Plant and Fungal Materials
The seeds of maize (B73), obtained from the Chinese Crop Germplasm Information System (CGRIS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, were surface sterilized by dipping in 2% Sodium Hypochlorite (NaOCl) solution followed by treatment with 70% Ethanol for 5 minutes each and washed at least three times with sterilized water. Sterile germination papers (30 × 45 cm; Anchor Paper Company, MN, USA) were used for the germination of the seeds. The germination papers were soaked in sterile Hoagland's solution. The seeds were allowed to germinate in a contamination-free and humidity-controlled growth facility at 25 oC with a 16 hours light/8 hours dark cycle and relative humidity of 80% for 72 hours. To apoplastic fluid was extracted from maize seedlings, which were grown in pots filled with solid substrate mixture and placed in a greenhouse at 25 oC temperature and 16 hours light/8 hours dark period. The seedlings were inoculated at 4 leaf stage, and the apoplastic fluid was extracted at 7 days post-inoculation. F. verticillioides (LNF15-11) strain was maintained on potato dextrose agar at 25 oC under 12 hours light/12 hours dark cycle for 7 days to induce conidiation. The conidia were collected by filtration using sterile water and filtered through a double layer of sterile Miracloth (Millipore Merck, MA, USA). All the experiments were repeated at least three times.
Pathogen colonization studies of maize roots
Sterilized maize seeds were allowed to germinate on the germination papers soaked in Hoagland's solution for 72 hours. After germination, the seeds were surface inoculated with the conidial suspension of F. verticillioides (20 μl) at a concentration of 106 conidia/ml and transferred to the 50 ml tubes containing 30 ml of sterile Hoagland's solution. Seedlings were supported by putting a piece of sterile cotton in 50 ml tubes and incubated at 25 oC in a humidity-controlled growth chamber. At 7 days post-inoculation, the seedlings were removed from the tubes, and roots were cut into small pieces of 2 cm length. Un-inoculated seedlings were taken as control. The samples were divided into two groups: fresh samples either washed with phosphate-buffered saline (PBS) or fixed using freshly prepared ethanol: acetic acid (3: 1, v/v) solution. Subsequently, the samples were stained using two different staining solution mixtures: for fresh tissues, we used a combination of wheat germ agglutinin WGA-Alexa FluorTM 488 (Thermo Fisher Scientific, Shanghai, China) mixed with FMTM 4-64 dye (Thermo Fisher Scientific, Shanghai, China). The fixed tissues were stained by using a mixture prepared by WGA-Alexa FluorTM 488 and propidium iodide (PI) (Sigma-Aldrich, Beijing, China). The fungal hyphae (Green Channel) was stained with WGA-Alexa FluorTM 488, whereas the plant tissues (Red Channel) were stained with PI (for cell wall staining) or FMTM 4-64 dye (for cell membrane staining)[56, 57]. For fresh plant tissues, the samples were washed with PBS solution and subsequently treated with the mixture of the staining solution, containing 10 μg/ml FMTM 4-64 dye, 10 μg/ml WGA-Alexa FluorTM 488 and 0.02 % Tween 20 in 1X PBS, twice for 15 minutes. The plant tissues, already fixed in ethanol: acetic acid solution (3: 1 v/v), were treated with 10 % KOH solution for 4 hours at 95 oC followed by submersion in PBS for 1 hour. The samples were infiltrated with the staining solution mentioned above after FMTM 4-64M dye was replaced with PI (20 μg/mL). The stained samples were stored in 1xPBS and placed at 4 oC in darkness. The stained samples were observed and photographed with the help of a Leica DM2500 microscope (Leica microsystems Inc., IL, USA).
Isolation of apoplastic proteins
For the extraction of apoplastic fluid, the stem of the maize plants was cut into small sections (5 cm) using a sterile razor blade. Three replicates were used for each condition: inoculated and un-inoculated plants at 7 days post-inoculation. The apoplastic fluid was collected using the vacuum infiltration-centrifugation (VIC) methodology with slight modifications. Briefly, the samples were washed using sterile water to remove any contamination from the surface and dried by gently blotting with tissue paper. The weight of the sample was measured, and the samples were then placed in a 20 ml syringe and filled with distilled water. The air in the syringe was removed by pushing the plunger. After that, the tip of the syringe was covered with a piece of parafilm, and negative pressure was generated by pulling the plunger. Then the plunger was carefully released to avoid any cell lysis and cytoplasmic contamination. Now the syringe was unplugged to eject any air inside and re-plugged. Then a modest positive pressure was created inside the syringe by pressing the plunger carefully. The process was repeated until the whole plant tissue was infiltrated. The sample was removed from the syringe and gently blotted with the piece of absorbent paper to remove the liquid outside the surface of the samples. The sample was weighed again, and approximate volume was calculated by the difference in weight before and after the infiltration. The samples were placed in a 20 ml syringe without plunger and inserted into a 50 ml centrifuge tube, and centrifuged at 2000×g for 15 minutes at 4 oC (Eppendorf, Hamburg, Germany). The harvested apoplastic fluid was further filtered through a cellulose acetate membrane (0.22 µm pore size) to ensure the removal of any cells or particulate matter in the fluid. The apoplastic fluids from un-inoculated and inoculated plants were concentrated by freeze-drying (Thermo Fisher Scientific, USA), and rehydration of the samples was achieved by adding a 20 µl buffer solution (25 mM Tris-HCL and 100 mM NaCl, pH=7.4). The samples were stored at -80 oC until the next use.
