Fusarium graminearum can cause very serious stalk rot disease in maize. Therefore, it was important to explore the resistance mechanism of stalk rot caused by F. graminearum in maize. To investigate the global proteomic changes in response to F. graminearum infection in maize stem, comparative proteomics-based TMT labeling technology was conducted with three independent biological replicates for each sample (Figure 1A). A total of 676,400 spectra were generated from TMT analysis, after searching the maize sequence database, yielding 85,386 matched peptides, 67,274 unique peptides, 8,471 matched proteins, and 8,044 quantified proteins (Figure 1B and Supplementary Table 1), and the biological replications in different infection stage showed good similarity (Figure 1C and Supplementary Figure 1). According to a criterion of 95% significance and a 1.3-fold cutoff (p-value < 0.05), 1,894 proteins were identified as differentially expressed proteins (DEPs) between 0, 1, and 2 days postinoculation (dpi; Supplementary Table 2). Then, we identified 1,064 DEPs in 1 dpi and 1,376 DEPs in 2 dpi that were significantly up or down expressed compared with 0 dpi (Figure 2). Among the 1,894 DEPs, we found 100 proteins were continuously upregulated (Supplementary Figure 2A), while 14 proteins were continuously downregulated after F. graminearum infection (Supplementary Figure 2B).

To further understand the identified and quantified functions and characteristics of proteins, we conducted GO enrichment analysis for these DEPs in response to F. graminearum infection. For upregulated DEPs, the GO terms “small molecule metabolic process” [GO:0044281, false discovery rate (FDR) < 8.30E-15], “response to biotic stimulus” [GO:0009607, FDR < 8.40E-5], “chitin catabolic process” [GO:0006032, FDR < 0.0026], “hydrogen peroxide metabolic process” [GO:0042743, FDR < 0.0085] in biological process (Figure 3A), “heme binding” [GO:0020037, FDR < 0.0025], “chitinase activity” [GO:0004568, FDR < 0.0058], “oxidoreductase activity” [GO:16491, FDR < 0.0095] in molecular function (Figure 3B), etc., were significantly enriched throughout the infection stage. In addition, some GO terms were significantly enriched from 1dpi to 2 dpi, such as “flavonoid biosynthetic process” [GO:0009813, FDR < 0.0027], “response to toxic substance” [GO:0009636, FDR < 0.00091], “phenylpropanoid biosynthetic process” [GO:0009699, FDR < 0.0018], “dioxygenase activity” [GO:0051213, FDR < 0.018] (Figures 3A,B), etc. GO terms in cellular component were significantly enriched cytoplasm [GO:0005737, FDR < 5.50E-14], whole membrane [GO:0098805, FDR < 0.0049], and extracellular region [GO:0005576, FDR < 7.20E-06] (Figure 3C), etc.

For downregulated DEPs, the carbohydrate metabolism related GO terms were significantly enriched, such as “carbohydrate metabolic process” [GO:0005975, FDR < 0.001], “cellular polysaccharide metabolic process” [GO:0044264, FDR < 0.0015], and “glucan metabolic process” [GO:0044042, FDR < 0.0056], etc (Figure 4A). Meanwhile, GO terms related to plant growth and development, such as “cell growth” [GO:0016049, FDR < 0.013], “cell wall organization or biogenesis” [GO:0071554, FDR < 0.0015], “plant-type cell wall” [GO:0009505, FDR < 0.00082], etc., were also significantly enriched (Figures 4A,C). Moreover, “protein complex subunit organization” [GO:0071554, FDR < 0.0015], “regulation of protein dephosphorylation” [GO:0071554, FDR < 0.0015], “glutathione transferase activity” [GO:0009505, FDR < 0.00082], etc., were also enriched in downregulated DEPs (Figures 4A,B). These results suggested that maize might modulate the immune system in response to the F. graminearum infection by slowing down plant growth and energy metabolism.

