Fungal hydrophobins are small, secreted, hydrophobic proteins that may be involved in the adhesion of spores to the leaf surface (Tucker and Talbot, 2001). In Magnaporthegrisea, the class I hydrophobin MPG1 and the class II hydrophobin MHP1 are both required for full pathogenicity (Talbot et al., 1993, 1996; Kim et al., 2005). In our data, a gene encoding MPG1 (CGGC5_8072) was expressed at high levels throughout the entire course of infection (Table S3). Another secreted fungal protein that binds to hydrophobic surfaces is encoded by HsbA in Aspergillus oryzae. This secreted protein binds to artificial polybutylene succinate-co-adipate (PBSA) hydrophobic surfaces and has been shown to recruit a polyesterase, which degrades PBSA and enables the fungus to use it as a carbon source (Ohtaki et al., 2006). Based on the occurrence of the HsbAPfam motif (PF12296), nine homologs of HsbA were discovered in the transcriptome of C. fructicola. Analysisof the transcriptomic data (Table S3) suggests that eight HsbA-encoding genes are up-regulated at 24 hpi and that the ninth gene is up-regulated only at a later stage (72 hpi). These data suggest that HsbA-like protein-mediated adhesion likely plays a role during C. fructicola appressorium development.

In the first step of invasion, pathogens need plant cell wall–degrading enzymes (PCWDEs) to degrade the plant cell walls and facilitate penetration and migration; meanwhile, remodeling of the pathogen's cell wall is necessary for evasion of PTI (Bellincampi et al., 2014; Oliveira-Garcia and Valent, 2015). The most prevalent PCWDEs during the early stages of C. fructicola infection (24 hpi) are pectin-degrading enzymes, such as carbohydrate esterase (CE) family 1, glycoside hydrolase (GH) family GH3, GH31, and several pectate lyases (PL1, PL3) (Table S4). Pectin is the main component of plant cell walls. The depolymerization of pectin makes the plant cell wall more vulnerable and accessible to other PCWDEs (Malinovsky et al., 2014). An increase in the number and diversity of the GH families that facilitate plant cell wall breakdown was observed at 96 hpi, including GH28 (polygalacturonase), GH31 (α-glucosidase), GH47 (α-mannosidase), and GH105 (rhamnogalacturonylhydrolase), as well as pathogen wall modification, such as GH-3 (β-1,3-glucan) and GH-18 (chitinase) (Figure S1, Table S4).

The transcription of an abundant class of genes encoding enzyme inhibitors was increased, including glucanase inhibitor proteins that bind to and inhibit host endo-β-1,3-glucanases of glycosyl hydrolase (GH) family 16; this was especially prevalent during 72 hpi. These proteins may impede the degradation of the fungal cell wall and prevent it from being recognized by the plant (Esquerré-Tugayé et al., 2000). Notably, three genes containing the carbohydrate-binding module 50 (CBM50), which binds to chitin (LysM domain-containing proteins), were up-regulated at 72 and 96 hpi (Table S4). The binding of chitin by pathogen proteins could help it to avoid recognition by host chitin receptors and suppress chitin-induced host immunity (de Jonge and Thomma, 2009).

During the infection process, pathogenic fungi usually encounter nutrient deprivation in the host before gaining access to sufficient nutrients to successfully colonize living tissue (Lee et al., 2009). We observed significant up-regulation of the key genes involved in two primary metabolic pathways (fatty acid oxidation and the glyoxylate cycle).

During the first step of infection, fungi produce extracellular degrading enzymes. We observed significant up-regulation of cutinase–coding genes during C. fructicola infection (Table S4). This process provides the materials needed for fungal fatty acid oxidation. Thereafter, fatty acid synthesis and subsequent transport were bothactivated at 24 and 72 hpi, respectively, and were characterized by the up-regulation of the genes encoding Acyl-CoA dehydrogenase and Enoyl-CoA hydratase. Acyl-CoA is the basic currency of carbon metabolism within the cell (Strijbis and Distel, 2010). In our study, two genes encoding key enzymes of the glyoxylate cycle (citrate synthase and isocitratelyase) were highly induced at 24 hpi. This indicates that the use of Acyl-CoA, which is derived from fatty acid β-oxidation, may provide carbon skeletons for anabolic processes via the glyoxylate cycle early in infection (Figure 4, Table S5).

