Molecular cloning and functional identification of an apple flagellin receptor MdFLS2 gene

QI Chen-hui, ZHAO Xian-yan, JIANG Han, LIU Hai-tao, WANG Yong-xu, HU Da-gang, HAO Yu-jin

1 National Key Laboratory of Crop Biology/Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, Ministry of Agriculture and Rural Affairs of China/College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, P.R.China

2 Shandong Yihui Detection Technology Co., Ltd., Tai’an 271000, P.R.China

Abstract The leucine-rich repeat receptor kinase flagellin-sensing 2 gene (MdFLS2; Gene ID: MDP0000254112) was cloned from Royal Gala apple (Malus×domestica Borkh.). This gene contained a complete open reading frame of 3 474 bp that encoded 1 158 amino acids. The phylogenetic tree indicated that Prunus persica FLS2 exhibited the highest sequence similarity to MdFLS2. The PlantCare database suggests that the promoter sequence of MdFLS2 contains several typical cis-acting elements, including ethylene-, gibberellin-, salicylic acid-, and drought-responsive elements. Quantitative real-time PCR analysis showed that MdFLS2 was widely expressed in the different tissues of the apple and most highly expressed in the leaves. Furthermore, MdFLS2 was signiflcantly induced by the flagellin elicitor peptide flg22. Treatment of the apple seedling leaves with flg22 resulted in an increase in leaf callose levels with increased treatment duration. An increase in the production of O2– along with the expression of disease-related genes was also observed. An oxidative burst was detected in the treated seedlings, but not in the control seedlings, indicating that flg22 had stimulated the expression of the MdFLS2 gene and its downstream target genes. Furthermore, the ectopic expression of MdFLS2 complemented the function of the Arabidopsis fls2 mutant and conferred enhanced flg22 tolerance to the transgenic Arabidopsis, suggesting that MdFLS2 acts as a positive regulator in the response to pathogens in apple.

Keywords: apple, flagellin receptor, flagellin elicitor peptide, MdFLS2, pathogen infection

1. Introduction

Plant-type pattern-recognition receptors (PRRs) play an important role in sensing pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) and defending against pathogen infection (Boller and Felix 2009; Takeuchi and Akira 2010). Common MAMPs contain flagellins and thermolabile elongation factors (Felixet al. 1999; Kunzeet al. 2004; Ottmannet al. 2009; Thommaet al. 2011; Dou and Zhou 2012; Winet al. 2012). Other MAMPs include lipopolysaccharides, peptidoglycans, and chitins, which constitute the major components of the fungal cell wall(Meyeret al. 2001; Gustet al. 2007; Miyaet al. 2007). In general, antigens, which contain such molecular patterns,provide ligands for receptors located on the plasma membrane. Different receptors can regulate different pattern recognition mechanisms; the regulation patterns of which may differ depending on the extracellular ligand binding region, cell mosaic, and intracellular plasma membrane regions. Recent immunochemistry studies have shown that the formation of ligand-induced immune receptor complexes is a common molecular model and cytoplasmic signal transduction system (Monaghan and Zipfel 2012).

Biometric phenomena involve ligands and receptors.For example, pathogen recognition by plants involves interactions between plant receptors and microbial ligands,called stimulators, which can stimulate phosphorylation in plants to trigger defense responses (Zipfel and Oldroyd 2017). Studies show that flagellin-sensing 2 (FLS2), a leucine-rich protein kinase, is a pattern-recognition receptor for bacterial flagellins localized in the plasma membrane inArabidopsis(Gómez-Gómez and Boller 2000). InArabidopsis, flagellins are the most active bacterial elicitors(Felixet al. 1999) and can trigger rapid defense mechanisms and activate the phosphorylation of receptors (Gómez-Gómezet al. 1999).

In 2000, Gómez-Gómez and Boller (2000) identifled the flagellin-sensing geneAtFLS2inArabidopsisusing sitedirected cloning.AtFLS2encodes a receptor kinase that is extracellularly rich in 28 leucine regions and contains tryptophan.AtFLS2is homologous to the rice resistance geneOsXa21, which enhances resistance to bacterial leaf blight (Liuet al. 2016). In addition, the leucine-rich repeats(LRR) region of theAtFLS2gene is similar to theCfgene family, which is resistant to tomato leaf mold (Zhaoet al.2016), suggesting that the recognition pathway of flagellins and other excitons is closely associated with the speciflc resistance that is determined by the interaction between genes.

