissue. Till now no transcriptomic analysis has addressed the specific contribution of the pathogen's pH regulation to the P. expansum colonization process. For this purpose total RNA from the leading edge of P. expansum-colonized apple tissue of cv. 'Golden Delicious' and from fungal cultures grown under pH 4 or 7 were sequenced and their gene expression patterns were compared.

Results

We present a large-scale analysis of the transcriptome data of P. expansum and apple response to fungal colonization. The fungal analysis revealed nine different clusters of gene expression patterns that were divided among three major groups in which the colonized tissue showed, respectively: (i) differing transcript expression patterns between mycelial growth at pH 4 and pH 7; (ii) similar transcript expression patterns of mycelial growth at pH 4; and (iii) similar transcript expression patterns of mycelial growth at pH 7. Each group was functionally characterized in order to decipher genes that are important for pH regulation and also for colonization of apple fruits by Penicillium. Furthermore, comparison of gene expression of healthy apple tissue with that of colonized tissue showed that differentially expressed genes revealed up-regulation of the jasmonic acid and mevalonate pathways, and also down-regulation of the glycogen and starch biosynthesis pathways.

Conclusions

Overall, we identified important genes and functionalities of P. expansum that were controlled by the environmental pH. Differential expression patterns of genes belonging to the same gene family suggest that genes were selectively activated according to their optimal environmental conditions (pH, in vitro or in vivo) to enable the fungus to cope with varying conditions and to make optimal use of available enzymes. Comparison between the activation of the colonized host's gene responses by alkalizing Colletotrichum gloeosporioides and acidifying P. expansum pathogens indicated similar gene response patterns, but stronger responses to P. expansum, suggesting the importance of acidification by P. expansum as a factor in its increased aggressiveness.

Keywords

Transcription profiling Penicillium expansum RNA-seq Gene expression pH regulation Pathogenicity pH-regulated genes Fungal genes regulated by pH Apple genes regulated by pH

Background

The genus Penicillium comprises a group of anamorphic fungi in the division Ascomycota [1]. Some Penicillium species are of economic importance because they are postharvest pathogens that cause spoilage in tropical and deciduous fruits. Penicillium expansum is the causal agent of blue mold, which is considered one of the most important postharvest pathogens [2] and that causes decay in deciduous fruits during postharvest handling and storage.

Penicillium expansum causes extensive maceration of the infected tissue by means of a common mechanism of tissue acidification [3]; it acidifies its host by activation of glucose oxidase 2 (gox2) and the catalyzed oxidation of glucose resulting in secretion of small molecules such as D-gluconic acid (GLA) [3, 4], which modulate the environmental pH and thereby activate several polygalacturonases that contribute to pectin depolymerization and consequently tissue maceration, at pH 3.5-4 [3, 5, 6]. Functional analysis of glucose oxidase 2-RNAi mutants showed a strong effect on pathogen interactions with their host: the greater the down-regulation of gox2, the stronger the impairment of GLA production, medium acidification, and apple fruit colonization [7]. More recently, Barad et al. [8] reported that P. expansum secreted not only GLA but also ammonia at the leading edge of colonization in apple fruits. Growth of the fungus in high-sucrose media induced rapid metabolism of sugar and increased GLA accumulation, and the decrease in available carbon reserves resulted in enhanced ammonia accumulation, probably because of amino acid catabolism, such as occurs with other pathogens [9, 10]. These results indicated that bidirectional pH regulation occurs in P. expansum-probably dependent on nutrient availability. The question concerns which metabolic processes are modulated by the pH level during colonization of apple fruits.

PacC is a transcription factor that activates genes of Aspergillus nidulans during the alkalization of the environment [11]. In a recent report [8], it was suggested that P. expansum's pacC gene was active also under acidic conditions, and that ammonia produced at the leading edge of the decaying tissue under low-pH conditions further contributed to the activation of pacC responsiveness. This may indicate that P. expansum uses ammonia accumulation during nutritional modification of the ambient environmental pH as a regulatory cue for activation of pacC, by signaling and activating alkaline-induced genes that contribute to pathogenicity and accumulation of secondary metabolites such as patulin [8]. This pH modulation under the influence of GLA and ammonia accumulation is important for understanding the fungal response to differential gene regulation by pH; however, the overall response remains poorly understood.

The diversity of recently described ecological interactions of Penicillium with fruits [3, 4] and with other resident microorganisms [5] have suggested a wide variety of genes that may contribute to the pathogenicity of this pathogen. However, the work of Barad et al. [8] has attributed to the pH regulation of the host by P. expansum specific importance for pathogenicity. In the present study we used a transcriptomic approach to analyze the effect of pH on fungal gene regulation and host responses to various pH-modulating pathogens. The effect of the fungal response was observed by comparing the responses of the apple to colonization by P. expansum as driven by expression of the fungal gene at pH 4 or 7. Our RNA-Seq data of fungal responses revealed nine differentially co-expressed gene clusters; they showed alterations of expression patterns that were associated with differential pH responses, and also with genes related to P. expansum colonization. The apple response to pH was elucidated by using qRT-PCR to analyze expressions of selected genes in response to acidifying and alkalinizing pathogens: P. expansum and Colletotrichum gloeosporioides, respectively. This analysis indicated similar patterns of host response to the respective pathogens, but the apple genes showed an extensive multifold greater response to infection by its natural pathogen P. expansum than to that by C. gloeosporioides. This may indicate the importance of the acidifying modulation of pH by Penicillium as a factor for greater aggressiveness of P. expansum.

Results and Discussion

Profiling the expression of P. expansum genes during colonization and growth at pH 4 and 7

Genomes of P. expansum [7] were sequenced by using paired-end reads by means of Illumina Hiseq 2500 [12]. For analysis of the P. expansum transcriptome we downloaded the reference draft genome accession JQFX00000000.1 from the Genbank site [13]. Ten libraries of single-end RNAseq (deposited in Genbank under accession SRP071104) were mapped to the reference genome by using the Bowtie2 software [14]. The 10 libraries contained the following features: (i) P. expansum grown in culture media at pH 4 for 3 h as well as a pooled sample collected at several time points-0.5, 1, 3, 10, and 24 h-with two replicates at each point; (ii) P. expansum grown in culture media at pH 7 for 3 h, as well as a pool collected at several time points-0.5, 1, 3, 10, and 24 h-with two replicates at each point; and (iii) the leading edge, comprising 1–2 mm of apple cv. 'Golden Delicious' tissue, colonized by P. expansum. For in vitro experiments, 106 spores of P. expansum were inoculated into germinating minimal medium (GMM) at pH 4.5 as described by Barad et al. [7] and transferred 2 days later to buffered inducing media at pH 4 or 7. At several different time points after transfer to inducing secondary media (SM), fungal mycelia were sampled and pooled for analysis. Pooled samples of RNA were compared with independent samples 3 h after transfer to inducing media.

Principal component analysis (PCA) of all expressed genes of P. expansum indicated that the replicates were very close to each other, and that the expression patterns of fungal genes exposed to the respective pH conditions for 3 h were almost identical to those of pooled samples of mycelia exposed for various intervals (Fig. 1a). Moreover, the general pattern of gene expression in decayed apple tissue was far different from those of cultures grown under different pH conditions, as could be expected (Fig. 1a). Hierarchical clustering analysis indicated differing patterns of gene expression between the in vivo and in vitro samples at both pH levels; and the colonized apple tissue contained more down- than up-regulated genes (Fig. 1b).
Fig. 1

Principal component analysis (PCA) and heat map analysis of all the samples: duplicates of leading edge of inoculated apple; duplicates of pooled samples from all time points treated in vitro at pH 4.0; two replicates of P. expansum mycelia treated in vitro at pH 4.0 for 3 h; duplicates of a pool of samples from all time points treated in vitro at pH 7.0; and two replicates of P. expansum mycelia treated in vitro at pH 7.0 for 3 h. a PCA based on 5,646 differentially expressed genes. The results are plotted as: (♦) decayed apple; (■) in vitro at pH 4 for 3 h; (▲) pooled samples at pH 4; (X) in vitro at pH 7 for 3 h; (ӿ) pooled samples at pH 7. Circles are drawn around clustered points. b Expression heat map of differentially expressed P. expansum genes in the various samples

Out of 11,019 annotated genes [12], 5,646 genes were differentially expressed according to an false discovery rate (FDR, [15]) threshold < 0.05 and the log fold change was greater than 1 or smaller than −1 implying at least 2 fold change in expression scale (Fig. 1b). We sorted the differentially expressed gene patterns into nine different co-expressed gene clusters, which fell into three major groups:

Group I comprised clusters of genes whose expression patterns in the colonized tissue differed with respect to growth at pH 4 or pH 7. This group comprised clusters 1, 2, 3, and 6, which contained genes that are important for P. expansum colonization within apple fruits.

Group II comprised clusters in which expression patterns of the colonized tissue were similar to those of the pathogen's growth in culture at pH 4, and differed from those of fungal growth at pH 7. This group includes clusters numbers 5, 7, and 8.

Group III comprised gene clusters in which expression patterns of the colonized tissue were similar to those expressed by the pathogen grown in culture at pH 7. This group includes clusters numbers 4 and 9 (Fig. 2).
Fig. 2

Expression patterns of 9 clusters of co-expressed differential genes. The normalized expression pattern (log 2-transformed, median centered) of 9 clusters that were derived from the hierarchical clustering algorithm by use of the hclust function algorithm in R [70, 71]. Each gene is plotted in gray, in addition to the mean expression profile for that cluster (blue). The figure was generated by using perl script define_clusters_by_cutting_tree.pl of the Trinity software package [67]

Group I includes clusters of expressed genes, whose level of expression under in vitro conditions differed from that exhibited during colonization. This group comprised clusters 1 and 2, with 2,248 and 233 genes, respectively, which exhibited higher expression under in vitro growth conditions at pH 4 or 7 than that manifested as down-regulation in the colonized apple tissue (Fig. 2). The genes expressed under both high and low pH are probably important for growth under in vitro conditions but not for pathogenicity. Group I also included clusters 3 and 6, with 2,220 and 72 genes, respectively, which comprise genes that are up-regulated in apple tissue colonization but down-regulated in vitro at both at pH 4 and pH 7. All the genes in this group probably are involved in growth in either nutritional environments or liquids, or both, that differ substantially from those within the apple tissue.

Group II comprised clusters 5, 7, and 8, in which the gene expression patterns in the colonized tissue were similar to those in vitro at pH 4 but different from those in vitro at pH 7 (Fig. 2). Cluster 7, with a gene count of 67 transcripts, showed similar overall expression levels during colonization of apple tissue and during in vitro growth at pH 4, which suggests that this cluster contained genes that are activated at pH 4, i.e., the usual fruit pH, therefore we consider that they may contribute to colonization at pH 4. Another two clusters present in this group were 5 and 8, with gene counts of 482 and 58, respectively; they include genes that were down-regulated both in colonized apple tissue and in vitro at pH 4, but maintained high expression levels in vitro at pH 7. These genes are probably necessary for growth under alkaline conditions.