To further analyze the key enriched metabolic pathways in response to F. graminearum infection, The KEGG pathway enrichment analysis was performed with pathogen-responsive proteins from the respective proteomes. The KEGG analysis demonstrated that phagosome, plant-pathogen interaction, and ribosome were significantly affected and upregulated by the early stage of F. graminearum infection (1 dpi). Compared with 2 dpi proteomic data, flavonoid biosynthesis, phenylpropanoid biosynthesis, amino sugar, and nucleotide sugar metabolism, diterpenoid biosynthesis, glyoxylate, and dicarboxylate mentalism, biosynthesis of secondary metabolites, terpenoid backbone biosynthesis, and fatty acid degradation were significantly enriched in upregulated DEPs (Figure 5). These results suggested that the disease resistance genes could mediate plant-pathogen interaction and the synthesis and metabolism of secondary metabolites in response to pathogen infection.

The KEGG pathway includes glutathione metabolism, starch and sucrose metabolism, oxidative phosphorylation, biotin metabolism, and photosynthesis that were enriched in downregulated DEPs (Figure 5), which suggested that energy metabolism and plant development were downregulated to resist F. graminearum infection. In addition, the results of KEGG analysis were relatively consistent with GO enrichment, and all of these results might reflect how plants resist fungal infection.

In KEGG pathway enrichment analysis, plant-pathogen interaction was significantly enriched in the upregulated DEPs with F. graminearum early infection, while terpenoid backbone and diterpenoid biosynthesis were enriched in later infection (Figure 5). To further examine relationships between mRNA expression and protein abundance under F. graminearum infection, the relative transcript level of proteins enriched in plant-pathogen interaction, terpenoid backbone, and diterpenoid biosynthesis was examined by quantitative real-time PCR (qRT-PCR). Transcript levels of these proteins (excepted Zm00001d024903) enriched in plant-pathogen interaction were successfully determined and mostly increased in both 1 and 2 dpi, compared with 0 dpi (Figure 6). The proteins enriched in terpenoid backbone and diterpenoid biosynthesis were also successfully determined. Except for Zm00001d038193 and Zm00001d021709, most of them were observably upregulated during F. graminearum infection, especially in 2 dpi (Figure 7). In addition, we also examined the transcript levels of proteins enriched in the benzoxazinoid biosynthesis pathway which were downregulated, and all transcript levels of these proteins were decreased by F. graminearum infection.

Then, we compared the expression level of the genes enriched in plant-pathogen interaction, terpenoid backbone, and diterpenoid biosynthesis pathway in susceptible (B73) and resistant (Mo17) lines. We found that the expression levels of these genes were also significantly upregulated in Mo17 (Supplementary Table 4). However, most of the genes in Mo17 plants had higher multiples of expression than in B73, especially in 1 dpi. Moreover, we further extend the expression analysis in 0, 1, 2, and 3 dpi (Supplementary Table 5). Compared with 0 dpi, qRT-PCR results showed that most genes enriched in plant-pathogen interaction, terpenoid backbone, and diterpenoid biosynthesis pathways were significantly upregulated in 3 dpi, whereas increase multiple was not as high as 2 dpi. These results indicated that these genes played important roles in disease resistance.

In the plant-pathogen interaction pathway, we found that ZmWRKY83 (Zm00001d038023), the homolog of AtWRKY33 in Arabidopsis, was always enriched with high protein abundance. Meanwhile, ZmWRKY83 was continuously upregulated from 0 dpi to 2 dpi both in transcript and protein levels (Figure 6), which indicated that ZmWRKY83 was induced by F. graminearum infection and might play an important role in disease resistance. As a transcription factor, transcriptional activity is necessary. Therefore, we used the yeast two-hybrid system to verify the transcriptional activity of ZmWRKY83. The transformed yeast strain grew well on SD/-Leu/-Trp and SD/-Leu/-Trp/-His medium and appeared blue on the selective SD/-Leu/-Trp/-His/x-α-gal medium, which indicated that the ZmWRKY83 transcription factor had the transcriptional activation activity (Supplementary Figure 3).