AtFLS2binds to bacterial flagellins, and this binding is facilitated by an antigen that consists of 22 conserved amino acids (Chinchillaet al. 2006). Although speciflc ligand recognition mechanisms are extensively used, research on FLS2 in plants has progressed greatly in recent years(Monaghanet al. 2012). Flg22 is a 22-amino-acid peptide,and is analogue of flagellin. TheArabidopsis AtFLS2can recognize the flg22 ofPseudomonas cepacia, which promotes plant growth, whereas the grapeVpFLS2cannot recognize flg22 (Trdáet al. 2013). ManyPseudomonas syringaestrains produce a mutated PAMP region flgII-28 that can be recognized by solanaceous plants (Caiet al.2011; Clarkeet al. 2013). The recognition of flgII-28 is restricted to a number of solanaceous species. Although the flgII-28 peptide does not trigger any immune response inArabidopsis, mutations in bothflg22andflgII-28have FLS2-dependent effects on virulence. However, the expression of a tomato allele ofFLS2does not confer toNicotiana benthamianathe ability to detect flgII-28,and tomato plants silenced for FLS2 are not altered in flgII-28 recognition (Clarkeet al. 2013). In addition, whenAtFLS2binds to flagellin ligands,AtFLS2can rapidly form heterodimers with other transmembrane receptor kinases,such as leucine-rich repeat receptor kinase (LRR-RK) and BRI1-associated receptor kinase 1/somatic embryogenesis receptor kinase (BAK1/SERK) (B?hmet al. 2014).AtFLS2is a co-receptor of diverse classes of LRR-RKs, including brassinosteroid receptor 1 (BRI1) (Chinchillaet al. 2009).Additionally, Albertet al. (2013) show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling.

The response ofFLS2to biotic stress in various plants has been preliminarily studied, but the function of theFLS2gene in apple has not yet been reported. Using Royal Gala apple as a test material,MdFLS2was obtained by cloning,and its expression in different tissues and under flg22 treatment was studied using quantitative real-time (qRT)-PCR.Arabidopsis thalianawas genetically transformed and subjected to biotic stress in order to identify the function of MdFLS2 and assess its regulatory mechanisms, with the aim of providing a theoretical basis for apple disease resistance.

2. Materials and methods

2.1. Materials and flg22-treated Arabidopsis seedlings

From May 2016, 8-year-old Royal Gala apple (Malus×domesticaBorkh.) seedling tree materials were obtained from the fruit trees planted in experimental fleld of the Shandong Pomology Research Institute (Taian, Shandong,China). Roots, young stems, new leaves (growing for 10 days), flowers (5 days after blooming), and fruit (30 days after flowering) were sampled and stored in liquid nitrogen.In addition, rootedRoyal Gala apple tissue culture seedlings were treated with H2O (control) and 10 μmol L–1flg22 for 0,1, 2, 4, 6, and 12 h, and then the entire plantlets were rapidly frozen in liquid nitrogen and stored at -80°C.

We flrst obtained thefls2mutant ofA.thaliana, after which the complemented strain with a35Spromoter,35S::MYC-MdFLS2/fls2, based on thefls2mutant, was obtained. The complemented strain was screened using MS+50 mg L–1hygromycin+50 mg L–1kanamycin-resistant medium, and positive transgenic plants were obtained by PCR. After screening for three continuous generations, T3 homozygotes were obtained, and the seeds were collected for phenotypic analysis. The seeds of the wild-type (WT)A.thaliana, mutants, and complementary strains were sown on Murashige-Skoog (MS) medium, and after 5 d the seedlings were transferred to MS and MS+50 μmol L–1flg22 medium and placed at 25°C in a light incubator for culturing. After 13 d, the root lengths of the seedlings were measured. Thirty samples of both the WT and transgenicArabidopsisseedlings were selected for average length calculations.