Group III comprises gene clusters that showed similar expression levels in colonized apple tissue and during in vitro growth fungal at pH 7, but showed altered gene expression at pH 4 (Fig. 2). This group included clusters 9 and 4, with 24 and 242 transcript counts, respectively. Genes in cluster 9 showed similar high expression levels in colonized fruit and in vitro at pH 7, but low levels in vitro at pH 4; these genes probably are expressed in fruits under induced alkaline conditions or during fungal ammonia accumulation [8]. Cluster 4 included genes that exhibited low expression levels both in colonized fruit and in vitro at pH 7; we hypothesize that these genes are repressed under alkaline conditions or during ammonia secretion [8].

Gene ontology analysis of decayed apple tissue compared with in vitro conditions

We analyzed functional enrichment in each cluster using blast2go software [16] and Fisher’s Exact Test [17].

Clusters with different gene expression levels under colonization than under in vitro conditions

Higher expression levels of fungal genes when grown in culture than in colonized tissue

Genes in clusters 1 and 2 showed higher expression levels under in vitro conditions than in colonized tissue. Functionality of these genes showed involvement in respiratory activity, in light of their easier growth in culture media than in colonized apple tissue. GO-enriched terms in clusters 1 and 2 are depicted in Additional file 1: Figure S1 and Additional file 2: Figure S2, respectively.

Lower expression levels of fungal genes when grown in culture than in colonized tissue

Genes in clusters 3 and 6 showed lower expression levels when grown in vitro at pH 4 or pH 7 than when grown in colonized tissue (Fig. 2). These clusters included genes encoding for vesicle transport, which might indicate activation of secretion process(es); probably of mycotoxin(s) and effectors (Additional file 3: Figure S3 and Additional file 4: Figure S6 respectively). Cluster 3 included most of the 15 genes involved in patulin biosynthesis-patB, D, E, F, H, I, J, K, L, and O. Unexpectedly, these genes were not highly expressed in vitro at either pH 4 or pH 7, which supports a previous report by Li et al. [18], which suggested the importance of specific nutritional growth conditions for induced expression of the patulin biosynthesis gene cluster (PatA-PatO) in P. expansum. However, at the same time, the question arises of the specific mechanism(s)-possibly nutritional and possibly others-that modulate the conditions for expression of the mycotoxin cluster in vivo [1820]. Cluster 3 also showed genes such as acetamidase, amidase-family proteins, and fatty-acid amide hydrolase, encoding for amidase activity (Additional file 3: Figure S3) these may indicate a possible source of NH4 + production during periods of limited sucrose levels and pathogenicity of P. expansum, which would support earlier suggestions by Barad et al. [8] that local pH modulation might result from ammonia accumulation in the leading edge during pathogenicity of P. expansum.

Cluster 6 shows enriched expression of genes associated with host-cell-wall degradation that are important for the virulence of P. expansum [5] (Additional file 4: Figure. S6). These genes have previously known functionalities associated with pathogenicity of fungi; these genes and activities include: chitinase-associated genes, pectin lyase, and polygalacturonase (PG) activities. Among these, PG was shown to be involved in maceration of apples by P. expansum, particularly in diseases characterized by tissue maceration or soft rot [21]; and pectin lyases contribute to the pathogenicity of many pathogens by degrading pectin polymers directly by means of a β-elimination mechanism that results in formation of 4,5-unsaturated oligo-galacturonides [22]. In addition, cluster 6 shows over-representation of genes encoding for aspartic endopeptidase-pep1-which is associated with pH modulation and pathogenicity of P. digitatum [23]. Aspartic endopeptidase catalyzes hydrolysis of elastin and collagen, the major structural proteins of basement membranes, and plays a significant role in virulence of P. digitatum on citrus fruits [23]. Aspartic endopeptidase was up-regulated during infection of citrus fruits, and contributed to fungal colonization, either by degradation of plant cell-wall components to provide a nitrogen supply, or even by inactivating defense proteins [24]. This type of response at low pH is probably a result of accumulation of GLA during the pathogenicity process of P. expansum, in order to ensure that secreted enzymes and metabolites are produced at the optimal pH to fully facilitate their physiological functions [3].

Clusters with similar expression levels in colonized apple and in vitro at pH 4

High expression level of fungal genes in colonized apple tissue and in samples grown in culture at pH 4

A single cluster-cluster 7-showed enhanced expression of genes both in colonized apple tissue and in vitro at pH 4 (Fig. 2). This cluster was enriched in genes involved in the glutamate metabolic process (Additional file 5: Figure S7); a process that includes high expression of glutamate decarboxylase (Table 1), which is required for normal oxidative-stress tolerance in Saccharomyces cerevisiae. γ-Aminobutyric acid (GABA) largely originates from decarboxylation of L-glutamate; it is associated with sporulation/spore metabolism [25] and in most fungi it serves as a carbon and nitrogen source. Also, γ-aminobutyric acid metabolism promotes asexual sporulation in Saccharomyces nodorum [26]. Moreover, in plants and fungi GABA synthesis has been associated with acidic pH, either in response to cytosolic acidification-probably as a pH-regulatory mechanism-or during growth under acidic conditions [25]. Since acidification of the host environment usually occurs during apple colonization by P. expansum, it is possible that γ-aminobutyric acid synthesis might be one of the mechanisms employed by the fungus to cope with this more acidic pH of the environment [6].
Table 1

Genes involved in each process of cluster 7

GO Term

Gene id

Genbank transcript id

Description

Nucleobase transport

PEXP_081890

KGO47390

Permease, cytosine/purine, uracil, thiamine, allantoin

 

PEXP_004400

KGO37467

Permease, cytosine/purine, uracil, thiamine, allantoin

 

PEXP_081870

KGO47388

Permease, cytosine/purine, uracil, thiamine, allantoin

Taurine metabolic process

PEXP_099510

KGO46394

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

 

PEXP_039450

KGO48081

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

Beta-alanine metabolic process

PEXP_099510

KGO46394

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

 

PEXP_039450

KGO48081

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

Transmembrane transport

PEXP_023020

KGO42773

Major facilitator superfamily domain, general substrate transporter

 

PEXP_104460

KGO36460

Major facilitator superfamily domain, general substrate transporter

 

PEXP_019050

KGO45782

C4-dicarboxylate transporter/malic acid transport protein

 

PEXP_083120

KGO38619

Major facilitator superfamily domain, general substrate transporter

 

PEXP_105230

KGO41447

Major facilitator superfamily domain, general substrate transporter

 

PEXP_012370

KGO49188

Major facilitator superfamily domain, general substrate transporter

 

PEXP_060340

KGO45245

Iron permease FTR1

 

PEXP_059590

KGO45345

Major facilitator superfamily domain, general substrate transporter

 

PEXP_094700

KGO43568

Major facilitator superfamily domain, general substrate transporter

 

PEXP_025230

KGO42652

Major facilitator superfamily domain, general substrate transporter

 

PEXP_030850

KGO40270

ABC transporter, integral membrane type 1

 

PEXP_078840

KGO37868

Major facilitator superfamily domain, general substrate transporter

Glutamate metabolic process

PEXP_099510

KGO46394

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

 

PEXP_039450

KGO48081

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

Glutamate decarboxylase activity

PEXP_099510

KGO46394

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

PEXP_039450

KGO48081

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

Nucleobase transmembrane transporter activity

PEXP_081890

KGO47390

Permease, cytosine/purine, uracil, thiamine, allantoin

PEXP_004400

KGO37467

Permease, cytosine/purine, uracil, thiamine, allantoin

 

PEXP_081870

KGO47388

Permease, cytosine/purine, uracil, thiamine, allantoin

Cofactor binding

PEXP_107000

KGO41320

Aldolase-type TIM barrel

 

PEXP_068570

KGO46687

Aldolase-type TIM barrel

 

PEXP_099510

KGO46394

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

 

PEXP_080260

KGO38010

Pyridoxal phosphate-dependent transferase, major region, subdomain 2

 

PEXP_043260

KGO39485

Thiamine pyrophosphate enzyme, C-terminal TPP-binding

 

PEXP_076130

KGO36094

D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding

 

PEXP_001130

KGO44422

Glyceraldehyde/Erythrose phosphate dehydrogenase family

 

PEXP_039450

KGO48081

Pyridoxal phosphate-dependent transferase, major region, subdomain 1

Glucosylceramidase activity

PEXP_081260

KGO47327

Glycoside hydrolase, family 30

3-isopropylmalate dehydratase activity

PEXP_074470

KGO48717

Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/alpha, subdomain 1/3

N-acylphosphatidylethanolamine-specific phospholipase D activity

PEXP_024950

KGO42624

N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D

Transmembrane transporter activity

PEXP_104460

KGO36460

Major facilitator superfamily domain, general substrate transporter

 

PEXP_034210

KGO40038

CDR ABC transporter

 

PEXP_019050

KGO45782

C4-dicarboxylate transporter/malic acid transport protein

 

PEXP_105230

KGO41447

Major facilitator superfamily domain, general substrate transporter

 

PEXP_081890

KGO47390

Permease, cytosine/purine, uracil, thiamine, allantoin

 

PEXP_004400

KGO37467

Permease, cytosine/purine, uracil, thiamine, allantoin

 

PEXP_030850

KGO40270

ABC transporter, integral membrane type 1

 

PEXP_081870

KGO47388

Permease, cytosine/purine, uracil, thiamine, allantoin

Integral component of membrane

PEXP_023020

KGO42773

Major facilitator superfamily domain, general substrate transporter

 

PEXP_104460

KGO36460

Major facilitator superfamily domain, general substrate transporter

 

PEXP_034210

KGO40038

CDR ABC transporter

 

PEXP_019050

KGO45782

C4-dicarboxylate transporter/malic acid transport protein

 

PEXP_083120

KGO38619

Major facilitator superfamily domain, general substrate transporter

 

PEXP_054930

KGO44009

Major intrinsic protein

 

PEXP_105230

KGO41447

Major facilitator superfamily domain, general substrate transporter

 

PEXP_012370

KGO49188

Major facilitator superfamily domain, general substrate transporter

 

PEXP_059590

KGO45345

Major facilitator superfamily domain, general substrate transporter

 

PEXP_094700

KGO43568

Major facilitator superfamily domain, general substrate transporter

 

PEXP_027630

KGO43014

Amino acid transporter, transmembrane

 

PEXP_025230

KGO42652

Major facilitator superfamily domain, general substrate transporter

 

PEXP_030850

KGO40270

ABC transporter, integral membrane type 1

 

PEXP_038200

KGO47945

Mitochondrial substrate/solute carrier

 

PEXP_078840

KGO37868

Major facilitator superfamily domain, general substrate transporter

Lysosome

PEXP_081260

KGO47327

Glycoside hydrolase, family 30

3-Isopropylmalate dehydratase complex

PEXP_074470

KGO48717

Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/alpha, subdomain 1/3

Low expression level of fungal genes in colonized apple and in samples grown in vitro at pH 4