To investigate the function of ZmWRKY83 in disease resistance, ZmWRKY83 overexpressing transgenic plants were generated in Arabidopsis by Agrobacterium-mediated transformation (Figure 8A). Then, we conducted a B. cinerea infection assay in Col-0, atwrky33 mutant, and ZmWRKY83 overexpressing transgenic plants. Compared with Col-0 and atwrky33 mutant, the resistibility of ZmWRKY83-OE plants to B. cinerea was relatively strong (Figure 8B and Supplementary Figure 4). In other words, the leaves of Col-0 and atwrky33 mutants were more sensitive to B. cinerea, whereas overexpression of ZmWRKY83 could enhance the resistance of Arabidopsis to this pathogen. Statistical analysis results showed that the size of the lesion area on Col-0 and atwrky33 mutant leaves after B. cinerea-infected was more than the lesion area on B. cinerea-infected ZmWRKY83-OE leaves (Figure 8C). In addition, we got a Mutator insertion line of ZmWRKY83, in which ZmWRKY83 showed low expression (Figure 8D). Then, we conducted F. graminearum infection assay in wild type B73 and zmwrky83 mutant. The results showed that the zmwrky83 mutant was more sensitive to F. graminearum infection (Figures 8E,F). These results suggested that ZmWRKY83 plays an important role in the plant defense system.

The maize inbred line B73 was grown in the greenhouse in Baoding (Hebei Agricultural University, Hebei province, China). Maize zmwrky83 mutant was obtained from the ChinaMu project (insertion site: chr 6, 145531076¹) (Liang et al., 2019). F. gramincarum PH-1 was grown on PDA plates at 28°C for 6 days. Conidial suspensions were harvested by adding sterile distilled water containing 0.05% (v/v) Triton X-100 and scraping the plates using a glass spreader. The concentration of conidia was quantified using a hemocytometer and diluted to 1 × 10⁶ spores/ml for inoculation. Maize stem was inoculated at the second or third internode using a sterile micropipet tip (10 mm hole depth) above the soil line, followed by injection of 20 μl macroconidia suspension. The plants were kept growing with the wounds being covered by sterile gauze to maintain moisture and avoid contamination from other organisms.

The sample was ground into powder by liquid nitrogen and then transferred to a 5 ml centrifuge tube. Then, lysis buffer (including 1% TritonX-100, 10 mM dithiothreitol, and 1% protease inhibitor cocktail, 50 μM PR-619,3 μM TSA,50 mM NAM, and 2 mM EDTA) was added to the powder, followed by sonication three times on ice using a high-intensity ultrasonic processor (Scientz). After an equal volume of Tris-saturated phenol (pH 8.0) was added, the mixture was further vortexed for 5 min. After centrifugation (4°C, 10 min, 5,000 g), the upper phenol phase was transferred to a new centrifuge tube. Proteins were precipitated by adding at least four volumes of ammonium sulfate-saturated methanol and incubated at −20°C for at least 6 h. After centrifugation at 4°C for 10 min, the supernatant was discarded. The remaining precipitate was washed with ice-cold methanol once, followed by ice-cold acetone three times. The protein was resuspended in 8 M urea (Sigma), and then the redissolved protein concentration was detected with a BCA kit (Beyotime) according to the instructions of the manufacturer.

The protein solution was reduced with 5 mM dithiothreitol (Sigma) for 30 min at 56°C and alkylated with 11 mM iodoacetamide (Sigma) for 15 min in darkness at room temperature. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2 M. Trypsin (Promega) was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4-h-digestion.

The peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried after trypsin digestion. According to the manufacturer’s protocol, peptide was reconstituted in 0.5 M TEAB and processed with TMT kit (Thermo Fisher Scientific²). Briefly, 1 unit of TMT reagent was thawed and reconstituted in acetonitrile. Then, the peptide mixture was incubated at room temperature for 2 h, and the pool was centrifuged in a vacuum, desalted, and dried. The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, and 250 mm length). Then, the peptides were combined into 18 components with a gradient of 8–32% acetonitrile (pH 9.0) and dried by vacuum centrifugation.

