Identification of a strawberry NPR-like gene involved in negative regulation of the salicylic acid-mediated defense pathway

Hormonal modulation plays a central role in triggering various resistant responses to biotic and abiotic stresses in plants. In cultivated strawberry (Fragaria x ananassa), the salicylic acid (SA)-dependent defense pathway has been associated with resistance to Colletotrichum spp. and the other pathogens. To better understand the SA-mediated defense mechanisms in strawberry, we analyzed two strawberry cultivars treated with SA for their resistance to anthracnose and gene expression profiles at 6, 12, 24, and 48 hr post-treatment. Strawberry genes related to SA biosynthesis, perception, and signaling were identified from SA-responsive transcriptomes of the two cultivars, and the induction of 17 candidate genes upon SA treatment was confirmed by qRT-PCR. Given the pivotal role of the non-expressor of pathogenesis-related (NPR) family in controlling the SA-mediated defense signaling pathway, we then analyzed NPR orthologous genes in strawberry. From the expression profile, FaNPRL-1 [ortholog of FvNPRL-1 (gene20070 in F. vesca)] was identified as an NPR-like gene significantly induced after SA treatment in both cultivars. With a conserved BTB/POZ domain, ankyrin repeat domain, and nuclear localization signal, FvNPRL-1 was found phylogenetically closer to NPR3/NPR4 than NPR1 in Arabidopsis. Ectopic expression of FvNPRL-1 in the Arabidopsis thaliana wild type suppressed the SA-mediated PR1 expression and the resistance to Pseudomonas syringae pv. tomato DC3000. Transient expression of FvNPRL-1 fused with green fluorescent protein in Arabidopsis protoplasts showed that SA affected nuclear translocation of FvNPRL-1. FvNPRL-1 likely functions similar to Arabidopsis NPR3/NPR4 as a negative regulator of the SA-mediated defense.


Introduction
Cultivated strawberry (Fragaria x ananassa) is one of the most economically important Rosaceae fruit crops. Genetically, F. x ananassa is a complex allo-octoploid plant derived from two diploid species, F. virginiana and F. chiloensis. The genome of the diploid woodland strawberry F. vesca (~240 Mb) was released in 2011, providing a good resource for studying Fragaria spp. SA-mediated defense responses. The functions of NPR1 orthologues in many crops [20] and potential roles of NPR3 orthologues in cacao and NPR3/NPR4 in citrus have been uncovered [34,35], but little is known about FvNPRLs. In the diploid strawberry genome sequence database [1], five FvNPRL genes were found, but none have been further characterized. We only know that overexpression of AtNPR1 in cultivated strawberry increased its resistance to anthracnose, powdery mildew, and angular leaf spot [36]. In this study, phylogenetic analysis and functional characterization revealed the role of FvNPRL-1 (gene20070). The results suggest that similar to NPR3/ NPR4, FvNPRL-1 may translocate into the nucleus and function as a negative regulator of the defense responses against biotic stresses.

SA treatment
Strawberry seedlings at the 4-5 leaf stage were sprayed with 5 mM SA or sterile ddH 2 O by airbrush (4 ml per plant, 10-15 psi) and kept in an incubator for 2 days under >90% relative humidity (RH) at 24˚C and 16-hr photoperiod. For Arabidopsis, 12-day-old seedlings were transferred to MS medium containing 200 μM SA and incubated for 24 hr under a 16-hr photoperiod at 22˚C. Seedlings transferred to MS medium without SA were used as control plants.

Evaluation of anthracnose resistance
Resistance to anthracnose was evaluated with a C. gloeosporioides isolate GL001 collected from an infected strawberry leaf from Miao-Li, Taiwan, in 2011. The inoculum was cultured on 1/4 potato dextrose agar (PDA) under a 12-hr photoperiod at room temperature for 7-10 days. Inoculation was conducted 72 hr after SA or ddH 2 O treatment by spraying the entire strawberry plants with 2-3 ml conidial suspension per plant (1 x 10 6 spores/ml in 0.02% Tween 20) with use of an airbrush at 10-15 psi. Sterilized ddH 2 O containing 0.02% Tween 20 was used as the control. The inoculated plants were kept in plastic boxes for 24 hr at >90% RH and 25˚C, then maintained in a growth chamber at 25˚C, 60% RH under an 8-hr photoperiod. Disease severity was evaluated at 3, 4, and 5 days post-inoculation (dpi). Individual plants were scored by using a self-defined disease severity index (DSI) with a 1-10 rating scale (S1 Table). The experiment was performed in three independent trials, each containing 3-4 plants per cultivar per treatment. Phenotypic differences were analyzed by Tukey's Honestly Significant Difference (HSD) test at p < 0.05 with SAS/STAT v9.3 (SAS Institute Inc., Cary, NC, USA).