In contrast to the single up-regulated cluster 7, two clusters-5 and 8-showed repressed expression of genes both in colonized apple tissue and in vitro at pH 4 (Fig. 2). Cluster 5 showed enrichment of genes that are involved in fungal pathogenicity and that are known to be active under alkaline conditions; they exhibit, for example, pectate lyase and hydrolase activities [27] (Additional file 6: Figure S5) and the transcription factor PacC (Table 2), which suggests that the acidic pH-repressed alkaline-expressed genes are involved in pathogenicity. It is likely that P. expansum adopted its acidifying life pattern characterized by production of organic acids-mainly gluconic acid secretion [7]-in order to optimize the pH of the media. These present findings support a previous publication by Barad et al. [28] which indicated that whereas pacC expression was inhibited under acidic conditions during the accumulation of gluconic acid, it might be activated in the leading edge of colonized tissue where ammonia is accumulated at pH 4 [8]. Part of the localized pH modulation may be regulated by the Na+ and Ca2+ transporters that are present in cluster 5, that modulate the internal equilibrium of hydrogen ions and contribute to cytoplasmic pH (Table 2).
Table 2

Genes involved in each process of cluster 5

GO Term

Gene id

Genbank transcript id

Description

Regulation of pH

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

 

PEXP_043720

KGO39291

Alkali metal cation/H+ antiporter Nha1, C-terminal

 

PEXP_009670

KGO49300

Cation/H+ exchanger

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

L-Arabinose metabolic process

PEXP_023030

KGO42774

Glycoside hydrolase, superfamily

PEXP_071150

KGO40611

Alcohol dehydrogenase superfamily, zinc-type

PEXP_089320

KGO41720

Glycoside hydrolase, superfamily

Calcium ion transport

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

 

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

Kynurenine metabolic process

PEXP_039010

KGO48037

putative cyclase

PEXP_045070

KGO39237

putative cyclase

PEXP_009350

KGO49268

Pyridoxal phosphate-dependent transferase, major region, subdomain 2

Glyoxylate cycle

PEXP_011610

KGO49112

Malate synthase A

 

PEXP_077890

KGO37773

Pyruvate/Phosphoenolpyruvate kinase

Tryptophan catabolic process

PEXP_039010

KGO48037

Putative cyclase

PEXP_045070

KGO39237

Putative cyclase

PEXP_009350

KGO49268

Pyridoxal phosphate-dependent transferase, major region, subdomain 2

Inorganic ion transmembrane transport

PEXP_059490

KGO45335

Sulfate anion transporter

PEXP_043720

KGO39291

Alkali metal cation/H+ antiporter Nha1, C-terminal

PEXP_048620

KGO39686

Nickel/cobalt transporter, high-affinity

 

PEXP_009670

KGO49300

Cation/H+ exchanger

 

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_094650

KGO43563

Ammonium transporter

Disaccharide metabolic process

PEXP_052570

KGO42305

Glycosyl transferase, family 20

PEXP_011870

KGO49138

Pectinesterase, catalytic

PEXP_069330

KGO46763

Glycoside hydrolase, family 61

 

PEXP_011880

KGO49139

Glycoside hydrolase, family 28

 

PEXP_066270

KGO46936

Hexokinase, N-terminal

 

PEXP_053240

KGO42372

Glycoside hydrolase, family 61

 

PEXP_107940

KGO41788

Alpha-amylase, C-terminal all beta

Substrate-specific transmembrane transporter activity

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

PEXP_059490

KGO45335

Sulfate anion transporter

PEXP_016910

KGO46049

Xanthine/uracil/vitamin C permease

 

PEXP_043720

KGO39291

Alkali metal cation/H+ antiporter Nha1, C-terminal

 

PEXP_052330

KGO42281

Major facilitator superfamily domain, general substrate transporter

 

PEXP_033600

KGO39977

Amino acid/polyamine transporter I

 

PEXP_094860

KGO43584

Major facilitator superfamily domain, general substrate transporter

 

PEXP_068270

KGO46657

Nucleobase cation symporter-1, NCS1

 

PEXP_006100

KGO36811

Major facilitator superfamily domain, general substrate transporter

 

PEXP_086200

KGO40808

Amino acid/polyamine transporter I

 

PEXP_003610

KGO37353

Major facilitator superfamily domain, general substrate transporter

 

PEXP_043410

KGO39260

Major facilitator superfamily domain, general substrate transporter

 

PEXP_048620

KGO39686

Nickel/cobalt transporter, high-affinity

 

PEXP_006360

KGO36837

Major facilitator superfamily domain, general substrate transporter

 

PEXP_000260

KGO44335

Amino acid/polyamine transporter I

 

PEXP_072400

KGO48510

Major facilitator superfamily domain, general substrate transporter

 

PEXP_009030

KGO48810

Na dependent nucleoside transporter

 

PEXP_068910

KGO46721

Major facilitator superfamily domain, general substrate transporter

 

PEXP_031270

KGO40312

Permease, cytosine/purine, uracil, thiamine, allantoin

 

PEXP_009670

KGO49300

Cation/H+ exchanger

 

PEXP_110420

KGO38764

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_095790

KGO43677

Major facilitator superfamily domain, general substrate transporter

 

PEXP_048590

KGO39683

Amino acid/polyamine transporter I

 

PEXP_104220

KGO36561

Cation/H+ exchanger

 

PEXP_019490

KGO45826

C4-dicarboxylate transporter/malic acid transport protein

 

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_076320

KGO37552

Major facilitator superfamily domain, general substrate transporter

 

PEXP_001370

KGO37112

Major facilitator superfamily domain, general substrate transporter

 

PEXP_084160

KGO41053

Amino acid/polyamine transporter I

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_062420

KGO45644

General substrate transporter

 

PEXP_022750

KGO42746

Major facilitator superfamily domain, general substrate transporter

 

PEXP_068690

KGO46699

Major facilitator superfamily domain, general substrate transporter

 

PEXP_034320

KGO40048

Arsenical pump membrane protein, ArsB

 

PEXP_074940

KGO36159

Major facilitator superfamily domain, general substrate transporter

 

PEXP_094650

KGO43563

Ammonium transporter

Solute:proton antiporter activity

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

 

PEXP_043720

KGO39291

Alkali metal cation/H+ antiporter Nha1, C-terminal

 

PEXP_009670

KGO49300

Cation/H+ exchanger

 

PEXP_104220

KGO36561

Cation/H+ exchanger

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

Cation:cation antiporter activity

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

PEXP_043720

KGO39291

Alkali metal cation/H+ antiporter Nha1, C-terminal

 

PEXP_104220

KGO36561

Cation/H+ exchanger

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

Metal ion binding

PEXP_046510

KGO39177

Cytochrome P450, E-class, CYP52

 

PEXP_036890

KGO35901

Transcription factor, fungi

 

PEXP_084560

KGO41093

4-Hydroxyphenylpyruvate dioxygenase

 

PEXP_055320

KGO44077

Polyketide synthase, enoylreductase

 

PEXP_042410

KGO39400

Manganese/iron superoxide dismutase, C-terminal

 

PEXP_072700

KGO48540

Annexin

 

PEXP_089160

KGO41704

Terpenoid synthase

 

PEXP_102680

KGO36423

Transcription factor, fungi

 

PEXP_042010

KGO48399

Protein of unknown function DUF3468

 

PEXP_011320

KGO49083

Zinc finger, C2H2

 

PEXP_095680

KGO43666

Protein of unknown function DUF3468

 

PEXP_050580

KGO42108

Cytochrome P450

 

PEXP_059140

KGO45293

Polyketide synthase, enoylreductase

 

PEXP_100050

KGO46448

Heavy metal-associated domain, HMA

 

PEXP_103630

KGO36296

Urease accessory protein UreD

 

PEXP_083550

KGO40992

Thiamine pyrophosphate enzyme, C-terminal TPP-binding

 

PEXP_070880

KGO40584

Polyketide synthase, enoylreductase

 

PEXP_027120

KGO42963

hypothetical protein

 

PEXP_040610

KGO48229

Transcription factor, fungi

 

PEXP_075350

KGO36016

Alpha-actinin

 

PEXP_011040

KGO49055

Cytochrome P450, E-class, group I

 

PEXP_005060

KGO36707

Transcription factor, fungi

 

PEXP_070870

KGO40583

Aldehyde dehydrogenase, C-terminal

 

PEXP_046450

KGO39171

Amidohydrolase 1

 

PEXP_008360

KGO37036

4-Hydroxyphenylpyruvate dioxygenase

 

PEXP_048620

KGO39686

Nickel/cobalt transporter, high-affinity

 

PEXP_101160

KGO38158

Pectin lyase fold/virulence factor

 

PEXP_022330

KGO42704

Transcription factor, fungi

 

PEXP_058230

KGO38945

Alcohol dehydrogenase superfamily, zinc-type

 

PEXP_053390

KGO42387

Polyketide synthase, enoylreductase

 

PEXP_017020

KGO46060

Cytochrome P450

 

PEXP_021500

KGO44639

Transcription factor, fungi

 

PEXP_043440

KGO39263

Polyketide synthase, enoylreductase

 

PEXP_102420

KGO36222

Protein of unknown function DUF3468

 

PEXP_108970

KGO41891

Polyketide synthase, enoylreductase

 

PEXP_095570

KGO43655

Transcription factor, fungi

 

PEXP_088350

KGO41611

Transcription factor, fungi

 

PEXP_056220

KGO38423

Cytochrome P450

 

PEXP_099120

KGO46300

Hypothetical protein

 

PEXP_028450

KGO43127

Transcription factor, fungi

 

PEXP_110420

KGO38764

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_050400

KGO42090

Zinc finger, C2H2

 

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_085230

KGO41160

Hypothetical protein

 

PEXP_000560

KGO44365

Class II aldolase/adducin N-terminal

 

PEXP_071150

KGO40611

Alcohol dehydrogenase superfamily, zinc-type

 

PEXP_090470

KGO45017

Pyruvate carboxyltransferase

 

PEXP_080790

KGO38063

hypothetical protein

 

PEXP_026670

KGO42918

Transcription factor, fungi

 

PEXP_084100

KGO41047

Transcription factor, fungi

 

PEXP_102160

KGO38273

Casein kinase II, regulatory subunit

 

PEXP_036970

KGO35909

Cytochrome P450, E-class, group I

 

PEXP_032030

KGO39788

Hypothetical protein

 

PEXP_089350

KGO41723

Transcription factor, fungi

 

PEXP_005230

KGO36724

Transcription factor, fungi

 

PEXP_057180

KGO44225

ATP adenylyltransferase, C-terminal

 

PEXP_002560

KGO37249

Hypothetical protein

 

PEXP_081210

KGO47322

ATP-grasp fold, subdomain 1

 

PEXP_071070

KGO40603

Molybdenum cofactor synthesis C-terminal

 

PEXP_095390

KGO43637

Transcription factor, fungi

 

PEXP_054870

KGO44003

Transcription factor, fungi

 

PEXP_042700

KGO39429

Hypothetical protein

 

PEXP_036990

KGO35911

Ureohydrolase

 

PEXP_041200

KGO48300

Transcription factor, fungi

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_048130

KGO39637

Transcription factor, fungi

 

PEXP_010010

KGO48952

Transcription factor, fungi

 

PEXP_085290

KGO41166

Transcription factor, fungi

 

PEXP_079380

KGO37922

Zinc finger, C2H2

 

