Plant culture (Fragaria × ananassa cultivar Camarosa) and growth conditions, C. acutatum (isolate CECT 20240) inoculation, and treatments with chemicals have been previously described (Encinas-Villarejo et al., 2009). In breaf, 8-week-old strawberry plantlets were placed in 20 cm diameter plastic pots containing sterilized peat and grown for a minimum of 6 additional weeks prior to mock or pathogen inoculation by spraying a spore suspension of 10⁴ conidia·ml⁻¹. Crown was collected 5 days after treatment (spray-infected and mock-treated) for microarray studies. At this time, under our experimental conditions, plants still looked healthy, and no visible disease symptoms were easily detected, even in petioles, crowns or leaves. For RTqPCR analysis, crowns and petioles were collected 1, 3, 5, 7, and 9 days after treatment, as previously described in Casado-Díaz et al. (2006). For treatment and hormonal content analysis, axenic in-vitro plantlets were used and aseptically sprayed with either MeJa (2 mM) or SA (5 mM) solutions, or inoculated with C. acutatum conidia suspension (10⁴ conidia·ml⁻¹), respectively. All collected plants were immediately frozen in liquid nitrogen and stored at −80°C until use. For transmission electron microscopy, pathogen was spot inoculated in crown by applying 50-μL droplets of conidia suspension (10⁶ conidia·ml⁻¹) (Arroyo et al., 2005). Control plants were similarly inoculated with 50 μL of sterile distilled water. The position of each infection site was marked for reference. Inoculated plants were enclosed in plastic bags for 48 h to maintain high relative humidity and incubated in similar conditions as described above. Hence, disease is forced to progress quicker than by spraying.

Light microscopy analyses of C. acutatum development were performed on strawberry leaf discs (10 mm diameter) randomly excised from infected leaflet at 1, 3, 5, 7, 9 dpi, using a method modified from Debode et al. (2009). In breaf, leaf discs were cleared in 0.15% trichloroacetic acid (TCA) in a 3:1 (v/v) mixture of ethanol and chloroform for 48 h, with at least three changes of the bleaching solution, rinsed briefly in lactoglycerol and incubated at room temperature for 1 h in lactophenol blue (Sigma), and washed 3 times in lactoglycerol. For ROS detection, leaf discs were infiltrated in a 10 mg/ml DAB (3,3′ -diaminobenzidine, Sigma) solution for 10 min and subsequently incubated overnight at room temperature in dark, and cleared for 24 h in 3:1 (v/v) ethanol: glacial acetic acid with three changes. Treated leaf disc was mounted in 50% fresh glycerol on glass slides and examined using a Leica DM5000B microscope. Images were captured with a Leica DC500 digital camera. Three leaftlets were sampled from each plant, and 3 plants were observed at each time point. The overall number of conidia (germinated and non-germinated) and appresoria were counted per leaf disc taken up to 9 dpi.

Strips of tissue ~1 mm thick and 1–2 mm long were removed from beneath the inoculation droplets and fixed in 4% (v/v) glutaraldehyde in 0.1 mol·L –1cacodylate buffer (pH 7.2) for 3 h at 4°C. Upon rinsing in the same buffer, tissues were post-fixed in 1% (w/v) osmium tetroxide for 2 h at 4°C and subsequently dehydrated in a graded acetone series and embedded in EMBED-812 (Polysciences, Warrington, Penn.) according to manufacturer's instructions. Slides with semithin sections (0.5 μm) were placed on a hotplate at 50°C, stained for 1 min with 0.1% aqueous toluidine blue O and examined using a light microscope (Leitz Aristoplan). Ultrathin 60–80 nm sections were made with a Reichert-Jung Ultracut E ultramicrotome and a diamond knife, and collected on 300-mesh copper grids (Dashek and Mayfield 2000). Grids were stained with 7% aqueous uranyl acetate and lead citrate. Sections were observed and images were collected using a Philips CM-10 transmission electron microscope (TEM).