The expression patterns demonstrate that in vitro, where abundant nutrition sources are available in the culture medium, C. fructicola tends to rely on glycolysis and the tricarboxylic acid cycle for energy production. By contrast, during the early stages of infection where there are limited sources of nutrition, C. fructicola tends to rely on the mobilization of stored lipids through fatty acid oxidation to form Acyl-CoA, which is further assimilated via the glyoxylate cycle to supply energyto cells (Table S6).

Secondary metabolites produced by Colletotrichum are known to contribute to pathogenicity (Gan et al., 2013). There were 11 secondary metabolite backbone genes up-regulated in our transcriptomic data (Table S7). Three polyketide synthases (PKSs)-encoding genes (CGGC5_9803, CGGC5_12144, and CGGC5_14485) were significantly up-regulated at 24 hpi. CGGC5_14485 was homologous with ALB1, which encodes a PKS that catalyze the first step in the melanin biosynthesis of M. grisea (Chumley, 1990). The other five secondary metabolite backbone-forming genes were highly up-regulated at 96 hpi; this coincides with the necrotrophic stage of C. fructicola (Table S7).

Secondary metabolites associated with sporulation can be placed into three broad categories, including pigment. Melanin, a pigment located in the cell wall of appressoria, can provide mechanical strength that aids in tissue penetration (Eisenman and Casadevall, 2012). Many fungi synthesize melanin via the dihydroxynaphthalene (DHN) pathway. DHN is produced from 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) via a sequence of reduction and dehydration reactions with the intermediates scytalone,1,3,8-trihydroxynaphthalene and vermelone (Eisenman and Casadevall, 2012). The expression profiles of C. fructicola genes encoding enzymes of the DHN melanin biosynthesis pathway were all up-regulated at 24 hpi (Table S8). Melanin biosynthesis is a pivotal process during appressorium development and seems to play a role in C. fructicola pathogenesis.

Plant pathogens must protect themselves, especially during infection when they are likely to encounter host defense mechanisms (Göhre and Robatzek, 2008). One way fungi accomplish this task while overcoming intercellular toxin accumulation is via efflux pumps, in particular ATP-binding cassette (ABC) transporters and transporters of the major facilitator superfamily (MFS) (Coleman and Mylonakis, 2009). In our data, several ABC-transporter-encoding genes were up-regulated at all three time points post infection (Table S9). The ABC transporters in other fungi have shown to play an important role in fungal resistance. For example, BcatrB from Botrytis cinerea (homolog of CGGC5_15191) and NhABC1 from Fusariumsolani f. sp. pisi (homolog of CGGC5_9023) were shown to be responsible for the efflux of plant-derived defense compounds (Stefanato et al., 2009; Coleman et al., 2011). A search for genes with the Pfam membrane transporter motifs PF00083 (a signature of saccharide) and PF07690 (MFS signature) identified 83 up-regulated genes (Table S10). Of these, 56 were up-regulated at 24 hpi. Genes encoding sugar and MFS transporters were also highly up-regulated; (Figure S2A). In Cercospora nicotianae, a MFS transporter gene CTB4 (homologs of CGGC5_9801), encodes a cercosporin efflux pump that contributes to self-protection from cercosporin, subsequently facilitating the virulence of these pathogens on their host plants (Choquer et al., 2007). The significant up-regulated ABC and MFS transporter genes at 24 hpi may suggest that these transporters play a critical role in protection against plant defense compounds during the early stage of infection (Del Sorbo et al., 2000).