PEXP_057190

KGO44226

Thiamine pyrophosphate enzyme, C-terminal TPP-binding

 

PEXP_069040

KGO46734

Cytochrome P450

 

PEXP_007560

KGO36956

Zinc finger, C2H2

 

PEXP_081220

KGO47323

Transcription factor, fungi

 

PEXP_087170

KGO40934

Transcription factor, fungi

 

PEXP_105830

KGO41509

Transcription factor, fungi

 

PEXP_043140

KGO39473

Cytochrome P450

 

PEXP_067370

KGO46568

hypothetical protein

 

PEXP_073070

KGO48577

Exonuclease, RNase T/DNA polymerase III

 

PEXP_051020

KGO42152

Amidohydrolase 1

 

PEXP_061400

KGO45512

Hypothetical protein

 

PEXP_101010

KGO38143

Glycoside hydrolase, family 71

 

PEXP_043150

KGO39474

Terpenoid synthase

 

PEXP_015320

KGO47645

ATP-grasp fold, subdomain 1

Pectate lyase activity

PEXP_101160

KGO38158

Pectin lyase fold/virulence factor

 

PEXP_080220

KGO38006

Pectate lyase, catalytic

Calcium ion transmembrane transporter activity

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

Sequence-specific DNA binding RNA polymerase II transcription factor activity

PEXP_102680

KGO36423

Transcription factor, fungi

PEXP_042010

KGO48399

Protein of unknown function DUF3468

PEXP_095680

KGO43666

Protein of unknown function DUF3468

PEXP_040610

KGO48229

Transcription factor, fungi

PEXP_070870

KGO40583

Aldehyde dehydrogenase, C-terminal

PEXP_021500

KGO44639

Transcription factor, fungi

PEXP_102420

KGO36222

Protein of unknown function DUF3468

 

PEXP_088350

KGO41611

Transcription factor, fungi

 

PEXP_099120

KGO46300

Hypothetical protein

 

PEXP_085230

KGO41160

Hypothetical protein

 

PEXP_080790

KGO38063

Hypothetical protein

 

PEXP_102160

KGO38273

Casein kinase II, regulatory subunit

 

PEXP_032030

KGO39788

Hypothetical protein

 

PEXP_005230

KGO36724

Transcription factor, fungi

 

PEXP_057180

KGO44225

ATP adenylyltransferase, C-terminal

 

PEXP_002560

KGO37249

Hypothetical protein

 

PEXP_095390

KGO43637

Transcription factor, fungi

 

PEXP_042700

KGO39429

Hypothetical protein

 

PEXP_048130

KGO39637

Transcription factor, fungi

 

PEXP_010010

KGO48952

Transcription factor, fungi

 

PEXP_085290

KGO41166

Transcription factor, fungi

 

PEXP_087170

KGO40934

Transcription factor, fungi

 

PEXP_067370

KGO46568

Hypothetical protein

 

PEXP_101010

KGO38143

Glycoside hydrolase, family 71

Hydrolase activity, hydrolyzing O-glycosyl compounds

PEXP_000940

KGO44403

Protein of unknown function DUF2985

PEXP_044190

KGO39338

Aldolase-type TIM barrel

PEXP_049330

KGO39518

Glycoside hydrolase, family 35

 

PEXP_023030

KGO42774

Glycoside hydrolase, superfamily

 

PEXP_101680

KGO38226

Concanavalin A-like lectin/glucanases superfamily

 

PEXP_023340

KGO42463

Glycoside hydrolase, family 43

 

PEXP_070780

KGO40574

Glycoside hydrolase, family 31

 

PEXP_072880

KGO48558

Glycoside hydrolase, superfamily

 

PEXP_048880

KGO39711

Concanavalin A-like lectin/glucanases superfamily

 

PEXP_069330

KGO46763

Glycoside hydrolase, family 61

 

PEXP_011880

KGO49139

Glycoside hydrolase, family 28

 

PEXP_089320

KGO41720

Glycoside hydrolase, superfamily

 

PEXP_076310

KGO37551

Glycoside hydrolase, family 32

 

PEXP_026640

KGO42915

Glycoside hydrolase, family 16, CRH1, predicted

 

PEXP_057570

KGO44264

Glycoside hydrolase family 3

 

PEXP_053240

KGO42372

Glycoside hydrolase, family 61

 

PEXP_042140

KGO48412

Concanavalin A-like lectin/glucanase, subgroup

 

PEXP_107940

KGO41788

Alpha-amylase, C-terminal all beta

ATPase activity, coupled to transmembrane movement of substances

PEXP_023060

KGO42777

CDR ABC transporter

PEXP_050880

KGO42138

ABC transporter, integral membrane type 1

PEXP_110420

KGO38764

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

PEXP_040720

KGO48240

ABC transporter, integral membrane type 1

PEXP_071990

KGO40700

ABC transporter, integral membrane type 1

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_104880

KGO41412

ABC transporter, integral membrane type 1

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_077310

KGO37715

CDR ABC transporter

 

PEXP_086490

KGO40844

ABC transporter, integral membrane type 1

 

PEXP_057530

KGO44260

ABC transporter, integral membrane type 1

4-Hydroxyphenylpyruvate dioxygenase activity

PEXP_084560

KGO41093

4-Hydroxyphenylpyruvate dioxygenase

PEXP_008360

KGO37036

4-Hydroxyphenylpyruvate dioxygenase

Integral component of membrane

PEXP_046990

KGO39741

Sodium/calcium exchanger membrane region

PEXP_024890

KGO42618

Major facilitator superfamily domain, general substrate transporter

PEXP_041670

KGO48366

Major facilitator superfamily domain, general substrate transporter

 

PEXP_059490

KGO45335

Sulfate anion transporter

 

PEXP_043720

KGO39291

Alkali metal cation/H+ antiporter Nha1, C-terminal

 

PEXP_052330

KGO42281

Major facilitator superfamily domain, general substrate transporter

 

PEXP_037770

KGO36126

Major facilitator superfamily domain, general substrate transporter

 

PEXP_102860

KGO36441

Mitochondrial carrier protein

 

PEXP_023060

KGO42777

CDR ABC transporter

 

PEXP_033600

KGO39977

Amino acid/polyamine transporter I

 

PEXP_014370

KGO47550

Amino acid transporter, transmembrane

 

PEXP_094860

KGO43584

Major facilitator superfamily domain, general substrate transporter

 

PEXP_006100

KGO36811

Major facilitator superfamily domain, general substrate transporter

 

PEXP_046430

KGO39169

Major facilitator superfamily domain, general substrate transporter

 

PEXP_086200

KGO40808

Amino acid/polyamine transporter I

 

PEXP_024440

KGO42573

Major facilitator superfamily domain, general substrate transporter

 

PEXP_003610

KGO37353

Major facilitator superfamily domain, general substrate transporter

 

PEXP_043410

KGO39260

Major facilitator superfamily domain, general substrate transporter

 

PEXP_048620

KGO39686

Nickel/cobalt transporter, high-affinity

 

PEXP_006360

KGO36837

Major facilitator superfamily domain, general substrate transporter

 

PEXP_044620

KGO39381

Tetracycline resistance protein, TetA/multidrug resistance protein MdtG

 

PEXP_072400

KGO48510

Major facilitator superfamily domain, general substrate transporter

 

PEXP_028370

KGO43119

Amino acid transporter, transmembrane

 

PEXP_009920

KGO48943

Peroxisomal biogenesis factor 11

 

PEXP_033410

KGO39958

Major facilitator superfamily domain, general substrate transporter

 

PEXP_050880

KGO42138

ABC transporter, integral membrane type 1

 

PEXP_056980

KGO44205

Major facilitator superfamily domain, general substrate transporter

 

PEXP_102420

KGO36222

Protein of unknown function DUF3468

 

PEXP_074410

KGO48711

Major facilitator superfamily domain, general substrate transporter

 

PEXP_068910

KGO46721

Major facilitator superfamily domain, general substrate transporter

 

PEXP_060610

KGO45433

Major facilitator superfamily domain, general substrate transporter

 

PEXP_009670

KGO49300

Cation/H+ exchanger

 

PEXP_017290

KGO46087

Major facilitator superfamily domain, general substrate transporter

 

PEXP_110420

KGO38764

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_059360

KGO45322

Mitochondrial carrier protein

 

PEXP_095790

KGO43677

Major facilitator superfamily domain, general substrate transporter

 

PEXP_040720

KGO48240

ABC transporter, integral membrane type 1

 

PEXP_071990

KGO40700

ABC transporter, integral membrane type 1

 

PEXP_048590

KGO39683

Amino acid/polyamine transporter I

 

PEXP_104220

KGO36561

Cation/H+ exchanger

 

PEXP_000120

KGO44321

Major facilitator superfamily domain, general substrate transporter

 

PEXP_019490

KGO45826

C4-dicarboxylate transporter/malic acid transport protein

 

PEXP_088420

KGO41618

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_076320

KGO37552

Major facilitator superfamily domain, general substrate transporter

 

PEXP_001370

KGO37112

Major facilitator superfamily domain, general substrate transporter

 

PEXP_001360

KGO37111

Major facilitator superfamily domain, general substrate transporter

 

PEXP_104880

KGO41412

ABC transporter, integral membrane type 1

 

PEXP_005240

KGO36725

Major facilitator superfamily domain, general substrate transporter

 

PEXP_108540

KGO41848

Major facilitator superfamily domain, general substrate transporter

 

PEXP_095400

KGO43638

Sodium/calcium exchanger membrane region

 

PEXP_046610

KGO39187

Sodium/calcium exchanger membrane region

 

PEXP_098820

KGO46270

Major facilitator superfamily domain, general substrate transporter

 

PEXP_001950

KGO37170

Mitochondrial carrier protein

 

PEXP_050270

KGO42077

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_023180

KGO42789

Major facilitator superfamily domain, general substrate transporter

 

PEXP_010400

KGO48991

Major facilitator superfamily domain, general substrate transporter

 

PEXP_098540

KGO46242

Major facilitator superfamily domain, general substrate transporter

 

PEXP_066250

KGO46934

Major facilitator superfamily domain, general substrate transporter

 

PEXP_062420

KGO45644

General substrate transporter

 

PEXP_026170

KGO42868

Major facilitator superfamily domain, general substrate transporter

 

PEXP_077310

KGO37715

CDR ABC transporter

 

PEXP_022750

KGO42746

Major facilitator superfamily domain, general substrate transporter

 

PEXP_068690

KGO46699

Major facilitator superfamily domain, general substrate transporter

 

PEXP_066860

KGO46517

Major facilitator superfamily domain, general substrate transporter

 

PEXP_086490

KGO40844

ABC transporter, integral membrane type 1

 

PEXP_067360

KGO46567

Major facilitator superfamily domain, general substrate transporter

 

PEXP_057530

KGO44260

ABC transporter, integral membrane type 1

 

PEXP_034320

KGO40048

Arsenical pump membrane protein, ArsB

 