Total RNA from strawberry tissues was isolated as described previously (Casado-Díaz et al., 2006), treated with DnaseI (Invitrogen) for residual DNA removal, and further purified with the RNeasy MinElute Cleanup Kit (QIAGEN). Purified RNA was quantified by NanoDrop 1000 Spectrophotometer (Thermo scientific). RNA integrity was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Deutschland). First-strand cDNA synthesis was carried out using 1 μg of purified total RNA as template for a 20 μL reaction [iScript cDNA Synthesis kit (Bio-Rad)]. RT reactions were diluted 5-fold with nuclease-free water prior to qPCR.

Specific primer pairs set were designed using Oligo Primer Analysis software version 6.65, tested by dissociation curve analysis, and verified for absence of non-specific amplification (Table S1). FaGAPDH2 gene was used for normalization (Khan and Shih, 2004; Amil-Ruiz et al., 2013). RTqPCR runs were performed using two technical replicates in the same run and three biological replicates in different runs, as described previously (Encinas-Villarejo et al., 2009), using SsoAdvanced™ SYBR® Green supermix, and MyIQ v1.004 and iCycler v3.1 real-time PCR systems (Bio-Rad).

For microarray analysis, strawberry samples were collected 5 days after treatment (spray-infected and mock-treated). Crowns were used to make biological replicates, and overall RNA was isolated from three independent biological replicates for hybridization against a proprietary microarray representing approximately 2529 predicted unigenes from F. vesca (Shulaev et al., 2011), previously identified from strawberry libraries (Casado-Díaz et al., 2006; and JL Caballero unpublished). Microarray data with accession GSE56296 were deposited in the NCBI Gene Expression Omnibus. Quality control, labeling, hybridization, and scanning were carried out by the SCAI, University of Córdoba (http://www.uco.es/servicios/scai/index.html), following the Genomic Unit guidelines. Microarray images were analyzed using GenePix 6.0 software (Molecular Devices). Data were transformed using an intensity-based Lowess function (Yang et al., 2002) with Acuity 4.0 software (Axon Instruments). Genes were considered as differentially expressed if they fulfilled both a FDR < 0.05 after a SAM test analysis (Tusher et al., 2001), and the fold-change (up or down) was above 1.75-fold.

To assign a putative biological function to every detected differentially expressed gene, their respective orthologous genes from the wild species F. vesca, which genome has been recently released (Shulaev et al., 2011), were identified by blasting the EST sequence associated with each singular spot within the array to the overall collection of F. vesca predicted genes (Altschul et al., 1990; Shulaev et al., 2011; http://www.rosaceae.org/). In order to enrich this process, A. thaliana putative orthologs were also identified for every F × ananassa gene as vast functional information is available for the former species (TAIR10: http://www.arabidopsis.org/). FunCat and GO terms assignments were first used to perform an automated functional categorization of differentially expressed genes (Ashburner et al., 2000; Ruepp et al., 2004). FatiGO tool (a web tool to find significant associations of Gene Ontology terms with gene groups; Ashburner et al., 2000; Ruepp et al., 2004) was used to perform a comprehensive Singular Enrichment Analysis (SEA) to extract relevant GO terms associated with up-regulated genes. Briefly stated, it takes two lists of genes (ideally a group of interest and the remaining genes in the experiment, while any two groups, formed in any way, can be tested against each other) and converts them into two lists of GO annotations using the corresponding gene or protein—term annotation table. A Fisher's exact test for 2 × 2 contingency tables is subsequently used to check for significant over-representation of GO annotations in one of the sets with respect to the other. Multiple test correction to account for the multiple hypotheses tested (one for each functional term) is applied. The terms are considered to be relevant by the application of statistical tests, as described in Al-Shahrour et al. (2004).