RNA extraction and quantitative real-time RT-PCR
Strawberry leaf samples and Arabidopsis leaf/seedling samples were collected, snap-frozen in liquid nitrogen, and ground into fine powder in liquid nitrogen. Total RNA was extracted from strawberry and Arabidopsis by using the cetyltrimethylammonium bromide (CTAB)based extraction method [37] and TRIzol Reagent (Life Technologies), respectively. To eliminate DNA contamination, strawberry and Arabidopsis RNA samples were treated with a TURBO DNase Kit (Ambion) following the manufacturer's instructions. The concentration of each RNA sample was measured by using the Nanodrop Spectrophotometer ND-100, then cDNA was synthesized by using the MMLV Reverse Transcriptase cDNA Synthesis Kit (Epicentre) following the manufacturer's instructions.
Each strawberry or Arabidopsis experiment involved 2-3 independent trials, with 3-4 strawberry plants or 3 Arabidopsis plants per treatment per trial. Relative gene expression was measured in three technical replicates with the StepOnePlus Real-Time PCR System (Applied Biosystems). Each qRT-PCR reaction contained 10 μl Fast SYBR Green Master Mix, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 0.5 μl cDNA, and 7.5 μl ddH 2 O. The primers listed in S2 Table and S3 Table for qRT-PCR were designed to amplify 100-to 150-bp fragments of targeted genes. qRT-PCR was performed under thermal cycling parameters of 95˚C for 20 sec followed by 40 cycles of 3 sec at 95 o C and 30 sec at 60˚C. The strawberry housekeeping gene FaActin and Arabidopsis AtActin2 were internal controls. The cycle threshold (C T ) values for each gene in different samples were normalized to C T values of the internal control gene in a given sample. The relative gene expression in different samples was calculated by the ΔΔC T method [38].

Transcriptome analysis
Equal amounts of total RNA from 6, 12, 24, and 48 hr post-SA or ddH 2 O treatment were pooled for strand-specific RNA sequencing (2 x 101 bp paired-end reads) on the Illumina HiSeq 2000 at Yourgene Bioscience (Taipei, Taiwan). At each time point, RNA was isolated from the first fully expanded leaves collected from 3-4 strawberry seedlings. RNA-seq data have been deposited in the NCBI Short Read Archive database under the accession numbers SRX4076953 (SJ-SA treatment), SRX4076954 (SJ-Control), SRX4076955 (TY3-SA treatment), and SRX4076956 (TY3-Control). The trimmed and filtered reads (error probability < 0.01, Q20; read length > 35 bp) from the same cultivar were first de novo assembled by the Trinity method (version r20121005) [39], then mapped to the diploid woodland strawberry database Fragaria vesca Whole Genome v1.0 (build 8) [1,40,41] by using Bowtie 2 [42]. Differential expression analysis was conducted with the R package DESeq [43]. The unigene sequences were annotated by a BLAST search of the Fragaria vesca Whole Genome v1.0 (build 8) database with E-value cutoff of 10 −5 . Confidence results of differential expression analysis were determined on the basis of FPKM values and contigs with at least 30 independent read counts from the same cultivar (from previous studies of Fragaria RNA-seq [37]). Less stringent criteria (fold change � 1.2 and � 0.8) were adopted for gene identification to gain an overview of the entire defense pathways.

3' rapid amplification of cDNA ends (3' RACE) of strawberry FvNPRL-3
The RNA of diploid strawberry was isolated and treated with DNase. Reverse transcription was conducted as described above, but the oligo-(dT) 20 primer was substituted by the 3'RACE adaptor [oligo-(dT) 20 anchored with a specific adaptor] (S3 Table). The cDNA template was used in nested-PCR for 3' exon and 3' UTR amplification. The forward primer targeting the 2 nd exon of FvNPRL-3 and the 3' RACE outer primer were used for the first PCR, then the forward primer targeting the 4 th exon of FvNPRL-3 and the 3' RACE inner primer were used for the second PCR. The~500-bp PCR product was ligated into the T&A Cloning Vector (Yeastern Biotech, Taiwan) and sequenced by using M13-F and M13-R primers at the Center for Biotechnology, National Taiwan University.