PEXP_074940

KGO36159

Major facilitator superfamily domain, general substrate transporter

A second cluster that was under-represented, both in colonized apple tissue and in vitro at pH 4 was cluster 8 (Fig. 2). This cluster mainly showed enrichment in carbon utilization and sugar-transport-related genes (Table 3, Additional file 7: Figure S8), which suggests that they contribute to a series of processes that induce carbon catabolism and thereby result in GLA accumulation [8]. This behavior may indicate that these processes are usually repressed during colonization unless there is a significant increase in ammonia accumulation.
Table 3

Genes involved in each process of cluster 8

GO Term

Gene id

Genbank transcript id

Description

Phosphate ion transport

PEXP_088430

KGO41619

Phosphate transporter

 

PEXP_030290

KGO40453

Phosphate transporter

Carbon utilization

PEXP_105770

KGO41503

Ribose/galactose isomerase

 

PEXP_105760

KGO41502

Aldolase-type TIM barrel

Transmembrane transport

PEXP_104850

KGO41409

Major facilitator superfamily domain, general substrate transporter

 

PEXP_048020

KGO39626

Major facilitator superfamily domain, general substrate transporter

 

PEXP_070650

KGO40561

Major facilitator superfamily domain, general substrate transporter

 

PEXP_011530

KGO49104

Major facilitator superfamily domain, general substrate transporter

 

PEXP_033090

KGO39926

Amino acid/polyamine transporter I

 

PEXP_078910

KGO37875

Cation efflux protein

 

PEXP_049390

KGO39524

Cation/H+ exchanger

 

PEXP_030290

KGO40453

Phosphate transporter

 

PEXP_034270

KGO40044

Major facilitator superfamily domain, general substrate transporter

Cation transport

PEXP_002160

KGO37191

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_078910

KGO37875

Cation efflux protein

 

PEXP_049390

KGO39524

Cation/H+ exchanger

 

PEXP_004260

KGO37441

Sodium\x3aneurotransmitter symporter

Neurotransmitter transport

PEXP_004260

KGO37441

Sodium\x3aneurotransmitter symporter

Triose-phosphate isomerase activity

PEXP_105760

KGO41502

Aldolase-type TIM barrel

Ribose-5-phosphate isomerase activity

PEXP_105770

KGO41503

Ribose/galactose isomerase

Cysteine dioxygenase activity

PEXP_086730

KGO40868

Cysteine dioxygenase type I

asparaginase activity

PEXP_042000

KGO48398

L-asparaginase, type II

Integral component of membrane

PEXP_002160

KGO37191

ATPase, P-type, K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter

 

PEXP_104850

KGO41409

Major facilitator superfamily domain, general substrate transporter

 

PEXP_025010

KGO42630

Hypothetical protein

 

PEXP_083360

KGO38643

FAD-binding 8

 

PEXP_048020

KGO39626

Major facilitator superfamily domain, general substrate transporter

 

PEXP_070650

KGO40561

Major facilitator superfamily domain, general substrate transporter

 

PEXP_011530

KGO49104

Major facilitator superfamily domain, general substrate transporter

 

PEXP_033090

KGO39926

Amino acid/polyamine transporter I

 

PEXP_078910

KGO37875

Cation efflux protein

 

PEXP_049390

KGO39524

Cation/H+ exchanger

 

PEXP_004260

KGO37441

Sodium\x3aneurotransmitter symporter

 

PEXP_034270

KGO40044

Major facilitator superfamily domain, general substrate transporter

Clusters with similar expression levels in colonized apple tissue and in vitro at pH 7

High expression level of fungal genes in colonized apple tissue and in fungi grown in vitro at pH 7

One of the most important clusters that showed high expression levels both in colonized apple tissue and in vitro at pH 7 was number 9 (Fig. 2), which showed significant enrichment of cellular amino-acid metabolism (Table 4, Additional file 8: Figure S9). Ammonia accumulation is induced under the limited-nutrient conditions present at the leading edge of the decay, in order to enable the pathogen to use a battery of pectolytic enzymes for tissue maceration [8]. Among the genes involved in cellular amino-acid metabolism process is glucose-methanol-choline oxidoreductase (GMC) (Table 4), whose families are clusters of FAD flavoprotein oxidoreductases-highly complex genes that contribute to several oxidation and reduction processes [29]. These enzymes include a variety of proteins, not all of which were present in cluster 9, such as choline dehydrogenase (CHD), which was present in clusters 1, 3, and 9, methanol oxidase (MOX), and cellobiose dehydrogenase, which was present in cluster 3; these are proteins that share a number of homologous regions that show sequence similarities. Since ammonia accumulation at the leading edge of the Penicillium-colonized tissue did not increase local pH [8], it is possible that the accumulated ammonia may activate gene expression, either directly or indirectly by generation of hydrogen peroxide and activation of RBOH in the killed cell [30]. To elucidate the role of ammonia, we analyzed the relative expression levels of several genes-MepB, CuAC and ACC-from cluster 9 in Penicillium-colonized fruits exposed to 22 μM of NH4 +, and found induction of their relative expression levels (Fig. 3), which indicates the capability of ammonia to activate this process.
Table 4

Genes involved in each process of cluster 9

GO Term

Gene id

Genbank transcript id

Description

Ammonium transmembrane transport

PEXP_025350

KGO42664

Ammonium transporter

Cellular amino acid metabolic process

PEXP_082650

KGO38572

Pyridoxal phosphate-dependent transferase, major region, subdomain 2

PEXP_109810

KGO38703

Glucose-methanol-choline oxidoreductase

PEXP_004920

KGO36657

Glutamate synthase, central-N

 

PEXP_053110

KGO42359

Catalase-peroxidase heme

 

PEXP_056170

KGO38539

1-aminocyclopropane-1-carboxylate deaminase

Amine metabolic process

PEXP_053110

KGO42359

Catalase-peroxidase heme

PEXP_056170

KGO38539

1-aminocyclopropane-1-carboxylate deaminase

 

PEXP_000620

KGO44371

Copper amine oxidase, N2-terminal

Hydrogen peroxide catabolic process

PEXP_053110

KGO42359

Catalase-peroxidase heme

Fig. 3

Effects of ammonia and pH on the relative expressions of genes involved in the pathogenicity process. Expressions were analyzed of: polygalacturonase (PG), Mep2, Copper amine oxidase (CuAO), ACC deaminase, PacC, and pectate lyase A (PelA). a Samples from the leading edge of infection in apples treated with exogenous NH4Cl at 0 and 22 μM, according to Barad et al. [8]; b Samples grown at pH 4 or pH 7, for 3 h in shaking secondary media (SM)

Another highly expressed family of genes in cluster 9 was the glucose-methanol-choline oxidoreductase family. Heat-map analysis of this family showed that genes with the same activity were differentially expressed between different clusters (Fig. 4). For example, 11 GMC oxidoreductase genes were detected in P. expansum: three in cluster 1, three in cluster 3, and one in cluster 9, whereas the remaining four were not differentially expressed. The GMC oxidoreductase family also exhibited close similarity to the glucose oxidase family. We were able to detect three glucose oxidase transcripts, as described by Ballester et al. [12], which showed differing expression patterns: gox1 of P. expansum was not differentially expressed under our conditions, gox2 was found in cluster 3, and gox3 was detected in cluster 1 (Fig. 4). Analysis of only the expression pattern at pH 4 compared with that at pH 7 showed that both gox2 and glucose oxidase 3 (gox3) were upregulated at pH 7 but not at pH 4 [7]. This differential expression pattern of the gox family suggests that genes were being selectively activated on the basis of their optimal conditions with respect to pH, in vitro, or in vivo, to enable the fungus to cope with varied conditions and to make optimal use of the inventory of available enzymes [31].
Fig. 4

Heat map of the expression pattern of GMC oxidoreductase gene family. The expression patterns of genes belonging to the GMC oxidoreductase gene family are shown as a heat map obtained with matrix2png software [72]. Gene identity, gene description, and its location in the differentially expressed gene clusters are indicated

Another process that was enriched in this cluster was amine metabolism (Additional file 8: Figure S9), which involves the gene copper amine oxidase (CuAO) (Table 4). In plants, wounding of tissue usually results in an increase in the steady-state levels of copper amine oxidase expression and H2O2 accumulation [32]. Activation of CuAO during Penicillium attack also may lead to enhanced accumulation of H2O2 at the wound site, thereby contributing to extension of necrotic lesions and extensive plant-cell damage [32]. Also, copper amine oxidase may catalyze oxidation of the aliphatic diamines putrescine and cadaverine at their primary amino groups [33]. The products of putrescine oxidation by CuAO are H2O2, NH3, and Δ1-pyrroline; and Δ1-pyrroline is further catabolized to γ-aminobutyric acid, which subsequently is transaminated and oxidized to succinic acid. Thus, in Penicillium copper amine oxidase may contribute to the balance of reactive oxygen species (ROS) produced in the cell wall extracellular matrix [34]. This specific regulatory mechanism and the molecular signals inducing modulation of copper amine oxidase in Penicillium during decay highlight the relevance of these enzymes as an H2O2-delivering system in colonized tissue. The overrepresentation of glucose-methanol-choline oxidoreductase- and copper amine oxidase-activated genes in cluster 9 may indicate their contribution to the ROS and oxidoreductase process at the leading edge of the colony; also, in the same cluster there was activation of the H2O2 catabolic process (Additional file 8: Figure S9) through catalase peroxidase, which suggests a hitherto unknown mechanism of Penicillium survival under oxidative stress. Interestingly, similar up-regulation of catalase peroxidase under pathogenic conditions was reported to account for the survival of P. marneffei, an intracellular pathogen that causes common opportunistic infections in humans, and of P. simplicissimum, a plant pathogen in which strong expression of the catalase peroxidase transcripts may contribute to survival of this fungus in host cells [35]. Considered together, the relative expressions of the oxidoreductase genes and copper amine oxidase under fruit pH levels ranging from 3.7 to 4.2, again may indicate the importance of local ammonification, as reported by Barad et al. [8], as a mechanism to induce activation of genes usually overexpressed at pH 7 (Fig. 3).

The overrepresentation in cluster 9, of several nitrogen-metabolism-regulating genes, such as glutamate synthase and MepB (Table 4) also is important for nitrogen metabolism in fungi. The ammonia transporter encoded by mepB can lead to an internal/external modulation of ammonia in the hypha, activation of pacC, and high-pH induced genes [36]. Accumulation of glutamate, followed by its transformation to glutamine by glutamate synthase activity may be the basis for accumulation of several amino acids, and both stages are activated by ammonia accumulation (Fig. 3). Overall, the nitrogen mobilization promoted by infection could be considered as part of a "slash-and-burn" strategy that deprives the pathogen of nutrients and modulates alkaline-expressed genes. Such nutritional and metabolic changes might occur as a differential-attack mechanism promoting pathogen development [37].

One interesting gene also activated in cluster 9 in cellular amino acid metabolism (Additional file 8: Figure S9) is the gene encoding for aminocyclopropane-1-carboxylate deaminase (ACC) (Table 4). The ACC functions as a deaminase, degrading aminocyclopropane-1-carboxylate deaminase to 2-oxobutyrate and ammonia, which is a precursor of the plant hormone ethylene. A similar reaction process catalyzing ACC synthase was found in Penicillium digitatum attacking citrus fruits [38, 39], which suggests that colonization may be associated with ethylene production and induced tissue senescence. Taken together these results indicate that ROS production by the pathogen may be accompanied by host tissue induced senescence.