Trypsin peptides are dissolved in 0.1% formic acid and directly loaded on a self-made reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient of solvent B (0.1% formic acid in 98% acetonitrile) was gradually increased from 6% to 23% through 26 min, increased from 23% to 35% over 8 min, climbing to 80% in 3 min, and then maintained at 80% for 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system. The separated peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM Plus (Thermo) coupled online to the UPLC. A 2.0 kV electrospray voltage was applied. At 70,000 resolutions, the intact peptides were detected in the Orbitrap, with 350–1800 m/z full scan range. Up to 20 most abundant precursors were then selected for further MS/MS analyses with 30-s dynamic exclusion. The HCD fragmentation was performed at a normalized collision energy of 28%. The fragments were detected in the Orbitrap at a resolution of 17,500. The fixed first mass was set as 100 m/z. The automatic gain control target was set at 5E4, with an intensity threshold of 1E4 and a maximum injection time of 200 ms.

Tandem mass spectrometry data were processed by the Maxquant search engine (version 1.5.2.8). MS/MS were searched against MaizeGDB (version 4, 131,585 sequences) database concatenated with reverse decoy database. Trypsin/P was designated as lyase, allowing up to two missing cleavages. The mass tolerance of precursor ions is set to 20 ppm in the first search, 5 ppm in the main search, and 0.02 Da in the fragment ions. The aminoformyl methyl on Cys was designated as a fixed modification, and the oxidation on Met was designated as a variable modification. The minimum score for peptides was set > 40 and FDR was adjusted to < 1%.

Functional annotations of DEPs were performed using agriGOv2 (Tian et al., 2017). The KEGG was used to predict the metabolic pathways and biochemical signals transduction pathways (Kanehisa and Goto, 2000). A p-value < 0.05 (Fisher’s exact test) was used as the threshold to determine the significant enrichment of GO and KEGG pathways.

The same samples used in proteomics were homogenized in liquid nitrogen before RNA isolation. Total RNA of the samples was isolated using TRIZOL (Invitrogen) and purified using Qiagen RNeasy columns. qRT-PCR was conducted using actin as an internal reference and cDNAs from samples collected at different time points as the template with TransStart Tip Green qPCR SuperMix according to the manufacturer’s instructions. Furthermore, comparative Ct analysis (2-^ΔΔCt) of each gene in B73 and its relative expression levels at different time points were employed, and quantitative data were expressed as mean ± SEM. Primers used in this study were designed with Primer 6.0 software and listed in Supplementary Table 3.

To detect the transcriptional activation of ZmWRKY83, the CDS of ZmWRKY83 were amplified and cloned into the yeast vector pGBKT7 (Clontech) to obtain BD- ZmWRKY83 construct. BD- ZmWRKY83 construct and empty vector pGBKT7 were, respectively, transformed into yeast strain AH109. A transformed single colony (2 mm diameter) grown on selection medium for 3 days was resuspended in 100 μl of autoclaved distilled H₂O, and 10 μl of resuspended cells was plated on SD/-Leu-Trp, SD/-Leu-Trp-His/x-α-gal for 3–5 days.

A full-length ZmWRKY83 CDS sequence was amplified by PCR with a specific primer (Supplementary Table 3). After sequence verification, the fragment was inserted into the binary vector pCAMBIA-1300. The recombinant plasmid was introduced into Columbia-0 (Col-0) plants by Agrobacterium using floral dipping. The transformed seeds were selected on a half-strength MS medium containing Basta.

Botrytis cinerea strain B05.10 was cultured on PDA (potato dextrose agar) media for 5–10 days at 18–22°C. Conidia inoculums of B. cinerea were prepared by collecting conidia in sterile water, filtered to remove mycelia, and suspended to a final concentration of 10⁶ conidia/ml. For resistance analysis, the single leaf of plants was inoculated with 5 μl conidia suspension of B. cinerea. Inoculated plants were kept in a growth chamber with high humidity for 24 h and then transferred to normal conditions. Lesion formation was monitored at 7 days postinoculation (dpi). Trypan blue staining was performed as described by the previous study (Hao et al., 2013).