Construction of the binary vector carrying FvNPRL-1 for Arabidopsis transformation
A DNA fragment of FvNPRL-1 was amplified from F. vesca var. vesca with the primers FvNPRL-1-F and FvNPRL-1-R carrying NcoI and PmacI sites (S3 Table) by Phusion High-Fidelity DNA Polymerase (Thermo Scientific). The PCR product was first ligated into the T&A Cloning Vector (Yeastern Biotech, Taiwan), resulting in the plasmid pyT&A-FvNPRL-1. FvNPRL-1 was released from pyT&A-FvNPRL-1 by NcoI-PmacI partial digestion, then ligated into NcoI/PmacI-treated pCAMBIA1301. The resulting pCAMBIA1301-FvNPRL-1 was transformed into Agrobacterium tumefaciens strain GV3101. The floral dip method [45] was used to transform Arabidopsis thaliana Col-0 wild type, and the transformed seeds were selected on MS medium containing 50 μg/μl hygromycin B. Each transformed line was re-selected by hygromycin B for two generations to obtain homozygous seedlings. The T 2 lines with all tested T 3 progeny (at least 90 seeds) surviving on hygromycin B-containing plates were considered homozygous.

Pseudomonas inoculation on Arabidopsis
Pseudomonas syringae pv. tomato (Pst) strain DC3000 was grown in King's B medium (KBM) containing 100 μg/ml rifampicin at 28˚C. Cells of Pst DC3000 from overnight cultures were collected, washed with sterilized 10 mM MgCl 2 and adjusted to O.D.600 = 0.05 in 10 mM MgCl 2 supplemented with 0.02% Silwet L-77. Arabidopsis plants at 25 days old were sprayinoculated with the bacterial suspension until water ran off and homogeneously distributed on the leaf surface. At days 0 and 3 after inoculation, the first fully expanded leaf from each plant was weighed, then homogenized in 1 ml cold 10 mM MgCl 2 . Serial dilutions were made and 10 μL of each dilution was spotted on the KBM plates containing 100 μg/ml rifampicin. After growing at 28˚C for 30 hr, colony-forming units (CFU) per milligram fresh leaf tissue were calculated [46]. Each transgenic line contained 6 plants, and the inoculation assay was repeated three times.
The isolation and transfection of Arabidopsis protoplasts were performed following the polyethylene glycol method [47,48]. After transfection with p2GWF7-FvNPRL-1, protoplasts were treated with 0, 1, 10, and 40 μM SA and harvested after incubation for 20 hr. The nucleus localization marker VirD2-NLS-mCherry was used [49]. Transfected protoplasts were examined by confocal microscopy (Zeiss LSM 510 META NLO DuoScan). GFP and mCherry in the protoplasts were excited by 488 and 543 nm laser beans and the detection spectrum ranged from 500 to 587 and 600 to 630 nm, respectively. This transient expression experiment was repeated three times.

SA-induced resistance to anthracnose in cultivated strawberry
To ensure that SA-mediated defense mechanisms exist in the two selected cultivars TY3 and SJ, strawberry plants treated with 5 mM SA or ddH 2 O (control) were inoculated with C. gloeosporioides and evaluated for disease severity. At 2 dpi, disease symptoms could be observed on the leaves of ddH 2 O-treated but not SA-treated strawberry plants. Both SJ and TY3 plants treated with SA showed delayed development of anthracnose symptoms as compared with mock-inoculated plants, and SJ plants exhibited higher resistance to anthracnose than TY3 plants (Fig 1).
Three previously characterized strawberry genes, FaPR5 (FaOLP2), FaWRKY1, and FaPR1 [11,50,51], were used as markers for detecting the activation of the SA-mediated defense pathway. In response to SA, the expression of the three genes was enhanced within 5 days [11,50,51]. Time-course gene expression was analyzed for the RNA samples extracted from TY3 or SJ plants on days 1, 3, and 5 after SA or ddH 2 O treatment. All three genes were triggered in both cultivars under 5 mM SA treatment, although the induction levels and expression patterns differed between the two cultivars (Fig 2). Of note, the induction of the three marker genes was faster and higher in the more resistant cultivar SJ than in TY3. The expression patterns also indicated that the SA-mediated defense pathway could be activated within 24 hr after SA treatment.