2.2. Cloning of MdFLS2

Using an RNA Plant Plus Reagent Kit (QIAGEN, Shanghai,China), RNA was extracted from the leaves of well-grown Royal Gala apple seedlings 5 cm in height and with 10–12 leaves. A PrimeScript? RT Reagent Kit with a gDNA Eraser(Perfect Real Time, TaKaRa, Japan) was used to obtain the cDNA, which was then stored at -20°C. PrimersMdFLS2-F(upstream primer): 5′-ATGGTGTCTCAGAGATTAAGTT-3′andMdFLS2-R (downstream primer): 5′-TGTTTCCCTTTT CAGCTTCAGAA-3′ were designed based on the sequence retrieved from the Apple Genome Database(MDP0000254112), using the Royal Gala tissue culture seedling cDNA as a template for the PCR ampliflcation.The reaction conditions were as follows: pre-denaturation at 94°C for 5 min; followed by denaturation at 95°C for 50 s,annealing at 58°C for 50 s, extension at 72°C for 2 min, 35 cycles; and a flnal extension at 72°C for 10 min. The PCR products were electrophoresed on a 1.5% agarose gel and the target band was recovered and ligated into the PMD18-T cloning vector for sequencing.

2.3. Bioinformatics analysis of MdFLS2

According to the amplifled DNA sequences, the corresponding nucleic acids and protein sequences of other species were searched and downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/), and a neighbor-joining evolutionary tree was constructed using MEGA 5.0 software (ver.5.1, http://www.megasoftware.net/) (Tamuraet al. 2011).SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) was used for structural domain prediction,and thecis-acting element on the promoter was analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

2.4. Plant expression vector construction and Agrobacterium-mediated genetic transformation of Arabidopsis

Expression vector construction andAgrobacteriummediated genetic transformation ofArabidopsisfollowed the method reported in Huet al. (2016). The overexpression vectorMYC-MdFLS2was constructed by inserting the DNA fragment ofMdFLS2open reading frame (ORF) into the transformed vector pXSN-MYC. TheAgrobacterium tumefaciensLBA4404 strain was grown in lysogeny broth(LB) media supplemented with 50 mg mL–1of hygromycin and 50 mg mL–1of rifampicin. TheMYC-MdFLS2consists of the coding sequence under the control of a35Spromoter.The35S::MYC-MdFLS2transgenicA.thalianawere generated using the floral dip transformation method (Huet al.2016).

2.5. qRT-PCR and RT-PCR analysis of gene expression

PrimeScript was used to detect the expression level ofMdFLS2in apple tissues and its response to biotic stress,using qRT-PCR and cDNA template detection.MdActin(GenBank accession number: CN938024) was selected as an internal control gene. Speciflc primer sequences ofMdFLS2for qRT-PCR analysis were the upstream primer 5′-GCACTTGCAGACTGGACAG-3′ and the downstream primer 5′-ATCTGACCGCTGAGCTGCT-3′.The upstream primer ofMdERF098(MDP0000930655) was 5′-GGTCTCCTCCTTATCCTCC-3′ and the downstream primer was 5′-CTTGCCTGCTTCCTTGTCC-3′, which was used for the detection of downstream diseases. The upstream primer ofMdWRKY11(MDP0000272940) was 5′-CATACAGGAAGCGGCATCAC-3′ and the downstream primer was 5′-CTTCTTGAACTTGGAGACGG-3′,upstream primer ofMdWRKY30(MDP0000767097) was 5′-ATGAGCTAGCACAAGGGAG-3′ and downstream was 5′-GGAGAGCGCCTTTTCGTAC-3′, UltraSYBR Mixture(with ROX) kit was used to produce fluorescent quantitative PCR analysis. A total of 20 μL of the reaction system was prepared as follows: 10.0 μL of 2× UltraSYBR Mixture,1.0 μL of the upstream primer (10 μmol L–1), 1.0 μL of the downstream primer (10 μmol L–1), 1.0 μL of cDNA and 7.0 μL of ddH2O. Per sample was performed 3 repetitions. The conditions of fluorescence quantitative PCR reaction were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 65°C for 10 s, 40 cycles, and fluorescence capture at step 3 of each cycle; 2–ΔΔCTmethod for quantitative data analysis.