Cytochrome P450s (CYP450) are involved in detoxification and contribute to host interactions (Nelson, 1999). We observed 36 genes encoding CYP450 (PF00067) that were simultaneously up-regulated at 72 hpi. A recent publication showed that P450s were specifically involved in virulence as well as both asexual and sexual development. Most importantly, P450s seem to play redundant roles in the degradation of xenobiotics in Fusarium graminearum; CYP51 genes in F. graminearum (homologs of CGGC5_10696, CGGC5_2390) mediate differential sensitivity to sterol demethylation inhibitors (Shin et al., 2017). CYP450 contributes to the production of mycotoxins and the detoxification of host metabolites in other fungi (Crešnar and Petric, 2011). The up-regulation of several P450 genes in our data may suggest a special role for theCYP450-mediated detoxification produced by the plant at this stage (Table S11, Figure S2B).

Fungal effectors are secreted molecules that modulate the interaction between the fungus and its host (Lo Presti et al., 2015). There are 2042 genes (13.2%) that encode secreted proteins in the genome of Nara gc5 (Gan et al., 2013). The current transcriptomic data show that some homologs of known effectors from other phytopathogens were differentially expressed after C. fructicola infection. For example, the gene encoding CGGC5_2199 (homolog of Cmu1) was up-regulated at 24 hpi. The effector Cmu1 counteracts salicylic acid (SA)-dependent immunity in the host, functioning as a chorismate mutase to reduce the levels of the SA precursor chorismate (Djamei et al., 2011). The genes encoding CGGC5_10914 (a homolog of Ecp6) were up-regulated at 96 hpi; this is consistent with the previously reported expression patterns of this gene. Previous reports showed that the expression of Ecp6 gene was gradually up-regulated and maximal at 13 dpi during the interaction of Cladosporium fulvum with tomato (Bolton et al., 2008). Ecp6 has identified to be a novel virulence factor through sequestering chitin oligosaccharides to prevent elicitation of plant immunity (de Jonge et al., 2010). The genes encoding for CGGC5_10914 (a homolog of CHEC30) were up-regulated at 72 and 96 hpi. C. higginsianum effector CHEC30 was appeared to be preferentially expressed at biotrophic stage (Kleemann et al., 2012). The expression patterns of other homologs of known effectors are listed in Table S12.

In addition to these known effectors, candidate effectors were identified in the transcriptome of C. fructicolavia a pipeline (Figure 5A) that shares common features with those described for filamentous plant pathogens (Petersen et al., 2011; Guyon et al., 2014). We first refined the prediction by combining the predictions from SignalP4.1 and TMHMM to identify 729 proteins that had secretion signals but did not have transmembrane helices (Petersen et al., 2011). Next, GPIsom removed 87 proteins harboring glycophosphatidyl inositol anchor motifs, which likely represent surface proteins rather than secreted effectors (Fankhauser and Mäser, 2005). This resulted in 642 predicted secreted proteins (SPs). A Circos plot was generated to visualize the peptide sequence similarities between these SPs and their homologs. These homologs show a 90% peptide sequence identity, which indicates that these SPs have striking similarities tothose of other Colletotrichum species (Figure 5B). In total, 294 of the 642 SPs were small (<300 aminoacid) secreted proteins (SSPs). We observed 85 SSPs that were highly expressed during infection. These 85 SSPs were evaluated with the EffectorP prediction tool, which is usable for both apoplastic and cytoplasmic effectors (Sperschneider et al., 2016). 52 SSPs were ultimately identified as the most likely set of C. fructicola effectors (Figure 5C). Post-translational modification of effectors influences their function. Glycosylation likely impacts effector function by modifying protein stability, as well as conformation or resistance to host proteases (Doehlemann et al., 2014). Here, 90.6% of candidate effectors carried at least one predicted N/O-glycosylation site (Figure 5C, Table S13).