Low expression level of fungal genes in colonized apple tissue and in fungus grown in vitro at pH 7

Group III also includes cluster 4, in which colonized apple tissue showed under-representation of transcripts similar to that in vitro at pH 7 (Fig. 2). This cluster shows enrichment of genes involved in the oxido-reduction process, such as cysteine dioxygenase (Table 5, Additional file 9: Figure S4). Among representatives of this group are the cytochrome P-450 (CytP) monooxygenases, which are enzymes that: catalyze conversion of hydrophobic intermediates of primary and secondary metabolic pathways; detoxify natural and environmental pollutants that are accumulated during colonization as phytochemical molecules belonging to the polyphenols; and enable fungal growth under varied colonizing conditions [40]. They do this by inserting one oxygen atom into the aliphatic position of an organic substrate, while the other oxygen atom is reduced to water.
Table 5

Genes involved in each process of cluster 4

GO Term

Gene id

genbank transcript id

Description

Oxidation-reduction process

PEXP_035940

KGO40210

Cytochrome P450, E-class, CYP52

PEXP_094610

KGO43560

Multicopper oxidase, type 1

PEXP_069560

KGO47265

Cytochrome P450

PEXP_047900

KGO39614

Cytochrome P450

PEXP_074010

KGO48671

Cytochrome P450

 

PEXP_065530

KGO46863

Short-chain dehydrogenase/reductase SDR

 

PEXP_073620

KGO48632

Polyketide synthase, enoylreductase

 

PEXP_079160

KGO37900

FAD-linked oxidase, N-terminal

 

PEXP_057820

KGO44289

Cysteine dioxygenase type I

 

PEXP_043230

KGO39482

Isocitrate and isopropylmalate dehydrogenases family

 

PEXP_101170

KGO38159

Oxoglutarate/iron-dependent dioxygenase

 

PEXP_060240

KGO45235

Short-chain dehydrogenase/reductase SDR

 

PEXP_034900

KGO40106

Aldolase-type TIM barrel

 

PEXP_106700

KGO41290

Isocitrate and isopropylmalate dehydrogenases family

 

PEXP_026690

KGO42920

Short-chain dehydrogenase/reductase SDR

 

PEXP_069740

KGO47756

Redoxin

 

PEXP_103230

KGO36363

Cytochrome P450

 

PEXP_091400

KGO45110

hypothetical protein

 

PEXP_027810

KGO43163

Polyketide synthase, enoylreductase

 

PEXP_023550

KGO42484

Indoleamine 2,3-dioxygenase

 

PEXP_077820

KGO37766

Polyketide synthase, enoylreductase

 

PEXP_043790

KGO39298

Dihydrolipoamide succinyltransferase

 

PEXP_082800

KGO38587

Cytochrome P450

 

PEXP_043420

KGO39261

Polyketide synthase, enoylreductase

 

PEXP_030200

KGO40444

Cytochrome P450

 

PEXP_061260

KGO45498

Short-chain dehydrogenase/reductase SDR

 

PEXP_078860

KGO37870

Dimeric alpha-beta barrel

 

PEXP_042100

KGO48408

NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain-3

 

PEXP_037510

KGO35963

Oxoglutarate/iron-dependent dioxygenase

 

PEXP_010380

KGO48989

Aldo/keto reductase

 

PEXP_000410

KGO44350

Acyl transferase/acyl hydrolase/lysophospholipase

 

PEXP_076770

KGO37511

3-oxo-5-alpha-steroid 4-dehydrogenase, C-terminal

 

PEXP_066880

KGO46519

FAD dependent oxidoreductase

 

PEXP_008580

KGO37058

hypothetical protein

 

PEXP_001620

KGO37137

Ubiquinone biosynthesis protein Coq7

 

PEXP_030860

KGO40271

NADPH-dependent FMN reductase

 

PEXP_107740

KGO41768

Polyketide synthase, enoylreductase

 

PEXP_049110

KGO39496

Monooxygenase, FAD-binding

 

PEXP_017320

KGO46090

Polyketide synthase, enoylreductase

Taurine metabolic process

PEXP_057820

KGO44289

Cysteine dioxygenase type I

 

PEXP_050220

KGO42072

Acetate/Proprionate kinase

Carbohydrate active enzymes (CAZy) cluster distribution

The diverse complex carbohydrates that contribute to Penicillium maceration capabilities are controlled by a panel of enzymes involved in their assembly (glycosyltransferases) and breakdown (glycoside hydrolases, polysaccharide lyases, carbohydrate esterases), which collectively are designated as Carbohydrate-Active enZymes (CAZymes) [41]. In plant pathogens CAZymes promote synthesis, degradation, and modification of carbohydrates that play important roles in the breakdown of plant cell walls and in host/pathogen interactions [42]. Penicillium uses these enzymes to macerate the colonized tissue by degradation of complex carbohydrates of the hosts to simple monomers that can be utilized as nutrients [43, 44].

The CAZymes analysis toolkit (CAT) [45] was used to identify 771 putative CAZymes in P. expansum within the various clusters (Fig. 5). CAZymes formed 8.00 % of all the transcripts in cluster 1, with 15, 24, 19, 57, and 65 of CAZymes families including auxiliary activity (AA), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 2 they formed 5.57 % of all the transcripts with 2, 3, and 8 of CAZymes families including carbohydrate esterases, glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 3 they formed 17.92 % of all the transcripts, with 24, 74, 28, 149, and 123 of CAZymes families including auxiliary activity (AA), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 4 they formed 14.46 % of all transcripts, with 2, 3, 4, 15, and 11 of CAZymes families including including auxiliary activity (AA), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 5 they formed 22.61 % of all transcripts, with 7, 11, 11, 5, 23, and 52 of CAZymes families including auxiliary activity (AA), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), polysaccharide lyases (PL), glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 6 they formed 29.16 % of all transcripts, with 2, 3, 2, 5, and 9 of CAZymes families including auxiliary activity (AA), carbohydrate-binding modules (CBM), PL, glycosyltransferases (GT), and glycoside hydrolases (GH), respectively. In cluster 7 they formed 22.61 % of all transcripts, with 4 of CAZymes families including glycosyl hydrolases. In cluster 8 they formed 12.06 % of all transcripts, with 2 and 5 of CAZymes families including carbohydrate esterases (CE) and glycoside hydrolases (GH), respectively. In cluster 9 they formed 16.67 % of all transcripts, with 2 of CAZymes families including each of auxiliary activity (AA) and glycoside hydrolases (GH) (Fig. 5).
Fig. 5

Comparison of the CAZyme repertories identified in each cluster of co-expressed genes. Enzyme families are represented by their class (GH-glycoside hydrolases; GT-glycosyltransferases; PL-polysaccharide lyases; CE-carbohydrate esterases; and CBM-chitin-binding modules) and family number according to the Carbohydrate-Active Enzyme Database. Abundance of the various enzymes within a family is represented on a color scale from 0 (dark blue) to 150 occurrences (dark red) per cluster

Carbohydrate esterases, glycoside hydrolases, and polysaccharide lyases are associated with the ability to utilize the diversity of carbohydrates present in the environment and within host fruits. Glycosyltransferases are mainly involved in the basal activities of fungal cells, e.g., fungal-cell-wall synthesis, glycogen cycle, and trehalose cycle [41, 44, 46]. The wide occurrence of CAZymes in the various Penicillium clusters, taken together with their importance in degradation of the plant cell wall, indicates their basic contribution to colonization of the host fruit.

Differentially expressed genes in Penicillium expansum- and Colletotrichum gloeosporioides-infected apple compared with healthy tissue

In order to analyze the effect of Penicillium expansum infection during acidification on the host (apple tissue) response, we used RNAseq to analyze the differentially expressed genes induced by P. expansum infection. Overall, in the P. expansum-infected apple tissue we found 4,292 differentially expressed genes with FDR threshold < 0.001 and with expression levels increasing or decreasing by a factor greater or less than 8, respectively, i.e., greater or less than +3 or −3, respectively, on a logarithmic (base 2) scale. We found 2,427 up-regulated and 1,865 down-regulated genes in colonized apple tissue. Analysis of the enriched pathways with MatGeneMap [47] revealed 21 significantly up-regulated pathways (Table 6) and 21 down-regulated ones (Table 7) with FDR < 0.05. Among the significantly up-regulated processes induced by Penicillium in apple tissue were: the jasmonic acid (JA), the mevalonate, and the flavonoid biosynthesis pathways, and the geranyl geranyldiphosphate biosynthesis I super pathway. Among the down-regulated pathways were the glycogen biosynthesis I and the starch biosynthesis pathways. Jasmonic acid is a lipid-derived signaling compound involved in regulation of diverse processes in plants such as fruit ripening, root growth, tendril coiling, senescence, and resistance to pathogens [48]; JA and related compounds are synthesized in plants via the octadecanoid pathway [49]. Biosynthesis of jasmonates starts with oxygenation of linolenic acid, which is thought to be released from membrane lipids through the action of a lipase, followed by several oxidation processes. This activation may indicate that there is a host response prior to fungal maceration induced by Penicillium, and this supposition is supported by findings that fruits pretreated with JA and related compounds showed enhanced resistance to pathogens [50].
Table 6

Apple up-regulated pathways by infection with P. expansum

Pathway name

p value (adjusted)

Jasmonic acid biosynthesis

5.73E-11

Mevalonate pathway

9.12E-08

Flavonoid biosynthesis

7.62E-05

Superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)

7.62E-05

Glutathione-mediated detoxification

0.00171

Trans,trans-farnesyl diphosphate biosynthesis

0.0032

Glutamate dependent acid resistance

0.0032

Chorismate biosynthesis

0.00325

Amygdalin and prunasin degradation

0.00988

Salicylate biosynthesis

0.01194

DIMBOA-glucoside degradation

0.01415

β-Alanine biosynthesis II

0.01456

Acetate formation from acetyl-CoA II

0.01456

Pyruvate fermentation to acetate III

0.01456

Glutamate degradation III (via 4-aminobutyrate)

0.01693

Superpathway of phenylalanine and tyrosine biosynthesis

0.01693

Phospholipid desaturation

0.03878

Glycolipid desaturation

0.03878

13-LOX and 13-HPL pathway

0.04864

Divinyl ether biosynthesis II (13-LOX)

0.04864

Superpathway of phenylalanine, tyrosine, and tryptophan biosynthesis

0.04864

The analysis was performed with MetGenMap

Table 7

Apple down-regulated pathways by infection with P. expansum

Pathway name

p value (adjusted)

Glycogen biosynthesis I (from ADP-D-Glucose)

9.064E-08

Starch biosynthesis

6.194E-07

C4 Photosynthetic carbon assimilation cycle

0.0002403

UDP-galactose biosynthesis (salvage pathway from galactose using UDP-glucose)