Extraction and purification procedures and chromatographic analysis have been previously described (Durgbanshi et al., 2005). In short, 3 grams of frozen green tissue were lyophilized and immediately homogenized in 5 mL of ultrapure water. After centrifugation (5000 g, 10 min), supernatant pH was adjusted to 2.8 with 15% (v/v) CH3COOH, and the supernatant was partitioned twice against an equal volume of diethyl ether. The aqueous phase was discarded and the organic fraction was vacuum evaporated at room temperature. The solid residue was resuspended in 1 mL of a 90:10 (v/v) water/methanol solution and subsequently filtered through a cellulose acetate filter (0.22 μm). A 20 μL aliquot of this solution was then injected into the high performance liquid chromatography (HPLC) system from Waters, Milford MA (Alliance 2690 system). Aliquots were injected on a Nucleosil ODS reversed-phase column. Phytohormones were eluted with a gradient of methanol and 0.01% CH3COOH in water that started from 10:90 (v/v) and linearly reached 60:40 (v/v) in 10 min. In the following 4 min, the gradient was increased to 80:20 (v/v). Isocratic conditions of 80:20 (v/v) were then retained during the last 2 min of the run. Initial conditions were restored and allowed to equilibrate for 5 min, for a total time of 21 min per sample. The solvent flow rate was 0.3 mL/min, with working pressures at around 70–100 bar.

Quoted plant hormone endogenous contents are mean values from 2 measurements of each of all 3 biological replicates. The One-way Analysis of Variance (ANOVA) with a Bonferroni Multiple Comparisons Test was performed using GraphPad InStat3 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com) to calculate the significant differences between control and inoculated plants.

The histopathology of the interaction strawberry-C. acutatum has been previously reported (Curry et al., 2002), and the transition to the necrotrophic phase was established after 4 dpi (Horowitz et al., 2002). However, fungal colonization in plant may vary with infection conditions. Thus, we monitored the infection progress of C. acutatum on strawberry plant after artificial inoculation (spraying) using microscopic analysis.

To identify the initial infection stages, conidial development was monitored. The number of ungerminated conidia, germinated conidia without forming appresoria, and germinated conidia with fully developed appressoria, was monitored 1, 3, 5, 7 and 9 dpi (days post-inoculation; Figure 1). No visual disease symptoms were detected in the plant during this period of time. At 5 dpi 44.76% of conidia registered fully developed appresoria (Figure 1A). Other conidia did not germinate (34.98%); others, while they did, they did not produce appresoria (20.24%). Early mycelium formation was observed microscopically at 5 dpi (Figure 1D), whereas more extended and abundant mycelium was detected at 7 and 9 dpi, respectively (Figures 1E,F).

As pathogen development in crown it is difficult to monitor by optical microscopy in the same way as aerial tissues, the progress of pathogen development in crown was monitored using TEM with the inoculation methods presented in Arroyo et al. (2005) (Figure 2). Such higher concentration of inoculum expedited infection development. Figure 2A shows penetration peg formation through the host cuticle and a small infection vesicle, 36 h post-inoculation (hpi), which reflects the establishment of the biotrophic stage of C. acutatum. No morphological signs of cuticular component degradation were observed during this penetration phase. Necrotic signals appeared as early as 4 dpi (Figure 2B), and pathogen development clearly expanded throughout vascular tissue at 7 dpi (Figure 2C).

ROS production was monitored by diaminobenzidine (DAB) staining throughout the plant infection time-course. No clear accumulation of DAB (reddish-brown color visible to the naked-eye) was evident at any stage of the infection analyzed here (data not shown). Furthermore, under light microscopy, DAB staining beneath fungal appressoria and surrounding the penetration pegs was very rarely observed and only after 7 dpi (Figures 3A–C). FaGST1 transcripts (the strawberry AtGST1 orthologous gene) did not increase across the time points examined, neither in crown (Figure 3D) nor in leaves (Figure 3E). A slight increase in the expression of this gene was detected in petiole tissue 3–5 dpi (Figure 3F).