Identification of defense-related genes differentially expressed upon SA treatment
To obtain a snapshot of the SA-responsive gene network in strawberry, we used the RNA pooled from 6, 12, 24, and 48 hr post-SA treatment for transcriptome analysis. About 20,000 unigenes were annotated from the transcriptomes of strawberry TY3 and SJ after SA/ddH 2 O treatment. The differentially expressed genes with predefined fold change � 1.2 or � 0.8 in the SA network are listed in S4-S10 Tables, and a set of 17 genes was selected from the transcriptome data and quantified by qRT-PCR (Fig 3 shows the results from one of two independent trials with a similar trend). These genes were chosen by their known functions in the SA-mediated defense pathway and their significant induction in response to SA treatment. They included four in the SA biosynthesis pathway [shikimate dehydrogenase (FaSD), shikimate kinase (FaSK), chorismate mutase (FaCM), and flavanone 3-dioxygenase (FaF3D)]; eight functioning in SA perception and signaling pathways (FaNPRL-1, FaTGA6, FaGrxC9, FaWRKY51, FaWRKY70, FaWRKY1, FaPR1, and FaPR5); the NBS-LRR gene resistance gene analog 1 (FaRGA1); two receptor-like genes [interleukin-7 receptor (FaIL7R) and brassinosteroid insensitive 1-associated receptor kinase (FaBIRK1)]; and two substantially induced genes [patatin T-5 (FaPT5) and hyoscyamine 6-dioxygenase (FaH6DO)]. All 17 selected genes were confirmed to be SA-inducible, with most induced to relatively higher levels in the more resistant SJ than in TY3. The induction was detected as early as 6 hr after SA treatment, then decreased rapidly in both cultivars. In particular, the expression of FaSK, FaCM, FaNPRL-1, FaTGA6, and FaRGA1 was induced at 6 and 12 hr, then returned to the basal level at 24 or 48 hr after SA treatment. Only FaWRKY70 and FaH6DO remained activated in both cultivars until 48 hr after SA treatment.

Structure features and phylogenetic analysis of strawberry NPR-like protein sequences
NPR genes are major components of the SA signal transduction pathway [20]. In some plants, such as Arabidopsis and grape, the expression of NPR genes can be activated by SA, SA analogs, and certain pathogens within a day [26,52]. By interacting with TGA transcription factors, NPR1 and NPR3/NPR4 positively or negatively regulate the downstream defense-related genes (e.g., PR genes), which is essential for balancing the complex immune responses in plant cells [30,53]. Among the five FvNPRL genes annotated in the diploid strawberry genome, the orthologs of FvNPRL-1, FvNPRL-2, FvNPRL-3 and FvNPRL-5 were identified in our transcriptome data, with FvNPRL-1 ortholog as the only NPRL exhibiting significant induction after SA  Table). The FvNPRL-4 ortholog was not detected in TY3 and SJ; this gene also showed infrequent and low expression in F. vesca [54]. The FvNPRL-3 ortholog was annotated as a protein consisting of 591 amino acids (similar to the predicted lengths of other FvNPRLs) in our library but annotated as 697 amino acids in the diploid strawberry genome database. Thus, 3' RACE was conducted to clarify the 3'-end sequences of FvNPRL-3. FvNPRL-3 actually coded for a protein with 591 amino acids (S1 Fig).

Ectopic expression of FvNPRL-1 in A. thaliana Col-0 wild type suppressed SA-mediated PR1 gene expression and resistance to P. syringae pv. tomato DC3000
The characteristics of early induction after SA treatment (Fig 3 and S5 Table) and the conserved NPR domains suggested that FvNPRL-1 may be a crucial regulatory component of SAdependent defense in strawberry. To investigate whether FvNPRL-1 is functionally similar to AtNPR1, AtNPR3 or AtNPR4, transgenic lines were generated to overexpress FvNPRL-1 driven by a 35S promoter in Arabidopsis wild type plants. FvNPRL-1 transcripts were accumulated in leaves of homozygous T 2 plants of wild-type/35S-FvNPRL-1 lines 1, 2, and 3 (WT-FvNPRL-1-ox-1, 2, 3) (Fig 5A). To assess whether FvNPRL-1 participates in the SA-mediated defense pathway, the downstream marker gene AtPR1 was first quantified in the wild type and three FvNPRL-1 overexpression lines after 24 hr of SA treatment. The expression of AtPR1 was significantly lower in the transgenic lines than the wild type (Fig 5B), which indicates that AtPR1 was negatively regulated by FvNPRL-1.