To identify the function of the candidate effectors, four highly expressed SSPs were chosen for a transient assay in Nicotiana benthamiana. The signal peptides of these candidate effectors were first removed to enable the genes to be expressed stably in plant cells. The four candidate effectors that partially or fully suppressed Bax-mediated programmed cell death in tobacco leaves (Figure 5D).

Improving cell wall lignin content is one of the common plant defense mechanisms. In our study, inoculation induced the expression of strawberry 4-coumarate-CoA ligase and disease resistance-responsive (dirigent-like) proteins. These proteins are involved in lignin biosynthesis (Lee et al., 1995; Davin et al., 1997). Several dirigent-like protein-encoding genes were up-regulated during the three stages of infection (Table S15).

A number of cell wall synthesis genes were also down-regulated during the course of C. fructicola infection. For example, six of the 10 cellulose-synthase-like genes, including CSLC5 and CSLD3 homologs (Dai et al., 2011; Almeida et al., 2015), were down-regulated at 72 and 96hpi (Table S15). Similar changes (i.e., replacement of cell wall components) have been observed in Arabidopsis CESA3-deficient mutants (Caño-Delgado et al., 2003). Reduced cellulose synthesis has been suggested to invoke a wide range of cellular responses, including activating lignin synthesis and defense responses mediated via JA and ethylene (ET) signaling pathways (Caño-Delgado et al., 2003; Hamann, 2012).

Through evolution, plants have developed a two-step innate immunity system to prevent pathogen invasion. The first step involves the recognition of pathogen-associated molecular patterns (PAMPs) through plant pattern recognition receptors (PRRs); this subsequently triggers the PTI (Nicaise et al., 2009). Plant PRRs are either surface-localized receptor-like kinases (RLKs) or receptor-like proteins (RLPs), which usually contain ligand-recognition domains, such asleucine rich repeats (LRRs) and lysine motifs (LysMs). Chitin is the major constituent of fungal cell walls and serves as a classic fungal PAMP that can be recognized by several typical plantPRRs (e.g., chitin elicitor receptor kinase 1 [CERK1], chitin elicitor-binding protein [CEBiP]) (Kaku et al., 2006; Miya et al., 2007). We did not observe significant up-regulation of genes encoding CERK1 or CEBiP in C. fructicola–infected plants. However, a set of strawberry RLKs were differentially expressed during infection. 53% (23/43) and 60% (26/43) of RLKs were significantly up-regulated at 72 and 96 hpi, while 23% (10/43) of RLKs were down-regulated at both 72 and 96 hpi. (Table S16).

ETI involves the direct or indirect recognition of effectors by plant resistance (R) proteins (Chisholm et al., 2006). The execution of ETI is mediated by intracellular immune receptors (e.g., NBS-LRR proteins) in order to perceive secreted virulence effectors (Zipfel, 2014). In our study, five CC-NBS-LRRs, two TIR-NBS-LRRs, and five NB-ARC domain-containing proteins were differentially expressed (Table S17). Among them, two NB-ARC genes were down-regulated while six were up-regulated during all three stages of infection. However, the successful infection of C. fructicola indicated that although some R genes were up-regulated at transcriptional level, they might not fully exert their function and block fungal infection.

