Plant growth and plant infection
Apple stock M9T337 was grown in an illuminated greenhouse at 20–30 °C (daytime) and 0–15 °C (night) with a relative humidity of 55–65%. The apple stock M9T337 was obtained from Shandong Horticultural Techniques Services Co., Ltd., Tai’an, Shandong, China. The ‘Orin’ apple callus was provided by Prof. Xue-sen Chen of the State Key Laboratory of Crop Biology (Tai’an, China). The appropriate permission was obtained for the plant collection and it’s use was executed in accordance with relevant guidelines. ‘Orin’ apple calli (Malus domestica cv. ‘Orin’) were cultured in subculture medium that contained MS, 2.5 mg·L− 1 2,4-D, and 0.5 mg·L− 1 6-indole-3-butyric acid at room temperature in the dark, and the subculture medium was renewed every 15 days for genetic transformation. F. solani (MG836251.1) was isolated from the roots of a replanted apple tree (Fig. S1) [52]. The hyphae of F. solani were inoculated in sterilized Potato Dextrose Broth (PDB) media and cultured in a shaker at 28 °C for one week. The hyphae were filtered with sterile gauze, and the number of plates was counted. The spores were adjusted to 105·mL− 1 with sterile water. Arbuscular mycorrhizal fungi used in this study was Paraglomus sp. SW1 (CGMCCNO. 20,744), which was provided by Shandong Agricultural University (Tai’an, China). The inoculant was a mixture of vermiculite, spores (spore density 28·g− 1), hyphae and colonized root segments. The systems of mycorrhizal plant roots were grown for 4 weeks using Paraglomus sp. SW1.
We established four treatments: (1) NM: non-mycorrhizal plants that had not been inoculated with F. solani; (2) AM: mycorrhizal plants that had not been inoculated with F. solani; (3) NM-F: non-mycorrhizal plants inoculated with F. solani; and (4) AM-F: mycorrhizal plants inoculated with F. solani. After the apple stock M9T337 plantlets grew for 4 weeks with 1% AMF inoculum, 50 mL of a solution of F. solani spores was used to treat them. After 5 days, the apple roots were collected for transcriptome sequencing, qRT-PCR verification, and an analysis of the enzymes involved in resistance. The apple seedlings were randomly collected after 10 days post-infection to determine the apple biomass, root morphology and mycorrhizal colonization rate.
Rate of mycorrhizal colonization
The rate of mycorrhizal colonization was determined as described by Giovannetti and Mosse [53]. A 1 cm root segment was digested with a solution of 10% KOH at 90 °C for 20 min, acidified with 2% HCl for 5 min, stained with 0.05% triphenyl blue lactic acid glycerin solution (lactic acid/glycerin =1/1) at 90 °C for 30 min, and decolorized overnight with a solution of lactic acid glycerin (lactic acid/glycerin/water = 1/1/1 [v/v/v]). The root segments were observed under a microscope. The rate of infection of each root segment was assessed by the number of root mycorrhizal structures in each section and expressed as 0, 10, 20, ..., 100% of the root. The mycorrhizal colonization rate (%) was calculated as follows:
$$\mathrm{Mycorrhizal}\ \mathrm{colonization}\ \mathrm{rate}=\Sigma\ \left(0\times \mathrm{root}\ \mathrm{segment}\ \mathrm{number}+10\%\times \mathrm{root}\ \mathrm{segment}\ \mathrm{number}+20\%\times \mathrm{root}\ \mathrm{segment}\ \mathrm{number}+\dots \dots +100\%\times \mathrm{root}\ \mathrm{segment}\ \mathrm{number}\right)/\mathrm{Total}\ \mathrm{root}\ \mathrm{segment}\ \mathrm{number}.$$
RNA extraction, cDNA library construction, and Illumina sequencing
Total RNA was isolated using a RNA Prep Pure Plant Kit (CWBIO, Beijing, China) according to the manufacturer’s instructions. Total RNA was tested with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The synthesis of cDNA was performed using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). cDNA fragments that were preferentially 250 ~ 300 bp long were purified with an AMPure XP system (Beckman Coulter, Brea, CA, USA). The PCR products were purified again after PCR amplification, and the cDNA library was finally obtained [54]. And cDNA library was assessed on an Agilent Bioanalyzer 2100 system (Agilent Technologies). The libraries were sequenced on a HiSeq® X Ten System (Illumina, San Diego, CA) by Novogene Co., Ltd. (Beijing, China).
Transcriptomic data analysis
To ensure the quality and reliability of the data analysis, it is necessary to filter the original data, including the removal of reads with an adapter, and remove the reads that contain N. N indicates that the nucleobase information cannot be determined. The low quality reads were then removed. The base number of qPHRED was ≤20, which comprised ≥50% of the whole read length. Moreover, the contents of Q20, Q30, and GC in the clean data were calculated. All follow-up analyses were based on the clean data high quality. Clean data obtained by filtering the raw data were aligned to the reference genome sequence of Malus × domestica (GDDH13 v. 1.1) by HISAT2 (v. 2.0.5).
The program FeatureCounts (v. 1.5.0-p3) was used to calculate the readings that mapped to each gene [55]. The FPKM of each gene was calculated based on the length of gene, and the reading that mapped to the gene was calculated. DESeq2 software (1.20.0) was used to analyze the differential expression between the four treatments [56]. DEGs with |log2(Fold Change)| ≥ 1 and P-value < 0.05 were considered as differentially expressed genes (DEGs) after multiple correction for subsequent analyses. The clusterProfiler R package (3.4.4) was used for the Gene Ontology (GO) enrichment analysis of DEGs [57].