0.0008945

Lipoate biosynthesis and incorporation I

0.0019429

Methylerythritol phosphate pathway

0.0031538

Acyl carrier protein metabolism

0.0034133

Trans-lycopene biosynthesis

0.0059809

Fatty acid biosynthesis initiation I

0.0070497

Pyridoxal 5'-phosphate biosynthesis

0.0087438

Superpathway of pyridoxal 5'-phosphate biosynthesis and salvage

0.0132283

Xylitol degradation

0.0161255

Starch degradation

0.0173149

Heme biosynthesis I

0.0178181

Heme biosynthesis from uroporphyrinogen I

0.0195451

Colanic acid building blocks biosynthesis

0.0199615

Xylose degradation I

0.0284483

Sucrose biosynthesis

0.0303525

5-Aminoimidazole ribonucleotide biosynthesis II

0.0332884

Biotin-carboxyl carrier protein

0.0357646

Methanol oxidation to formaldehyde

0.0437164

The analysis was performed with MetGenMap

The second process that was activated was the mevalonate pathway, which produces isoprenoids that are vital for diverse cellular functions; these isoprenoids include sterols, carotenoids, chlorophyll, plant hormones, and defense isoprenoids. The penetration of Penicillium probably induces defense isoprenoids such as were found in damaged plant leaves [51]. These comprise a wide variety of defense-related genes, including those that activate biosynthesis of JA and ethylene, as a possible response to pathogen penetrations, as was reported for Botrytis cinerea [52]. qRT-PCR analysis of the ethylene-responsive transcription factor 2-like showed a significant induction of this gene in the apple tissue as a result of the Penicillium infection (Table 8).
Table 8

Relative expression (Pe/Cg) of selected genes in apple tissue infected with P. expansum or C. gloeosporioides

Apple down-regulated genes

Apple (Pe)/apple (Cg)

Apple up-regulated genes

Apple (Pe)/apple (Cg)

Programmed cell death protein 4-like

0.025/0.112 = 0.223

Histone deacetylase hdt3-like

0.70/1.21 = 0.583

Auxin-repressed kda isoform x1

0.002/0.009 = 0.222

Phenylalanine ammonia-lyase 1

9.41/3.38 = 2.781

ap2-like ethylene-responsive transcription factor at2g41710 isoform x2

0.015/0.063 = 0.238

Chalcone synthase

16.33/24.23 = 0.670

udp-glucose:glycoprotein glucosyltransferase

0.104/0.291 = 0.357

Peroxidase 47

98.83/10.55 = 9.368

Senescence-associated carboxylesterase 101-like

0.079/0.192 = 0.411

Respiratory burst oxidase homolog protein d-like

185.14/24.15 = 7.664

1-Aminocyclopropane-1-carboxylate oxidase 1

0.034/0.064 = 0.544

Lipoxygenase

490.05/205.74 = 2.381

Anthocyanidin 3-o-glucosyltransferase 5-like

0.007/0.041 = 0.169

Indole-3-acetic acid-induced protein arg2

9.97/6.49 = 1.535

Expansin 1

0.002/0.040 = 0.067

Zinc finger an1 domain-containing stress-associated protein 12-like

17.66/17.78 = 0.993

ap2-like ethylene-responsive transcription factor at2g41710 isoform x2

0.012/0.045 = 0.282

NADPH--cytochrome p450 reductase isoform x2

8.18/4.04 = 2.022

Lysine-specific histone demethylase 1 homolog 1-like

0.075/0.192 = 0.394

Ethylene-responsive transcription factor 2-like

33.81/33.83 = 0.999

  

Serine threonine-protein kinase-like protein ccr4

11.12/1.83 = 6.049

The relative expression of the apple genes was compared with healthy tissue

The third up-regulated process was related to biosynthesis of flavonoids, which are linked to fungal potential cytotoxicity and capacity to interact with enzymes through protein complexation [53]. Some flavonoids provide stress protection, for example, by acting as scavengers of free radicals such as reactive oxygen species (ROS), as reported by Falcone Ferreyra et al. [53]. During P. expansum pathogenicity, the fungus produces gluconic acid, with H2O2 as a by-product. The host reacts to ROS by activating the flavonoids biosynthesis pathway and thereby initiating a resistance response to fungal penetration. Production of secondary metabolites such as anthocyanins, isoflavonoids, and flavonol glycosides may contribute to this resistance. Plants such as Arabidopsis respond to the combination of biotic, i.e., bacterial, and abiotic, e.g., UV-B radiation, stresses through synthesis of defense-related compounds such as phytoalexins and lignin, which serve as structural barriers that restrict the spread of pathogens. These responses modify the expression of genes involved in the production of protective metabolites such as flavonols [53]. This behavior suggests that there is a significant apple response to Penicillium penetration under these susceptible conditions. This activation matches the fourth activated pathway of P. expansum colonization, in which the geranyl-geranyl diphosphate-mediated processes are activated, probably in biosynthesis of essential compounds such as chlorophylls, carotenoids, tocopherols, plastoquinones, and gibberellins, but mainly in production of a variety of secondary metabolites. All of which indicates the significant response of apple fruits in coping with the colonization process.

Two significantly down-regulated processes that modulate carbohydrate metabolism were found: glycogen biosynthesis I from ADP-D-glucose, and starch biosynthesis. Glycogen and starch are both multibranched polysaccharides of glucose that serve as a means of energy storage for fungi; their downregulation may indicate their importance as energy stores that rapidly can be mobilized from the cytosol/cytoplasm and that perform an important function during glucose consumption by Penicillium during the acidification process [7]. Down-regulation of these processes probably results from catabolism of the substrates during attack by P. expansum and strongly supports previous findings of Prusky et al. [3], Hadas et al. [4], and Barad et al. [7] that indicate acidification of the tissue as a factor in pathogenicity of P. expansum.

In order to analyze the responses of apple fruits to fungal acidification or alkalization, the differential expressions of specific genes induced during colonization by Penicillium were compared to those expressed during colonization of the same host by Colletotrichum. For that purpose, the relative expressions of 21 specific key genes, of which 10 were up-regulated and 11 down-regulated by Penicillium (Table 8), were compared in apple RNA extracted from the leading edges of tissue colonized by the two respective pathogens. Genes that were up-regulated in the fruit showed the same response pattern when fruits were colonized by either Penicillium or Colletotrichum, but the relative expression obtained in Penicillium-inoculated apples was always significantly higher than that in Colletotrichum-inoculated apples: the ratio of P. expansum: C. gloeosporioides relative expression responses ranged from 0.58 to 9.36. This indicates a stronger host response to Penicillium colonization than to Colletotrichum colonization. Analysis of the down-regulated genes also showed similar patterns of host responses to the two respective pathogens, but lower ratios of P. expansum: C. gloeosporioides relative expressions, ranging from 0.067 to 0.54, which indicates that, although the response patterns were similar, the alkalizing pathogen induced lower expression levels. These results indicate that host responses to Penicillium and to Colletotrichum attack did not show opposite senses of gene modulation, as might be expected from their contrasting pH modulation directions, but only reduced expression levels in the Colletotrichum-inoculated apples.

Conclusions

Overall, gene-expression profiling of pH-dependent genes of P. expansum during colonization of apple fruits revealed three major effects: (1) a pattern of high gene expression during colonization that had no connection with pH response; (2) gene expression patterns during colonization similar to those obtained under in vitro growth conditions at pH 4; and (3) gene expression patterns during colonization similar to those obtained under in vitro growth conditions at pH 7. These three main trends indicate the existence of pH-regulated genes, expressed at pH 4 and pH 7 that may contribute to P. expansum colonization. One of the key processes overrepresented in cluster 7 at pH 4 was high expression of glutamate decarboxylase (GAD). One of the key products of this enzyme-GABA-largely originates from decarboxylation of L-glutamate [54], and it had been found to function in communication between tomato (Lycopersicon esculentum var. commune Bailey) plants and the fungus Cladosporium fulvum [55]. Hyphae of C. fulvum are restricted to the apoplast, therefore the fungus is dependent on the contents of the apoplastic nutrients. During infection, the GABA concentration in the apoplast increased from about 0.8 mM to 2–3 mM; this can be attributed to stimulation of glutamate dehydrogenase activity by decreased pH and increased cytosolic calcium, which are associated with pathogen attack [56]. In the present study, similar conditions were present in the Penicillium/apple interactions, where acidification by GLA accumulation may enhance the consumption of this amino acid.

Accumulation of ammonia, induced under the limited-nutrient conditions present at the edge of the decaying tissue, may contribute to gene expression, either by alkalization or by direct induction of ammonia during gene activation [8]. Modulation of ammonia levels by MepB transporters and/or the amine metabolic process (both overrepresented at pH 7 and decayed tissue in cluster 9) may contribute to the alkalizing effects and pH increase. Under these pH conditions also an over-representation of glucose-methanol-choline (GMC) oxidoreductase was observed. GMC shows similar activity to the GOX of P. expansum that contributed to oxidation and reduction processes [29] by providing the H2O2 required by ligninolytic peroxidases in Pleurotus species [57]. This combination of GMC genes [58], described here for the first time in P. expansum under alkaline conditions, indicates activity of new undescribed mechanisms that contribute to cell-wall degradation by generation of H2O2 during fungal colonization at pH 7.

In addition, the copper amino oxidase (CuAO) contributes to the enhanced accumulation of H2O2 at the wound site which, in turn, contributes to the extended necrotic lesions and extensive plant cell damage [32]. The CuAO catalyzes oxidation of aliphatic diamines of the primary amino groups that contribute to H2O2 and NH3 accumulation, which emphasizes the relevance of the H2O2-delivering system in colonized tissue [33]. This over-representation of GMC and CuAO indicates the contribution to ROS production and oxidoreductase processes at the leading edge of the developing colony, and attributes the presence of fungal catalase peroxidases the need to protect colonizing hyphae.

These oxidative processes were concurrent with activation of Cyt P-450 monoxygenase-in group 3, cluster 4-which catalyzes the conversion of hydrophobic intermediates of primary and secondary metabolic pathways, thereby detoxifying natural and environmental pollutants and allowing fungi to grow under difficult oxidative conditions [40], which accounts for the activation of genes coding for proteases, cell-wall-related enzymes, redox homoeostasis, and detoxification processes that are expressed during the infection process.

Exploiting these strong oxidative conditions during its necrotrophic development, the fungus further advances the colonization process by activation of aminocyclopropane-1-carboxylate deaminase (ACC) (cluster 9), a precursor of the plant hormone ethylene. A similar reaction process catalyzing ACC synthase was found when P. digitatum attacked citrus fruits [38, 39], which indicates that the colonization process involves induction of senescence of the colonized tissue, in conjunction with the strong oxidative process.

Out of the 771 putative CAZymes identified in P. expansum, eight were found to be expressed in clusters 7 and 9, which were the most important clusters modulating pathogenicity (Fig. 5). Given the importance of CAZymes during cell-wall degradation, it is clear that they contribute strongly to the apple maceration process. The present transcriptome analysis showed induced ammonification, and strong oxidative and senescence processes, accompanied by strong activation of pectolytic enzymes, all of which indicate the pH dependence of the tools used by the pathogen to colonize the environment. All these induced metabolic changes indicate the significant role of pH modulation in the pathogenicity of P. expansum in fruits.