In this study, 50, 88, and 100% of SA-signaling genes were significantly up-regulated at 24, 72, and 96 hpi, respectively. The nonexpressor of pathogenesis-related genes 1 (NPR1) is the key gene that mediates defense gene expression induced by SA (Mou et al., 2003). We observed an up-regulation of NPR1 at 72 and 96 hpi. The SA pathway activates many downstream defense related genes, including PR genes (van Loon et al., 2006). The PR4 family encodes chitinase and can be induced by F. culmorum infection, SA treatment, or JA treatment in wheat (Bertini et al., 2003). In our study, two homologous PR4 genes, PR4a and PR4b, were up-regulated (by 2.3- and 2.4-fold, respectively) at 24 hpi. By 72 hpi, they were up-regulated by 29- and 38-fold, respectively; this increased further to 49- and 60-fold by 96 hpi (Table S18). In addition, one gene coding the cysteine-rich secretory protein (CRISP) was highly expressed at 96 hpi. The homolog of the CRISP in plants is the PR-1 protein; this protein plays an important role in anti-fungal activities (Niderman et al., 1995). These highly up-regulated PR genes seem to indicate their potential role in C. fructicola resistance. However, in our previous reports, several relevant immune-related genes were chosen for qRT-PCR assay to evaluate their gene expression level between susceptible (JiuXiang, JX) and less-susceptible plant (Sweet Charlie, SW) (Zhang et al., 2016). Specifically, several SA-related genes, including PR1a and PR5 were up-regulated in both JX and SW. Notably, the gene expression level in less-susceptible plant were significantly higher than that in susceptible plant. For example, at 96 hpi, PR1a was up-regulated by ~30- and 400-folds in susceptible and resistant plant, respectively. Similarly, PR5 was up-regulated by ~10- and 600-folds in susceptible and resistant plant, respectively. These results suggest that although several SA-related genes seem to be up-regulated in successful infection, the extent of the up-regulation is significantly less than that in resistant infection. The relative low level of these genes may not be able to inhibit fungal infection in susceptible infection.

JA is an important plant hormone which plays a role in regulating plant development and stress responses. The largely antagonistic functions of JA and SA have been suggested for plant response to pathogen infection (Verma et al., 2016). There also exists cooperative interplay between JA and SA during early ETI. In parallel to SA accumulation in Arabidopsis, the endogenous JA level also increases during ETI induction, which may reduce plant susceptibility to necrotrophic pathogens in the neighboring tissue; this elevation of JA is dependent on the degradation of JAZ mediated by direct interaction with NPR3-NPR4 (Liu et al., 2016). Lipoxygenases (LOX), especially LOX2, are required for wound-induced JA accumulation in Arabidopsis (Bell et al., 1995). In this study, the gene encoding LOX1 was reduced by 10.5-, 3.6-, and 4.3-fold at 24, 72, and 96 hpi, respectively. In addition, the gene encoding LOX2 was significantly down-regulated at 96 hpi. JA biosynthesis is possibly regulated by post-translational modifications of pre-existing enzymes, such as allene oxide cyclase (AOC) (Schaller and Stintzi, 2009). Similar to LOX, the gene encoding AOC4 was reduced by 3.1-, 3.0-, and 4.2-fold at 24, 72, and 96 hpi, respectively (Table S19). These results indicate that JA signaling maymainly inhibited by C. fructicola during the entire infection process.

JA and ET operate synergistically in regulating defense responses after pathogen infection (Verma et al., 2016). ET and the related signaling pathway can positively or negatively regulate plant immunity; additionally, this pathway exhibits extensive cross-talk with the SA and JA pathways (Sato et al., 2010; Shakeel et al., 2013). The gene encoding ethylenereceptor2 was down-regulated at 24 hpi. Ethylene response factors (ERFs) act downstream of the intersection between the ET and JA pathways. These transcription factors, such as ERF1, are key elements regulating defense responsive genes (Lorenzo et al., 2003). In our study, the genes encoding the ethylene responsive element–binding factor 2 (EREBF2) and the ethylene-forming enzyme (EFE) were mildly up-regulated after C. fructicola infection. Meanwhile, the other eight genes encoding the ethylene-responsive transcription factor were significantly down-regulated at three time points after infection (Table S20).

The endogenous hormone contents of strawberry leaves during C. fructicola infection were analyzed to further confirm the role of plant hormone signaling in fungal infections. The results showed significant up-regulation of SA content at 72 and 96 hpi. Meanwhile, JA content was mildly up-regulated at 24 hpi, but then down-regulated at 72 and 96 hpi (Figure 6). Our results regarding SA and JA signaling were consistent with previous reports, which demonstrated that the primary mode of interaction between SA and JA pathways was mutual antagonism (Doares et al., 1995; Clarke et al., 2000; Gupta et al., 2000). SA is generally involved in the activation of defense responses against biotrophic and hemi-biotrophic pathogens. By contrast, JA and ET are required for resistance against necrotrophic pathogens (Glazebrook, 2005; Bari and Jones, 2009). In our study, the inhibition of ET and JA signaling may be triggered by C. fructicola infection. Alternatively, C. fructicola induced the activation of SA signaling, which may indirectly inhibit ET and JA signaling.