Mutantfls2and its complementary strain inArabidopsiswere identifled by PCR and semi-quantitative RT-PCR.The expression level ofMdFLS2was detected using cDNA template. Semi-quantitative RT-PCR primers were designed according toMdFLS2.MdFLS2upstream primer was 5′-TGCATGCATTCAAGAAATCG-3′, and downstream primer was 5′-TAGAATTGGCACCAAACTGTGA-3′.AtFLS2upstream primer was 5′-CTGCGATAGTACCGGACATG-3′,and downstream primer was 5′-TTTTCCCGGTTAAG TTGTTG-3′.AtACTINupstream primer was 5′-TTTGGA GCCTGGGACTATGGAT-3′ andAtACTINdownstream primer was 5′-ACGGGGGAATGGGATGAGAT-3′. All the primers were used equal amounts for loading reference in semi-quantitative PCR detection. A total of 20 μL reaction system: 2× EsTaqMasterMix 10.0 μL, 1.0 μL upstream primer (10 μmol L–1), 1.0 μL downstream primer (10 μmol L–1), 1.0 μL cDNA, and 7.0 μL ddH2O. Reaction conditions:pre-denaturation at 95°C for 5 min, denaturation at 95°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 20 s and 35 cycles.

2.6. Assessment of reactive oxygen species (ROS)production and determination of H2O2 and O2–

Five similar green leaf buds from the WT apple plantlets were dissected into 0.25-cm2leaf discs and then placed in 96-well plates and incubated with 100 μL H2O overnight to eliminate any wound effects. The H2O was replaced with 100 μL of reaction solution containing 50 μmol L–1of luminol and 10 μg mL–1of horseradish peroxidase (Sigma, St. Louis, Missouri,USA) supplemented with 10 μmol L–1of flg22. Immediately after adding the solution, measurements were taken with a luminometer with 1-min interval reading for a period of 20 min. The measurement values for ROS production from 40 leaf discs per treatment were indicated as means of relative light units. The experiments were repeated in triplicate.

Arabidopsis thalianaleaves treated with MS and MS+50μmol L–1flg22 and Royal Gala apple leaves treated with MS and MS+10 μmol L–1flg22 were evaluated using a hydrogen peroxide test box (spectrophotometry) and superoxide anion test box (spectrophotometry) (Suzhou Keming Biotechnology Co., Ltd., China).

2.7. Callose staining

Four 5-week-old leaves of each WTA.thaliana,fls2mutants,complemented strains were soaked in 50 μmol L–1flg22, and the growing green apple plantlets were soaked in 10 μmol L–1flg22, while the control group was treated with water. After soaking for 6 and 12 h, the leaves were washed overnight with 95% ethanol:lactophenol (phenol:glycerol:lactic acid:H2O=1:1:1:1)=2:1. The samples were subsequently rinsed with 50% ethanol and H2O. The clean leaves were stained with 0.01% aniline blue in 0.15 mol L–1phosphate buffer (pH=9.5) and the callose deposits were visualized under a UV fllter using a fluorescence microscope. Callose deposits were counted using ImageJ software. At least three independent experiments were conducted, and similar results were obtained.

2.8. Data presentation and statistical analysis

All results were based on the average of three parallel experiments. We use DPS 7.05 data analysis software,and single factor Tukey method to analyze the relative expression level. Different lowercase letters indicate that there are signiflcant differences (P＜0.05) between different tissues at the same treatment.

3. Results

3.1. Cloning of MdFLS2 and conserved domain analysis

Using the cDNA of Royal Gala as the template, we cloned a 3 000-bp gene (Fig. 1-A). Sequencing analysis showed that it contained a complete ORF of 3 474 bp that encodes a polypeptide of 1 158 amino acids. This gene has the same sequence as MDP0000254112, and was thus namedMdFLS2.

SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) was used to analyze the conserved domains of its protein, which revealed that theMdFLS2-predicted protein was a leucine-rich domain (Fig. 1-B).Additionally, the transmembrane and signal peptide domains suggested that it may function similarly toAtFLS2inArabidopsisby involving the immune response of the plant,which is associated with the transmembrane.

3.2. Phylogenetic analysis of MdFLS2

MEGA5.0 software was used to construct a phylogenetic tree in order to compare the MdFLS2 protein family between different species, including apple. The results showed that MdFLS2 was most closely related to, and had the highest homology with PpFLS2 inPrunus persica(Fig. 2).