Based on these results, and the observation that spray inoculation with 10⁴ conidia·ml⁻¹ more closely mimics natural infection conditions, the 5 dpi time point was chosen for transcriptome analysis. The assumption was that most of the changes in transcript accumulation in response to early C. acutatum infection would be detected around this time point, including those in response to early stages of switching to necrotrophic growth. In addition, analysis of known defense-related transcripts was performed at 1, 3, 5, 7, and 9 dpi to assess plant response to a wider range of fungal developmental stages and disease progression.

Transcript abundance was measured in crown tissue 5 days after C. acutatum infection and in mock-treated plants. Tables 1A,B and Tables S1, S2 show a summary of identified transcripts with the highest induction or repression levels after crown infection. A total of 147 transcripts varied in accumulation more than 1.75-fold following criteria described under Material and Methods. Of these, 118 genes were induced, and 29 genes were repressed. Verification of gene expression changes by real-time RTqPCR was performed on eleven up-regulated genes and two down-regulated genes, representing different categories shown in Tables 1A,B (marked with #). The expression pattern of analyzed genes after C. acutatum inoculation was consistent with that obtained by microarray analysis.

Automated functional analysis by FunCat and GO terms assignments showed that many of these up- and down-regulated genes described in Tables 1A,B belong to plant defense and stress response-related categories (Figure S1 and Tables S3–S5). However, when no obvious functional role was annotated within the corresponding F. vesca ortholog genes, a thorough search through the references available in the database from many plant species was performed (see Table S3). A wider range of the strawberry transcripts matched defense and biotic stress annotations with the number of up- and down-regulated genes changing 89.93 and 65.51%, respectively.

Transcripts representing five subsets of putative molecular function were identified. These include plant receptors, signal transduction mechanisms under hormonal control (protein modification and degradation), transcriptional changes (transcription factors), new protein synthesis, and secretion of active defense components (PR proteins, degradative enzymes or chemical defenses).

To further investigate how SA- and JA- hormone-dependent pathways are responding to C. acutatum infection, transcript levels of their described signaling pathways were measured by RTqPCR. Transcripts corresponding to JA-associated genes FaWRKY33-1 (M8H3^EST) and FaWRKY33-2 (M1C12^EST) (two orthologs to AtWRKY33), FaAOS-1 (M28A2^EST, AtAOS ortholog), FaLOX2-1 (AtLOX2 ortholog), FaJAR1 (AtJAR1 ortholog), and FaPDF1 (AtPDF1.2 ortholog), and SA-associated transcripts such as FaEDS1 (EDS1-936^EST, AtEDS1 ortholog), FaPAD4 (M4E10^EST, AtPAD4 ortholog), FaGRX1 (M30F8^EST, AtGRX480 ortholog), FaWRKY70-1 and FaWRKY70-2 (M17H1^EST, and M12E12^EST, respectively), FaPR1-1 and FaPR1-2 (AtPR1 ortholog), FaPR2-1 and FaPR2-2 (AtPR2 ortholog), were analyzed in crown tissue in response to C. acutatum inoculation (Figures 5B,C), and after MeJA or SA exogenous applications (Figures 6B,C).

None of known JA inducible pathways, except for gene regulators FaWRKY33-1, FaWRKY33-2, and FaAOS-1 (whose induction was also detected by microarray, see Tables 1A,B) were infection induced (Figure 5B). Similar results were also obtained on petiole tissue analysis after infection (Figure S2B). On the contrary, all transcripts were induced (FaAOS-1, FaWRKY33-1, FaWRKY33-2, FaLOX2-1, FaJAR1, and FaPDF1) in response to MeJA treatment (Figure 6B).

On the other hand all SA-pathway associated orthologs were induced in crown after C. acutatum infection (Figure 5C) except for FaPR1-1, a well-known SA-pathway-associated gene. The same result was found in petiole (Figure S2C). Nearly all strawberry SA-associated transcripts including FaPR1-1, increased in abundance in response to SA treatment. Two classical SA-associated PR orthologous genes, FaPR1-2 and FaPR2-2, are shown to be mainly JA-dependent in strawberry (Figure 6C).