ABA prominently contributes to plant defense response against both abiotic and biotic stress conditions (Fitzpatrick et al., 2011). ABA shows negative interaction with JA-signaling in the modulation of Arabidopsis resistance to the necrotrophic fungi but it can also positively regulate the resistance to some biotrophic pathogens (Denance et al., 2013). Glycosyl hydrolases (GH) belong to a large family of proteins implicated in carbohydrate metabolism andthe remobilization of sugars. ABA significantly increased the activity of α-L-Arabinofuranosidase, one member of glycosyl hydrolase (GH) family, suggesting that some GH might be associated with ABA mediated processes (Alayon-Luaces et al., 2008). Two glycosyl hydrolase superfamily genes and one glycosyl hydrolase family 32 gene were up-regulated at 72 hpi. Meanwhile, five other GH genes were down-regulated at 72 and 96 hpi (Table S21).

Emerging evidence indicates that auxin-signaling genes regulate resistance to different plant pathogens (Denance et al., 2013); additionally, it is possible that components of the auxin synthesis, signaling, and transport pathways could be utilized by pathogens to increase colonization efficiency (Denance et al., 2013). In our study, the temporary up-regulation of auxin related genes, such as auxin-responsive GH3 family protein and indole-3-acetic acid 7 (IAA7), was observed at 24 hpi. However, these auxin-related genes were significantly down-regulated during the middle and late stages of infection. For example, the expression of the early auxin-responsive gene (IAA7) was increased by 7.1-fold at 24 hpi, but was then decreased by 1.6- and 1.8-fold at 72 and 96 hpi, respectively (Table S22). An analysis of phytohormones levels revealed that ABA content was only mildly inhibited at 24 hpi. Other than this, ABA and IAA content were not significantly different at any point during infection (Figure 6). ABA play a negatively role in plant resistance to certain necrotrophic fungi (Audenaert et al., 2002; AbuQamar et al., 2006). A negative effect of auxin signaling on plant resistance to Necrotrophic Fungi was recently described (Llorente et al., 2008). In our present study, since ABA and auxin pathways were not significantly triggered during infection, these two pathways may not play an equally important role as SA, JA, and ET pathways. However, the precise function of these two hormone pathways needs to be further analyzed.

Reactive oxygen species (ROS) in plants are normally generated as by-products of oxidative phosphorylation and other diverse biosynthetic pathways (Almeida et al., 2015). The acute accumulation of ROS leads to an oxidative burst that induces cell death and restricts further pathogen infection (Apel and Hirt, 2004). Therefore, generation of ROS is usually accompanied by visual symptoms (i.e., hypersensitive responses or necrosis) in plants. In our study, there were obvious pathological changes at 96 hpi. Seven peroxidases (PODs) were significantly up-regulated at 96 hpi. Of the seven, two were up-regulated by 52- and 131-fold at 96 hpi. Meanwhile, another was up-regulated by 30- and 52-fold at 72 and 96 hpi, respectively (Table S23). The significant up-regulation of POD-encoding genes at 96 hpi may indicate the activation of plant defense mechanisms.