3.3. MdFLS2 promoter cis-acting element analysis

Analysis of the upstream promoter sequence ofMdFLS2using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) indicated that theMdFLS2promoter sequence contained multiple hormone-related response elements including the ethylene response element(ERE), gibberellic acid response element (GARE)-motif gibberellin response element, tricarboxylic acid (TCA)-salicylate response element, and auxin response elements(TGA-box), indicating thatMdFLS2may be involved in hormonal regulation (Table 1).

Fig. 1 Cloning of MdFLS2 and its analysis of conserved domains. A, ampliflcation of the full length of MdFLS2 gene with PCR.B, prediction of the conserved domains of MdFLS2 protein by SMART. LRR, leucine-rich repeats.

Fig. 2 Phylogenetic tree analysis between different species and apple FLS2 proteins. The phylogenetic tree was constructed using the neighbor joining (NJ) method by the MEGA (ver. 5.1) software. The bootstrap values of 100 replicates were calculated at each node.

3.4. Tissue expression analysis of MdFLS2 and its biotic stress response

qRT-PCR showed thatMdFLS2was expressed in the roots,stems, leaves, flowers, and young fruits of apple, and was most highly expressed in the leaves (Fig. 3-A).

Table 1 cis-acting regulatory elements in the upstream regulatory sequences of MdFLS2

The expression ofMdFLS2was induced by flg22. After treatment with 10 μmol L–1flg22 for 1, 2, 4, 6, and 12 h, the expression ofMdFLS2was signiflcantly up-regulated in comparison to the water-treated control (Fig. 3-B).

Fluorescence microscopy was used to observe the effects of flg22 treatment after 6 and 12 h in the leaves,and as time progressed, the amount of callose was found to increase (Fig. 3-C). Furthermore, the expression of the disease-related genes includingMdERF098,MdWRKY11,andMdWRKY30, as well as O2–production and ROS burst showed a signiflcant increase after flg22 treatment compared to the control (Fig. 3-D–F), suggesting that flg22 can trigger a series of immunity responses in apple.

3.5. The response of MdFLS2 to biotic stress

RT-PCR was used to test the complementary lines35S::MYC-MdFLS2/fls2, which had been obtained from theA.thalianamutantfls2with a35Spromoter. Three lines(L1, L2, and L3) were used to verify thatMdFLS2had been transformed into thefls2mutant (Fig. 4-A).

Under control conditions in MS medium, there were no signiflcant differences in the root lengths of the WTArabidopsis,fls2mutants, and three complementary lines(L1, L2, and L3) (Fig. 4-B and C). And theseArabidopsistreated by MS+10 μmol L–1flg22 medium had no obvious phenotype after 13 d. However, the root length of the WT and three complementary lines were signiflcantly shorter compared to thefls2mutant after 13 d of growth on MS+50μmol L–1flg22 medium (Fig. 4-B and C). Additionally, the application of flg22 resulted in lower yields of O2–and H2O2infls2mutant compared to WT and complementary lines (L1, L2, and L3) (Fig. 4-D and E). meanwhile, the WTArabidopsisand complemented lines (L1, L2, and L3)treated with flg22 exhibited increased callose production with time, while no callose was observed in thefls2mutant(Fig. 4-F). These results suggest thatMdFLS2performed a similar function in immunity responses withAtFLS2inArabidopsis.

4. Discussion

ArabidopsisAtFLS2, a pattern-recognition receptor for flagellins, is involved in the innate immune response of plants and is important for the detection of pathogens and bacteria (B?hmet al. 2014). When bacteria infect plants,AtFLS2recognizes the bacterial-associated molecular patterns on the flagellins, which can result in short-term reactions, including ROS bursts, increased respiration,ethylene production, and a heightened response to some immune-related marker genes. Callose is produced when plants are subjected to bacterial attack for a long period of time. In general,AtFLS2elicits a series of immune-related responses that resist bacterial infections in plants (Haoet al. 2016).

Apple is widely planted and is of great economic signiflcance. However, some fungal infections not only affect tree growth but also cause considerable economic losses by decreasing apple production outputs. In this paper, an appleMdFLS2gene that is homologous toAtFLS2was obtained by comparison with theAtFLS2gene fromArabidopsison GenBank.AtFLS2is expressed in the roots, stems, leaves,and flowers ofArabidopsis(Gómez-Gómez and Boller 2000), and qRT-PCR analysis of gene expression indicated thatMdFLS2was mainly expressed in the leaves of apple.The expression ofMdFLS2was induced by flg22, which indicated thatMdFLS2can respond to pathogen infection in all apple tissues, primarily in the leaves.