MapMan software (version 3.6.0RC1) (http://mapman.gabipd.org/web/guest/mapman) was used to generate functional assignments in the different pathways [58] for each input gene and the data visualization/interpretation of apple gene expression. Gene annotation in the pathway analysis was prepared via Mercator online software within the PlabiPD website (https://www.plabipd.de/portal/mercator4) based on the DNA sequence and followed the default annotation parameter.
Measurement of root physiological parameters
The root physiological parameters included the activities of SOD, CAT, and POD, the contents of PRO, SUG, MDA, H2O2 and O2·−. Each physiological parameter was measured using kits obtained from Suzhou Keming Biotechnology Co., Ltd. (Suzhou, China). All the measurements were performed three times, and the average was calculated for further analysis.
Gene cloning and function validation of MdWRKY40
MdWRKY40 (MD15G1039500) sequence information was obtained from the apple genome database (http://www.rosaceae.org). Total RNA extraction and cDNA synthesis were conducted as described by RNA Extraction, cDNA library construction, and Illumina sequencing section. A fragment of 912 bp was obtained by PCR amplification, cloned into the pLB vector (TianGen, http://www.tiangen.com/).
The CDS of MdWRKY40 was inserted into the PRI101-AN vector, which has a green fluorescent protein (GFP) tag. The constructed overexpression vector was transformed into Agrobacterium tumefaciens LBA4404. The positive A. tumefaciens was obtained by PCR and used to infect ‘Orin’ callus. The infected calli were cultured on MS solid media at 24 °C in the dark for 1–2 days. It was then transferred to a screening medium that contained 50 mg·L− 1 kanamycin and 250 mg·L− 1 carbenicillin. The overexpression of MdWRKY40 was confirmed by PCR.
Yeast one-hybrid test
The target gene fragments MdWRKY40, MdWRKY40-N (0–120 aa), MdWRKY40-C (121–303 aa) were ligated into the pGADT7 vector. The fragment of MdGLU promoter was cloned into the pHIS2 vector. A yeast one-hybrid test was conducted according to the manufacturer’s instructions of Yeast transformation system 2 (TaKaRa, Dalian, China). The yeast one-hybrid strain was Y187 (Clontech, Takara Bio USA, San Jose, CA, USA). The yeast strain Y187 that contained the recombinant pHIS2 vectors was grown on –Trp/−His (−T/−H) screening media with different 3-AT concentrations to determine the optimal concentrations. To determine the interactions, the Y187 yeast strain that harbored the recombinant pGADT7 and pHIS2 plasmids was spotted onto media that lacked Trp, His, and Leu. The control was an empty pGADT7 plasmid. The primers used are listed in Supplemental Table S2.
Electrophoretic mobility shift assays
The sequence of MdWRKY40 was inserted into the pET-32a (+) expression vector (Novagen, Madison, WI, USA). The recombinant MdWRKY40 protein was expressed in E. coli BL21 (DE3), and the fusion protein MdWRKY40-His was purified by a His-Tagged Protein Purification Kit (CWBIO, Beijing, China). The biotinylated probe was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The fusion protein, probe, and binding buffer were mixed in a centrifuge tube and incubated at 24 °C for 30 min. After 50% glycerol and 5 × loading buffer were added to the sample, non-denaturing acrylamide gel electrophoresis was performed, and the protein-nucleic acid strip was transferred to film placed on nylon. After the completion of UV crosslinking, the preheated Blocking Buffer closure was added, then HRP Conjugate and 20 mL new Blocking Buffer (ThermoFisher, Shanghai, China) were incubated at room temperature for 15 min. The Washing Buffer (ThermoFisher) was used after washing and developing.
LUC activity
MdWRKY40 full length CDS inserted into the pHBT-AvrRpm 1 carrier and promoter segments of MdGLU into the pFRK1-LUC-nos carrier. Both plasmids were converted simultaneously from the protoplasm of apple callus and then expressed for 6 h at 24 °C. Subsequently, the protoplasm was suspended in 100 μL of cell lysate. The 5 μL cell extract and 20 μL 1 mmol·L− 1 4-MUG were incubated at 37 °C at 1 h, and the 100 μL of 0.2 mol·L− 1 sodium acetate was added to the termination reaction. The LUC activity was determined using the Luciferase Reporting Analysis System (Promega, Madison, WI, USA).
Quantitative reverse transcription-PCR (qRT-PCR)
The cDNA was synthesized using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The reverse transcription reactions began with 500 ng of total RNA. The resulting first stand cDNA was diluted 10-fold with ddH2O, and then used as templates for qRT-PCR assays. The primers were designed for qRT-PCR by Primer 6.0 software (Premier Biosoft, Palo Alto, CA, USA). The internal reference gene was actin. The reaction system in the PCR of each primer was SYBR Green Mix 5 μL, primer (10 μM) 0.3 μL, cDNA 1 μL, and dd H2O 3.4 μL. The PCR procedures were 50 °C 2 min, 95 °C 10 min, 95 °C 15 s, 65 °C 60 s, 72 °C (30 cycles), and 72 °C 10 min. The primers were synthesized by Sangon Biotech Co., Ltd. The relative quantitative method used the 2-∆∆CT method [59]. Each sample had three biological replicates. The primer sequences are shown in Supplementary Table S2.
Data analysis
The experimental data was expressed as the means and standard deviation (SD) of three biological replicates. The plant growth, mycorrhizal colonization rate and physiological data were analyzed by Duncan’s test at the 0.05 level using SPSS v. 19.0 (IBM, Inc., Armonk, NY, USA). The qRT-PCR data were analyzed by t-test. An asterisk (*) indicates significant differences, where ‘*’ represents significant differences at P < 0.05, and ‘**’ represents highly significant differences at P < 0.01. The graphs were constructed using GraphPad Prism 8 (San Diego, CA, USA). The annotations of the DEGs were based on the databases of GO and MapMan software.