Salicylic acid (SA) and jasmonic acid (JA) content in strawberry plantlets cv. Camarosa was measured at 3 and 5 days after inoculation with C. acutatum. A significant 272% increase in free SA concentration was detected in aerial tissues only at 3 dpi (202.21 ng/g dw, in infected plantlets vs. 74.42 ng/g dw, in mock-treated plantlets; Figure 7A). In addition, free SA in infected plantlets increased up to 678% at 5 dpi compared with that of mock treatment (354.77 ng/g dw vs. 52.33 ng/g dw, respectively). A significant 241% increase in free JA concentration was detected in infected plantlets at 3 dpi compared with that of mock treatment (771.39 ng/g dw, vs. 320.42 ng/g dw, respectively; Figure 7B). This increase was even higher after 5 dpi (425%), compared with that of mock treatment (1707.03 ng/g dw vs. 401.93 ng/g dw, respectively).

This report characterizes the molecular events related to strawberry response to C. acutatum infection largely prior to plant disease symptom occurrence. In other pathosystems, main changes in gene expression are known to occur before significant lesion development (Windram et al., 2012). Previous research indicates that C. acutatum may persist and grow extensively on strawberry tissues without causing visible symptoms (Freeman et al., 1998; Leandro et al., 2001; Debode et al., 2009). Under inoculation conditions (10⁴ conidia·ml⁻¹) used herein, no symptoms were visible during the time course of the experiment. Conditions mirror those used in previously published work (Casado-Díaz et al., 2006; Encinas-Villarejo et al., 2009; Amil-Ruiz et al., 2012, 2013). Transmission electron microscopy results (Figure 2) are roughly consistent with the timing of pathogen development observed after spray inoculation, taking into account that spot inoculation with a higher spore concentration (10⁶ conidia·ml⁻¹) is forcing a much quicker infection process. Indeed, this inoculum concentration is also used by Horowitz et al. (2002) to study the developmental stages of this pathogen in strawberry, and necrotrophic transition was first identified after 4 dpi, which is consistent with our TEM observations. In addition, the penetration phase of C. acutatum on strawberry tissues is not a synchronous process, as previously described for this pathogen and other Colletotrichum spp. on other hosts (Makowski and Mortensen, 1998; Arroyo et al., 2005). Furthermore, at 5 days after inoculation, less than fifty percent of the conidia showed fully formed appresoria, and limited primary hyphae formation was observed (Figure 1). To strengthen the transcriptomic analysis, a more detailed gene expression study (RTqPCR) was performed throughout the C. acutatum infection stages from 1 to 9 days after inoculation, which certainly should reflect changes in host in response to most pathogen development stages. A similar time-window for gene expression has been monitored in other pathosystems (Doehlemann et al., 2008; Marcel et al., 2010; Vargas et al., 2012). Previous studies on C. acutatum-strawberry interaction have also shown that main transcript changes occur in host along the period of time herein analyzed (Casado-Díaz et al., 2006; Encinas-Villarejo et al., 2009). All in all, these observations informed about the appropriate time points for transcriptomic analyses.

Following C. acutatum infection, the transcriptomic analysis shows a wide range of responses to this pathogen. Many of the identified transcripts encode proteins with demonstrated roles in resistance and defense functions. Other transcript classes identified include members of plant pathogen perception and sensing apparatus, signal transduction machinery, transcriptional factors and regulatory genes, and protein synthesis and secretion mechanisms. For the purposes of this report, only components belonging to known SA and JA signaling pathways are discussed.