Fig. 3 Tissue expression analysis of MdFLS2 in wide type (WT) and its response to biotic stress. A, expression of MdFLS2 gene in different tissues of apple by qRT-PCR analysis. B, expression pattern of MdFLS2 gene under biotic stress. The Royal Gala apple seedlings were treated with 10 μmol L–1 flg22 or H2O (Ctrl) for 0, 1, 2, 4, 6, and 12 h, respectively. C, callose deposition. Royal Gala apple leaves were treated with 1 μmol L–1 flg22 for 0, 6, and 12 h, respectively. Scale bar is 10 μm. D, E, and F, expression of pathogenesis-related gene, burst of reactive oxygen species (ROS) and determination of O2–. Royal Gala apple leaves were treated with H2O (Ctrl) or 10 μmol L–1 flg22. The above experiments were repeated three to four times with similar results. The means and standard deviations were calculated from the results of three independent experiments. Bars with different letters are signiflcantly different at P＜0.05 according to Duncan’s multiple range test.

Furthermore, in WTA.thaliana, FLS2 can respond to pathogen infection as well increase callose deposits and ROS bursts with time (Luet al. 2011). qRT-PCR was used to detect the expression ofMdERF098,MdWRKY30,andMdWRKY11in apple. The results showed thatflg22 influenced the expression ofMdFLS2downstream genes.Moreover, it was able to increase callose deposits and O2–,as well as ROS bursts, when the apple seedling leaves were treated with flg22. This shows that flg22 can stimulate the expression of theMdFLS2gene inRoyal Gala apple seedlings.

Because over-expression ofFLS2inA.thalianaresults in a dwarf phenotype (Gómez-Gómez and Boller 2000), we hypothesized that the heterologous expression ofMdFLS2would have the same effect. Using theA.thalianamutantfls2and complementary strains35S::MYC-MdFLS2/fls2, we discovered that flg22 treatment resulted in the complementary strains possessing a similar phenotype to the WTA.thaliana; both exhibited shorter root lengths than the mutantfls2,while the root length of the mutant was similar to that of untreatedA.thaliana. We also found that flg22-treatedfls2exhibited decreased O2–and H2O2compared to WTArabidopsisand complementary strains(L1, L2, and L3), and almost no callose was produced,which indicated thatMdFLS2responded to the biotic stress.

Fig. 4 The response of MdFLS2 to biotic stress. A, RT-PCR analysis of MdFLS2 and AtFLS2 in wide type (WT) Arabidopsis and transgenic Arabidopsis lines with fls2 background. B and C, growth status and root length of 13-day-old WT Arabidopsis thaliana,fls2 mutant, complementary lines (L1, L2 and L3) treated by flg22, respectively. D and E, determination of O2– and H2O2. F, callose deposition. Arabidopsis WT, fls2 and L1, L2, L3 leaves were treated with 50 μmol L–1 flg22 for 0, 6 and 12 h, respectively. Scale bar is 10 μm. The above experiments were repeated three to four times with similar results. The means and standard deviations were calculated from the results of three independent experiments. Bars with different letters are signiflcantly different at P＜0.05 according to Duncan’s multiple range test.

5. Conclusion

Flg22 can trigger a series of immunity responses in apple. The expression ofMdFLS2was induced by flg22,which indicated thatMdFLS2can respond to pathogen infection. Our study flnd MdFLS2 can recognize flg22 and act as a positive regulator in the response to pathogens in apple.

Acknowledgements

We would like to thank Prof. Wu Shujing of State Key Laboratory of Crop Biology, Shandong Agricultural University, China for providingArabidopsis thalianamutantfls2, and LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript. This work was supported by the National Natural Science Foundation of China (31601728 and 31430074), the Ministry of Education of China (IRT15R42), the Natural Science Foundation of Shandong Province, China (ZR2016CQ13 and SDAIT-06-03), the Young Scientists Funds of Shandong Agricultural University, China (564024 and 24024).