The protein sample for each treatment was mixed with a lysis buffer containing 4% SDS, 100 mM Tris-HCL, and 1mM DTT (pH 7.6) (Bio-Rad, USA) and boiled for 15 minutes. The debris was removed by centrifugation at 14000×g for 40 minutes. After centrifugation, the supernatant was retained and quantified by the help of the BCA Protein Assay Kit (Bio-Rad, Beijing, China). The protein solution was added with 30 µl SDT buffer containing 4% SDS, 150mM Tris-HCL and 100mM DTT (pH=8.0) (Bio-Rad, USA). The sample volume was reduced by ultrafiltration (Microcon Units, 10 kD) after adding UA buffer (8 M Urea, 150mM Tris-HCL, pH=8.0) to the samples. For alkylation, the samples were treated by 100µl iodoacetamide and were placed in complete darkness for 30 minutes at room temperature. The filters were washed with 100 µl UA buffer three times, followed by washing with 100 µl of 25 mM ammonium bicarbonate (NH4HCO3). Finally, the protein suspension was diluted by adding 40 µl of 25 mM NH4HCO3 buffer and digested by adding trypsin (Promega, Madison, USA). The sample to trypsin mass ratio was adjusted as 50:1 for the first round of digestion at 37 oC overnight, and 100:1 for the second round of digestion for 4 hours. The resulting peptides were collected by filtration. About 200 µg of each protein sample was digested by trypsin. The peptides for each sample were desalted using Empore C18 solid-phase extraction column (Sigma-Aldrich, Beijing, China) and concentrated by vacuum centrifugation. The resulting peptides were reconstituted in 40 µl of 0.1% (v/v) of formic acid before analysis.
HPLC and LC-MS/MS Analysis
The HPLC and LC-MS/MS analysis were done by Shanghai Applied Protein Biotechnology, Ltd. Shanghai, China. Briefly, the peptides mixture was added to the buffer A (0.1% Formic acid) and loaded onto a C18 trap column (Thermo Scientific Acclaim PepMap100, 100 μmx2 cm, nanoViper C18, 3 μm, 100 Å). The reverse-phase trap column was connected to the C18 analytical column (Thermo scientific EASY column, 10 cm, ID 75 μm, 3 μm, C18-A2). For the separation of peptides, a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) was injected at a flow rate of 300 nl/min using IntelliFlowTM technology. The time for the linear gradient of buffer B was set to 120 minutes (0-55% for 110 min, 55-100% for 5 min, hold in 100% for 5 min).
For LC-MS/MS analysis, the Q ExactiveTM mass spectrometer coupled with Easy nLC Liquid Chromatograph (Thermo Fisher Scientific Co. Ltd., Shanghai, China) was used for 120 minutes. The instrument was operated on positive ion mode and run with peptide recognition enabled. MS data was acquired by series of cyclic scans at a high resolution of 70,000 using a data-dependent method dynamically choosing the most abundant precursor ions from the survey scans (300-1800 m/z) followed by scans at a relatively low resolution of 17,500 at 200 m/z. The survey scan width was set as 2 m/z. The automatic gain control (AGC) target was set to 3e6, whereas the maximum time for injection was set to 10 ms. The dynamic exclusion duration was set as 40 sec. The normalized collision energy was 30 eV, and the underfill ratio was defined as 0.1%. The underfill ratio specifies the minimum percentage of the target value to reach the maximum fill time.
Database search and label-free quantification analysis
The MS data were searched against the Maize Uniprot proteome database (https://www.uniprot.org/proteomes/UP000007305), including other contaminants (total number of entries 132460) using Andromeda connected with the MaxQuant Software version 220.127.116.11 (Max Plank Institute of Biochemistry, Martinized, Germany) using the default parameters. To quantify the proteins, Carbamidomethylation was set as a fixed modification, whereas the oxidation as a variable modification. A mass error of 20 ppm was allowed with 2 min retention time for shift tolerance. False Discovery Rate (FDR) threshold for proteins and peptides was less than 1%, the peptides having less than 7 amino acids were not included. Besides, proteins having unique peptides were considered for quantification.
For protein annotation, the Blast2GO bioinformatics platform was used to search the sequences. For pathways analysis, the online Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg) database was searched, and the proteins were mapped for KEGG pathways. Subsequently, the corresponding GO terms and KEGG pathways were extracted. The secreted proteins were identified using different online servers including SignalP (http://www.cbs.dtu.dk/services/SignalP/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). For subcellular localization, WolfpSort (https://wolfpsort.hgc.jp/) and TargetP (http://www.cbs.dtu.dk/services/TargetP/) were used, whereas, SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP/) was used to predict the leaderless secretory proteins (Additional file S3).
Reverse transcription-quantitative PCR validation (RT-qPCR)
To validate the protein quantification results, RNA from each sample was extracted using the commercial RNA extraction Kit (Easy Spin, NBFB, Beijing, China) by following the manufacturer's instructions. The first-strand cDNA was synthesized from 3 µg of total RNA with the help of a commercial cDNA synthesis kit (Takara, Dalian, China) according to the manufacturer's instructions. RT-qPCR was done using the SYBR Green kit (NovoStart® SYBR qPCR SuperMix Plus) on QuantStudio (TM) 6 Flex System (Thermo Fisher Scientific, Shanghai, China). The experiment was conducted in triplicate. The maize actin gene was used as an internal standard to calculate relative fold-changes based on comparative cycle threshold (2−ΔΔCt) values. The primers were designed using the DNASTAR laser gene version 7.5. (Additional file S4).