The SA and JA signaling pathways are activated in strawberry challenged with C. acutatum. However, results indicate that expression of well-known components of both SA- and JA-dependent defense pathways are not activated during this interaction. FaPR1-1 has been described as SA inducible in strawberry in cv. Pájaro, challenged with the avirulent strain M23 of C. fragariae but not after infection with virulent strain M11 of C. acutatum (Grellet-Bournonville et al., 2012). Infection with the avirulent strain M23 induced temporary SA accumulation (nearly 2-fold) in strawberry plants that was accompanied by induction of FaPR1-1 transcripts and protection against a subsequent infection with C. acutatum.

Here the infection of cv. Camarosa with C. acutatum induced the SA accumulation to a higher level (nearly 3-fold), but no significant accumulation of FaPR1-1 transcript above basal levels in crown and petiole was observed (Figure 5C and Figure S2C). In contrast, many other SA-responsive PRs (FaPR1-2, FaPR2-1, and FaPR2-1) were upregulated. The FaPR1-1 transcript did increase in abundance after SA treatment in “Camarosa,” which means that strawberry cv. Camarosa has the ability to induce this gene (Figure 6C).

No significant increase in FaGST1 expression (the strawberry AtGST1 heterolog, a well-known oxidative stress inducible transcript) was observed in crown and leaf tissue (Figures 3D,E) at any time after pathogen inoculation. Only a slight increase was observed in petioles after 5 dpi. These results suggest that little, if any, ROS production occurs during pathogen development. No significant DAB staining was evident in strawberry tissue during this time course, which is consistent with the results. It is known that ROS is generated in response to abiotic stress, particularly H₂O₂, which is an active signaling molecule triggering a variety of defense responses, with GST induction being the most significant (Bhattacharjee, 2012).

Production of H₂O₂ and activation of gene PR-1 gene occur simultaneously, and it has been suggested that H₂O₂ acts downstream from SA in the pathogenesis-related (PR-1) gene induction [73]. Intriguingly, no ROS production and no significant FaGST and FaPR1.1 transcript induction were detected in strawberry after pathogen inoculation, even though SA increased. Our results strongly correlate with those of Grellet-Bournonville et al. (2012), who also reported that virulent C. acutatum strain M11 showed no ROS accumulation and no FaPR1.1 gene induction after strawberry infection.

On the other hand, expression of strawberry orthologs to JA-associated defense related genes, such as FaPDF1, FaLOX2-1, and FaJAR1, also remained unchanged after infection with C. acutatum, despite the fact that many other components of the JA-mediated signaling pathway were induced (FaAOS-1, FaWRKY33-1, FaWRKY33-2; Figure 5B and Figure S2B).

Our results indicate that a number of known components of both SA- and JA-dependent defense pathways are not activated in strawberry during interaction with C. acutatum. Furthermore, our results support a hypothesis that C. acutatum might be interfering with certain branches of both SA- and JA-dependent defense pathways in strawberry. Absence of a significant plant defense response would allow successful colonization of host tissue by this pathogen. In this sense, recent results reported on the tomato-Botrytis system have shown that the exopolysaccharide production by this pathogen [EPS, known as b-(1,3)(1,6)-D-glucan] acted as elicitor of the tomato SA biosynthesis pathway and that inappropriate induction of SA by this pathogen impaired tomato JA-dependent defenses by interrupting the JA signaling pathway downstream of JA production (El Oirdi et al., 2011; Rahman et al., 2012). Consequently, the fungus could gradually spread through tomato plant tissues.