Subcellular localization of FvNPRL-1
To investigate the subcellular localization of FvNPRL-1, GFP was fused to the C-terminus of FvNPRL-1 (FvNPRL-1-GFP) for transient expression in protoplasts of Arabidopsis. Both FvNPRL-1-GFP and free GFP were observed in the cytoplasm and nucleus (Fig 7). In the normal condition (no SA),~21% of the cells transformed with FvNPRL-1-GFP showed a GFP signal in the nucleus. The incidence of GFP signal in the nucleus was reduced to approximately 13%, 10%, and 8% after treatment with 1, 10, and 40 μM SA for 20 hr, respectively (Table 1 and S2 Fig). In contrast, the incidence of GFP signal in the nucleus remained at 58% to 63% with different SA concentrations in our positive control (free GFP) (Table 1 and S3 Fig). Thus, in Arabidopsis cells, nuclear translocation of FvNPRL-1 was negatively affected by SA.

Discussion
Much effort has been put into the elucidation of the SA-mediated defense network in model plants, but the hormonal modulation of immunity responses in non-model crops remains mostly unknown. In strawberry, previous defense-related studies mostly focused on diploid strawberry [57,58] or the genes and mechanisms in Colletotrichum-or Botrytis-inoculated plants of octoploid strawberry [12,57,[59][60][61]. In this study, we hypothesized that exogenously NPR-like genes in strawberry applied SA could activate strawberry defenses against anthracnose and possibly other diseases in strawberry leaves, and vital SA-mediated defense regulators could be explored in this process [11]. We directly sprayed two strawberry cultivars with SA, checked the induction of anthracnose resistance and defense marker genes, and analyzed the transcriptomes. Profiling of the SA-induced gene expression in cultivated strawberry has previously involved analysis of 1847 expressed sequence tags from whole-plant vegetative tissues at 24 hr after SA treatment [62] and by low-coverage Roche-454 sequencing (2G per cultivar) of pooled RNA samples from diverse tissues under various conditions [37]. Despite the lack of enough replicates in our transcriptome analysis, the results provide a preliminary overview of the defense network and allowed for identifying defense-related genes that are worthy of further investigation.
The SA-mediated defense mechanisms in strawberry may be similar to those in the model plant Arabidopsis. In our transcriptome data, we discovered orthologs of a variety of genes known to play critical roles in defense mechanisms as well as in SA biosynthesis, perception, and signaling in Arabidopsis and other plants [63][64][65][66][67]. Time-course qRT-PCR analysis of 17 selected genes validated the induced expression of 5 previously investigated marker genes (FaGrxC9, FaWRKY70, FaWRKY1, FaPR1, and FaPR5) [11,12,50,51] in TY3 and SJ cultivars. Amil-Ruiz et al. (2016) found FaGrxC9 (FaGRX1), FaWRKY70, FaWRKY1, FaPR1, and FaPR5 induced at an early time point, although at a smaller magnitude, in 14-week-old plants of a strawberry cultivar Camarosa sprayed with 5 mM SA; the five genes showed increased expression at 3 days after C. acutatum inoculation. The more enhanced transient induction detected in our study may be due to different cultivars and younger plantlets (4-5 leaf stage). For the first time, the remaining 12 genes were found associated with SA-mediated defense in strawberry. These genes were induced earlier and with greater expression in the moderately resistant cultivar SJ than in TY3. This result corresponded to the study of Casado-Diaz et al. [59], in which a relatively more resistant cultivar Andana exhibited a quicker and stronger induction of defense-related genes in response to Colletotrichum infection.