During PTI and ETI, plants trigger a diverse array of immune responses, such as hormone signaling, ROS generation, and MAP kinase (MAPK). The signal transduction of these immune responses relies on a regulatory network of transcription factors (TF) (Moore et al., 2011). A subset of TF families, such as WRKY, NAC, bHLH, ERF/AP2, MYB, and bZIP, play a role in the regulation of immune responses. WRKY transcription factors belong to one of the largest families of transcriptional regulators in plants, and are key regulators of PTI and ETI (Eulgem and Somssich, 2007). In this study, we identified a total of 12 differentially expressed WRKY factors (Table S24). Nine genes encoding WRKY factors, including WRKY75, WRKY50, and WRKY23, were significantly up-regulated during the middle-to-late stages of C. fructicola infection. Genes encoding three members of the WRKY family, including WRKY33, were down-regulated at all three stages during infection. Arabidopsis WRKY33 is a key regulator of host immunity and also regulates the expression of some other WRKY factors as well as defense-associated genes (Birkenbihl et al., 2012). Interestingly, a strawberry homolog of the upstream regulatory gene, mitogen-activated protein kinase 3 (MPK3), was also down-regulated at all stages during infection.

In addition to the WRKY family, the NAC family of proteins is another group of transcriptional regulators that have been identified as important immune components (Jensen et al., 2008). Our study identified six NAC domain-containing proteinsthat were mainly induced at the middle-to-late stages of infection (Table S25). Like WRKY and NAC, other TFs, such as bHLH and ERF, were also differentially expressed after C. fructicola infection (Tables S20, S26). The differential expressions of these TFs as observed in the present study suggest that they may play a potential role in the interaction between C. fructicola and strawberry.

The transcriptomic data give a picture of how adapted C. fructicola invades host cells and completes the infection process as strawberry works to mitigate immune suppression and slow the infection rate, albeit at a serious price. It should be noted that C. fructicola ultimately, successfully infects the plant and causes symptoms; this means that C. fructicola wins the “battle” against host resistance. Our transcriptomic data demonstrate that C. fructicola utilizes a series of strategies to antagonize or escape host resistance mechanisms at every stage of infection (Figure 7).

The establishment of a successful infection is dependent on C. fructicola invasion, which is the first step of infection and includes the degradation of the plant cell wall and subsequent penetration into the cell. The top 100 up-regulated genes at 24 hpi provide a glimpse into C. fructicola–strawberry interactions (Tables S27, S28). Specifically, at this stage, we found that 15 of the top 100 up-regulated C. fructicola genes were identified to encode putative effectors, one of which was a chitin–binding protein (CGGC5_3464). The literature shows that the pathogen is likely to initiate its adhesion to the host cells by expressing accessory molecules, such as chitinases and/or chitin-binding proteins (Chaudhuri et al., 2010). Four homologs of HsbA were within the top 100 up-regulated genes at 24 hpi. As previously mentioned HsbA binds to PBSA hydrophobic surfaces and recruits a polyesterase for the degradation of PBSA (Ohtaki et al., 2006). It prompts us to speculate that four homologs of HsbA are involved in attachment to the strawberry surface and the degradation of constituents from the plant epidermis; however, this requires further elucidation. Meanwhile, the host expresses a series of proteins to stabilize the cell wall to cope with C. fructicola invasion. Among the top 100 up-regulated strawberry genes, the most prevalent genes (n = 5) encode proteins involved in cell wall reinforcement and catabolism. This includes the glycine-rich cell wall structural protein, pectin acetylesterase family protein, wound-induced protein WI12, chitinase and AMP-dependent glycosyl hydrolase. WI12 preferentially accumulates in the cell wall, which suggests that it plays a role in cell wall reinforcement after wounding and during plant development (Yen et al., 2001). Glycine-rich proteins (GRPs) form a major part of the highly extensible and specialized cell walls. However, there has been no genetic evidence indicating a specific function for GRPs (Ringli et al., 2001). Although we did not thoroughly examine the function of these proteins, their high level of expression in our data indicates that the plant cell wall seems to be reinforced to prevent fungal invasion. However, most of the genes encoding cellulose synthase-like proteins, which participate in cellulose biosynthesis and cell wall modification, were down-regulated (Table S15 and Figure 7, upper panel). We conclude that this down-regulation might betargeted by C. fructicola, which served to help the fungus break down the cell wall and invade the host cells.