It is tempting to speculate that C. acutatum may be able to interact with strawberry defense response. Based on our results, and considering the canonical SA JA crosstalk model proposed in Arabidopsis (Spoel et al., 2007; Spoel and Dong, 2012), an integrated model has been devised (Figure 8). In this model, in which activation of both SA and JA pathways and increased amount of SA and JA signal molecules occurs after C. acutatum infection, negative crosstalk between these two pathways should somehow be expected. Spoel et al. (2007) showed that simultaneous A. thaliana inoculation with a biotrophic and a necrotrophic pathogen resulted in impaired resistance to the necrotrophic pathogen, and demonstrated that the SA pathway that was activated by the biotrophus suppressed the level of JA-dependent resistance against the necrotroph. Indeed, SA-mediated suppression of JA-responsive gene expression has been reported to be targeted downstream of JA biosynthesis (Leon-Reyes et al., 2010). Thus, GRX480 is an NPR1 dependent-SA-inducible class III glutaredoxin (Rouhier et al., 2006; Krinke et al., 2007) specific to land plants (Ziemann et al., 2009), which interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription (Ndamukong et al., 2007; Zander et al., 2011). In addition, NPR1 and WRKY70 also acts downstream of the SA molecule as a node of convergence for JA-mediated and SA-mediated signals (Dong, 2004; Li et al., 2004; Wang et al., 2006; Ren et al., 2008), balancing the JA- and SA-dependent responses (Li et al., 2006). Interestingly, strawberry orthologs FaGRX1, FaWRKY70-1 and FaWRKY70-2 were specifically induced during interaction with C. acutatum, but no induction on FaPDF1 and other major components of JA-dependent signaling pathway, such as genes FaLOX2-1 and FaJAR1, was detected. Moreover, an increase in JA synthesis and upregulation of FaAOS-1, the strawberry ortholog to Arabidopsis AtAOS, a well-known JA-associated marker gene encoding a key enzyme for JA synthesis, was also reported after C. acutatum infection, supporting the idea that in strawberry repression of some JA-responsive genes is targeted downstream of the JA biosynthesis. Indeed, AtAOS has been described as a MeJA-inducible gene but not suppressed by WRKY70 (Li et al., 2006). Very interestingly, the expression of a second group of known JA-responsive genes, such as FaGLN-2, FaCHI4-2, FaPR10-4, FaPR5-1, FaPR5-2, FaPR5-3, increased after being challenged with this pathogen, suggesting the presence in strawberry of a second GRX480/WRKY70-independent JA-dependent defense branch.

Importantly, two other JA-associated AtWRKY33-like genes, FaWRKY33-1 and FaWRKY33-2, were also upregulated in strawberry by C. acutatum. The JA-associated component AtWRKY33 has recently been reported as a key transcriptional regulator of defense responses to necrotrophus (Birkenbihl et al., 2012). Indeed, AtWRKY33 acts as a negative regulator of the SA-defense pathway upon pathogen infection and negatively controls expression of many important genes, including those responsible for SA biosynthesis and accumulation, positive regulatory proteins EDS1 and PAD4, and SA responsive genes PR1, PR2, and PR3. Interestingly, in strawberry, expression of the SA-dependent orthologous genes FaGST and FaPR1-1 remained unaltered but very intriguingly, the synthesis of SA and the expression of orthologs to components of SA-mediated signaling pathway acting upstream (FaEDS1 and FaPAD4), and downstream of SA (FaGRX1, FaWRKY70-1, FaWRKY70-2, FaPR1-2, FaPR2-1, and FaPR2-2), was remarkably induced during the infection with C. acutatum, despite the fact that FaWRKY33-1 and FaWRKY33-2 were clearly upregulated. Unlike what has been previously described for AtWRKY33 (Birkenbihl et al., 2012), these results suggest that repressive control of many known components of the SA-pathway through these FaWRKY33 transcription factors does not work in strawberry during interaction with C. acutatum (Figure 8).

In conclusion, our results demonstrate both that SA and JA increase in strawberry upon C. acutatum infection, and that known plant defenses through SA and JA dependent signaling pathways are partially promoted during such interaction. Indeed, major plant defense marker genes are not up-regulated in strawberry after infection with this pathogen, which might evidence a putative molecular strategy used by this pathogen to overcome strawberry plant defense. This is in line with the new emerging paradigm that a key pathogen virulence strategy involves modulation of plant hormone signaling (El Oirdi et al., 2011; Chung et al., 2013).

Results from our research are of great use to further our understanding of the strawberry immune system to enable future disease control through biotechnological and breeding strategies.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.