Among the 17 differentially expressed genes, FaSD, FaSK, FaCM, and FaF3D are involved in early steps of the shikimate-phenylpropanoid pathway, from which SA and phenolic  NPR-like genes in strawberry secondary metabolites are derived. Application of benzothiadiazole (BTH; chemical analog of SA) on strawberry leaves induced the accumulation of phenolics and resistance to powdery mildew [68], which supports that the shikimate-phenylpropanoid pathway is SA-responsive and defense-related in strawberry. Pattern recognition receptors (PRRs) and resistance (R) proteins recognize the elicitors or effectors of microorganisms and trigger various downstream immune responses. Treating strawberry plants with SA alone was able to activate FaRGA1, FaIL7R, and FaBIRK1, which implies a feedback control mechanism in the pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) systems of strawberry. In Arabidopsis, positive SA regulation of certain NBS-LRR transcripts, such as RPP1 and RPS4, has been reported [69]. Among the 133 genes highly induced (fold change � 5 in the transcriptome data) in response to exogenous SA in either or both of the strawberry cultivars (S10 Table), FaH6DO (gene01857 ortholog) and FaPT5 (gene09059 ortholog) were confirmed by qRT-PCR. H6DO was found involved in alkaloid biosynthesis in a study of transgenic sugarcane lines [70], and PT5 was reported as a potato storage protein [71]. These two genes may be new components in SA-mediated plant immunity. FaNPRL-1 (FvNPRL-1 ortholog) was identified as an NPR-like gene significantly induced after SA treatment in the strawberry cultivars TY3 and SJ. The transcript level of FaNPRL-1 peaked at 6 hr, remained activated at 12 hr, and returned to an inactive state at 24 and 48 hr after SA treatment. NPR genes were highly induced shortly after exogenous SA treatment in Arabidopsis and other plant species. For instance, the AtNPR3/AtNPR4 transcripts in Arabidopsis remained at a high level from 30 min to 48 hr after treatment with SA and SA analogs such as INA and BTB [26,72]. TcNPR1 in cacao was dose-dependently induced at 24 hr after SA treatment [73], GhNPR1 expression in cotton was greatly increased at 8 hr after SA treatment [74], and MaNPR1-B in banana was highly induced at 12 hr after SA treatment [75]. Although FaNPRL-2, FaNPRL-3, and FaNPRL-5 seemed not significantly induced in our transcriptome data, additional experiments are required to clarify their time-course expression patterns under SA regulation.
FvNPRL-1 was phylogenetically closer and functionally more similar to AtNPR3/AtNPR4 than AtNPR1. In this study, the potential role of FvNPRL-1 was characterized by ectopic expression in wild-type Arabidopsis. Such a strategy has been used to validate the similarity between AtNPR1 and its orthologous genes in cacao [73], soybean [76] and grapevine [52]. Overexpression of FvNPRL-1 decreased the level of AtPR1 transcripts and increased leaf susceptibility to Pst DC3000. The level of increased susceptibility observed in this study was relatively minor, which may be due to different inoculation methods and the effectiveness of FvNPRL-1 in the ectopic system. Similar disease reactions were observed when AtNPR3 was overexpressed in wild-type Arabidopsis [77]. The overexpressed AtNPR3 and AtNPR4, coupled with TGA transcription factors, repressed the expression of WRKY70, which acts as an inducer of SA-activated genes and a repressor of JA-mediated defense [30]. As well, overexpression of FvNPRL-1 in the Arabidopsis npr1 mutant did not recover its resistance to Pst DC3000 (data not shown), so FvNPRL-1 could not complement the function of AtNPR1. Taken together, similar to AtNPR3 or AtNPR4, FvNPRL-1 likely functions as a negative regulator of the SA-mediated defense. The rapid induction of FvNPRL-1 after SA treatment implied its role as a repressor to quickly balance the effect caused by excessive SA and to fine-tune the overall defense responses.
With the C-terminal NLS, FvNPRL-1 showed the ability to translocate into the nuclei of Arabidopsis protoplasts. Similarly, AtNPR3 was found to interact with TGA2 in the nuclei of onion epidermal cells [77]. The translocation and accumulation of FvNPRL-1 seemed affected by SA concentration. Previous studies showed that SA directly interacted with AtNPR1, AtNPR3 and AtNPR4 in the nucleus and affected the resistance of Arabidopsis to pathogens [28,31,77]. Ding et al. also reported that SA directly suppressed the functions of AtNPR3 and AtNPR4 [30]. Although how an increase in SA suppresses the nuclear localization of FvNPRL-1 remains to be resolved, lines of evidence from previous studies suggest that plants can utilize SA to modulate the functions of NPR proteins or other regulators involved in the SA-mediated defense pathway. We need to further investigate the biological functions of FvNPRL-1 and other FvNPRLs during strawberry-pathogen interactions.