Analysis of the fruit transcriptome suggested that even if apple fruits are susceptible to Penicillium, the fungus activates significant gene processes related to fruit resistance. This may indicate that the necrotrophic maceration of the tissue occurs, not as a senescence response, but as an active fungal process that supports its pathogenicity. Also interesting is the stronger response of Penicillium than of Colletotrichum, which indicates that the pH adjustment effected by the fungus did not affect the pattern but the level of host response. At the same time, these findings indicate that the fungal matching to the host is the main factor activating the maceration process.

Methods

Fungal strains and culture conditions

The WT P. expansum isolate Pe-21 was obtained from decayed apples (Malus domestica cv. Golden Delicious) purchased from a local market in Israel [4]. Cultures were grown at 27 °C in the dark, and maintained on PDA plates (Difco, MD, USA) unless otherwise indicated. Conidia were harvested with 10 mL of sterile distilled water supplemented with 0.01 % (v/v) Tween 80 (Sigma-Aldrich, Copenhagen, Denmark). Cells were visualized with a model BX60F-3 microscope and a model SZ-60 stereoscope (both from Olympus America Inc., Melville, NY, USA) and counted with a hemocytometer (Brand, GMBH, Werheim, Germany).

Assay for colonization and disease development

'Golden Delicious' apples were inoculated by placing 5 μL of a conidial suspension containing 106 spores mL−1 on each of six 2-mm-deep, 2-mm-diameter wounds spaced evenly in a circle around the upper part of the stem end of the fruits. Following inoculation, the fruits were incubated for 5 days at 25 °C in covered plastic containers containing wet paper towels. Samples of healthy tissue and of the leading edge of each wound were collected and frozen with liquid nitrogen for subsequent RNA analysis.

In vitro procedure of P. expansum

Fungal spores were inoculated at 106 spores mL−1 into 40 mL of a primary medium, i.e., glucose minimal medium, in 125-mL flasks containing (per liter) 10 g sucrose, 5 g yeast extract (Difco Laboratories, MD, USA), 50 mL nitrate salts, and 1 mL trace elements, at pH 4.5. The cultures were incubated at 25 °C with shaking at 150 rpm for 48 h. Cultures were harvested by vacuum filtration through a sterile Büchner funnel fitted with a Whatman number 1 filter paper, and the remaining mycelia were washed twice with 50 mL of sterile distilled water. The washed mycelia were resuspended in 50 mL of 0.2 M phthalate-buffered or phosphate-buffered liquid secondary medium (SM) at pH 4 or pH 7, respectively. This SM contained (per liter) 60 g sucrose, 7 g NaNO3, 3 g tryptone (Difco Laboratories, MD, USA), 1 g KH2PO4, 0.5 g MgSO4 · 7H2O, and 0.5 g KCl. The cultures were incubated at 25 °C on a rotating shaker at 150 rpm for 0.5, 1, 3, 10, or 24 h. A sample of mycelia from the cultures was collected into a 1.5-mL Eppendorf at each time point, and the mycelia were frozen with liquid nitrogen for RNA extraction.

RNA extraction

RNA was extracted from the in vitro samples with the SV Total RNA Isolation kit (Promega, Madison,WI, USA). Purity of the extracted RNA was assayed with an ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA), and the extracts were stored at −80 °C pending further analysis.

Total RNA of apple fruits was extracted according to Yang et al. [59], with minor changes: aliquots were taken from pooled samples from the leading edges of the six inoculation areas of each apple. The samples were ground to a fine powder in liquid nitrogen and transferred into 50-mL centrifuge tubes with 10 mL of CTAB RNA extraction buffer comprising 100 mM Tris-borate pH 8, 2 M NaCl, 25 mM ethylenediaminetetraacetic acid (EDTA) pH 8, 2 % (w/v) CTAB, 2 % (w/v) polyvinylpolypyrrolidone, and 2 % (v/v) β-mercaptoethanol. The mixture was shaken for 3 min and then incubated at 65 °C for 15 min. Samples were extracted twice with an equal volume of 24:1 (v:v) chloroform:isoamyl alcohol, and the phases were separated by centrifugation at 10,000 × g for 10 min. Following centrifugation, LiCl was added to a final concentration of 2.5 M and RNA was allowed to precipitate overnight at 4 °C. The precipitated RNA was pelleted at 4 °C for 30 min at 10,000 × g, washed with 70 % ethanol, and resuspended at 65 °C for 3 min in SSTE buffer comprising 10 mM Tris pH 8, 1 M NaCl, 1 mM EDTA pH 8, and 0.5 % (w/v) SDS. Samples were extracted with equal volumes of 24:1 (v:v) chloroform:isoamyl alcohol, and with equal volumes of 24:1:25 (v:v:v) chloroform:isoamyl alcohol:water-saturated phenol, and the phases were separated by centrifugation at 10,000 × g for 10 min. The RNA was ethanol-precipitated overnight, and resuspended in diethyl-pyrocarbonate-treated water. The RNA was further treated with Turbo DNAse (Ambion, Austin, TX, USA).

Preparations of libraries

A 500-ng of total RNA from 11 samples was processed with the TruSeq RNA Sample Preparation Kit v2 (RS-122-2001) (Illumina, San Diego, CA, USA). Libraries were evaluated with the Qubit and TapeStation software (Agilent Technologies, Palo Alto, CA, USA), and sequencing libraries were constructed with barcodes to enable multiplexing of pools of the eight in vitro samples-duplicates of pH 4 3 h, pool pH 4, pH 7 3 h and pool pH 7-in one lane. The results showed 14.5–32.1 million single-end 50-bp reads.

Three individual lanes were used for the in vivo samples and yielded 199.5–253.3 million single-end, 100-bp reads from the duplicates of the leading edges of infected apple tissue and 31.7 million single-end 100-bp reads from healthy apple tissue. All samples were sequenced on a HiSeq 2500 The transcriptome of the healthy apple tissue and the leading edge of infected tissue were sequenced with Trueseq protocols at the Genome Center of the Life Sciences and Engineering Faculty, Technion-Israel Inst. Technology, Haifa, Israel.

Bioinformatic analysis of RNAseq Data

Three libraries with total single-end, 100-nucleotides-long RNA-seq reads were generated from the in vivo samples and eight libraries with 50-nucleotides-long total single-end RNA-seq reads were generated from the in vitro samples. The libraries contained the following sequences: 1) duplicates of in-vitro P. expansum mycelia in pH 4 for 3 h with 21,506,394 and 26,786,825 reads, respectively; 2) duplicates of in-vitro P. expansum mycelia in pH 7 for 3 h with 16,745,224 and 32,103,824 reads, respectively; 3) duplicates of pooled samples from all time points during in vitro exposure in medium at pH 4, with 19,665,360 and 14,581,483 reads, respectively; (4) duplicates of pooled samples from all time points during in vitro exposure in medium at pH 7, with 17,324,922 and 19,406,152 reads, respectively; (5) duplicates of leading edge samples of inoculated apple tissue with 253,299,784 and 199,419,262 reads; (6) one sample of healthy apple cv. 'Golden Delicious' tissue, with 31,719,018 reads.

The datasets are available at the NCBI Sequence Read Archive (SRA) under accession number SRP071104.

The Bowtie2 softweare [14] was used to align the RNA-seq outputs against the transcriptome of P. expansum. The libraries were aligned against the P. expansum genome (downloaded from NCBI Accession no. JQFX00000000.1). The apple samples were also aligned against the Malus × domestica. Whole Genome v1.0 that was downloaded from GDR (Genome Database for Rosaceae) [60]. RSEM software [61] was used for transcript quantification of the RNAseq data and then the edgeR package [62] was used to calculate differentially expressed genes.

The genes of P. expansum were annotated by using BLASTx [63] against the non-redundant NCBI protein database, after which their GO term [64] was assigned by combining BLASTx data and interproscan analysis [65] by means of the BLAST2go software pipeline [16]. GO-enrichment was analysed by using Fisher’s Exact Test with multiple testing correction of FDR [15]. Heatmap and clustering of the genes were visualized by using the R software ggplots2 package [66]. We used a threshold of FDR < 0.05 [15] and the criterion that expression level increased or decreased by a factor greater or less than 2, respectively, i.e., greater than or less than +1 or −1, respectively, on a logarithmic (base 2) scale. The normalized expression value was centered and log 2 transformed for visualization purposes with a script taken from trinity pipeline [67]. The CAZY analysis was done with the CAZymes Analysis Toolkit (CAT) [45]. A heat map of Cazymes in each cluster was plotted by using the R package pheatmap [68].

Ammonia atmosphere analysis

To create an ammonia vapor atmosphere, 250-mL aliquots of 5 N NaOH with 0 or 50 μM of NH4Cl were placed in closed containers for 24 h before use, after which, five apple fruits were placed inside each container for 5 days. Concentrations of 0 and 50 μM NH4Cl in NaOH solutions resulted in detected ammonia concentrations of 0 and 22 μM, respectively. All presented gene-expression results are relative to 0 μM NH4Cl in 5 N NaOH.

Gene-expression analysis by qRT-PCR

Real-time qPCR was performed with the StepOnePlus System (Applied Biosystems, Grand Island, New York, USA). PCR amplification was performed with 3.4 μL of cDNA template in 10 μL of reaction mixture containing 6.6 μL of the mix from the SYBR Green Amplification Kit (ABgene, Surrey, UK) and 300 nM of primers; Additional file 10: Tables S1 and Additional file 11: Table S2 (see Supporting Information) list the forward and reverse primers for each of the indicated genes. The PCR was carried out as follows: 10 min at 94 °C, and 40 cycles of 94 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. The samples were subjected to melting-curve analysis: efficiencies were close to 100 % for all primer pairs, and all products showed the expected sizes of 70 to 100 bp. All of the samples were normalized to 28S expression levels, and the values were expressed as the change (increasing or decreasing) of levels relative to a calibrator sample. Results were analyzed with the StepOnePlus software v.2.2.2 (Applied Biosystems, Grand Island, New York, USA). Relative quantification was performed by the ∆∆CT method [69]. The ∆CT value was determined by subtracting the CT results for the target gene from those for the endogenous control gene-18S for apple analysis and 28S for P. expansum analysis-and normalized against the calibration sample to generate the ∆∆CT values. Each experiment was performed in triplicate, and three different biological experiments were conducted. One representative set of results is presented as mean values of 2-∆∆CT ± SE for each treatment.

Availability of data and material

All data presented in this manuscript were deposited in the NCBI Sequence Read Archive (SRA) database under accession number SRP071104.

Declarations

Acknowledgements

We acknowledge the financial support for D.P. from the US/Israel Binational Agricultural R&D (BARD) fund; project I-IS-4773-14. We appreciate the contributions of the Bioinformatics units of the Weizmann Institute and the Technion – Israel Institute of Technology, in preparing libraries and providing sequencing services.

Funding

We acknowledge funding by the US/Israel Binational Agricultural Research Fund, BARD, IS-4773-14. SB was funded by a scholarship of the Volcani Center, Israel.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center
(2)
Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem
(3)
Department of Plant Pathology and Weed Research, ARO, the Volcani Center
(4)
Genomics Unit, ARO, the Volcani Center

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