After C. fructicola invasion, fungus PAMPs are sensed by host PRRs. A number of strawberry genes encoding RLKs were up-regulated (Figure 7, left panel). In addition, several plant chitinase-encoding genes (n = 7) were significantly up-regulated, especially during the late stage of C. fructicola infection (Table S29). Chitinases were identified to be induced in various plants after fungal infection (Punja and Zhang, 1993). Furthermore, the antifungal activity of chitinases was shown by large number of transgenic plants raised for over expression of chitinase genes (de las Mercedes Dana et al., 2006). To evade recognition by plant PRRs and subsequent elimination, the fungus utilizes numerous effectors to suppress PTI. Typically, secreted LysM Protein1 (SLP1) can bind to chitin and suppress chitin-induced plant immune responses (Mentlak et al., 2012). Our results show that the SLP1-encoding gene was up-regulated at 72 and 96 hpi. The highly-expressed SLP1 may play an important role in the suppression of PTI, such as chitin-induced immunity; however, this requires further investigation. A subset of these effectors might be perceived by R proteins resulting in a second layer of host defense called ETI. A total of 10 R genes were significantly up-regulated during C. fructicola infection; this suggests that they may play a role in antagonizing effectors, although their functions still need to be characterized (Figure 7 right panel, blue shape).

After recognition of the fungus by PRR and R proteins, the plant initiates downstream anti-fungal innate immunity. In our study, hormone signaling, especially the SA, JA, auxin, ABA, and ET signaling pathways, displayed obvious changes in transcriptional activity (Figure 7, middle panel). The early activation of ABA and auxin signaling, the delayed activation of the SA pathway, and the continuous suppression of the JA and ET pathways might contributed to the deployment of basal defenses and the insufficient induction of the defense system in strawberries. The literature showed that the secreted chorismate mutase protein (Cmu1) of fungus Ustilago maydis is proposed to suppress SA-dependent plant defense response (Djamei et al., 2011). Therefore, fungi could formulate efficient strategies against strawberry hormone-related defense systems.

A recent report on the strawberry leaf transcriptomes of “Yanli” and “Benihoppe” cultivars infected with C. gloeosporioides consistently showed that PR1 expression was up-regulated after infection, thus supporting the SA pathway response (Wang et al., 2017). Similarly, various defense-related genes, such as those encoding PR proteins, peroxidases, and lipoxygenases, were up-regulated in bean leaf following inoculation with C. lindemuthianum, which is in accordance with our transcriptomic data (Padder et al., 2016). A dual RNA-seq analysis on C. graminicola-maize leaf pathosystem during appressorial maturation, penetration, and colonization revealed that genes encoding secreted proteins, secondary metabolism enzymes, and receptors were over-represented among the differentially expressed genes. Several genes encoding homologs of BAS2, SLP1, and GAS1 were significantly up-regulated following C. graminicola infection. The up-regulation of these genes werealsoobserved in the current study, which suggests that these effectors may play important conserved roles in Colletotrichum infection (Torres et al., 2016).

In conclusion, the RNA-Seq technique was used to obtain a comprehensive characterization of infection-responsive DEGs from both C. fructicola and strawberry. The data revealed the metabolic, host/pathogen-responsive genes, and transcriptional networks associated with C. fructicola–strawberry interactions. This study resulted in several novel findings regarding C. fructicola–strawberry interactions: (1) an overview of strawberry defense and C. fructicola evasion mechanisms during the infection process; (2) a large number of up-regulated fungal genes encode candidate effector proteins during plant invasion; (3) the pivotal role of SA signaling during C. fructicola infection; and (4) other plant defense-related genes involved in cell wall fortification and ROS production were differentially regulated in response to C. fructicola infection. Future functional studies are needed to confirm the role of these candidate genes in C. fructicola–strawberry interactions; in particular, these studies will focus on the C. fructicola-specific effector genes and the putative susceptibility-related genes.