Shunxi Wng, Wencheng Liu, Zn Chen, Jinghu Zhng, Xingmeng Ji,Mingyue Gou, Xueyn Chen, Yuqin Zhng,c, Hehun Li, Ynhui Chen, Liuji Wu,

a National Key Laboratory of Wheat and Maize Crop Science, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, Henan, China

b State Key Laboratory of Crop Stress Adaptation and Improvement, State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University,Kaifeng 475004, Henan, China

c School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia

Keywords:Maize stalk rot Zmprx5 Transgenic Mutant Disease resistance

A B S T R A C T Maize (Zea mays L.) stalk rot is a devastating disease worldwide, causing severe yield losses. Although previous studies have focused on the genetic dissection of maize resistance to stalk rot,the mechanisms of resistance remain largely unknown.We used a comparative proteomics approach to identify candidate proteins associated with stalk rot resistance.Statistical analyses revealed 763 proteins differentially accumulated between Fusarium graminearum and mock-inoculated plants.Among them,the antioxidant protein ZmPrx5, which was up-accumulated in diseased plants, was selected for further study. ZmPrx5 transcripts were present in root, stalk, leaf, ear, and reproductive tissues. The expression of ZmPrx5 in three inbred lines increased significantly upon F. graminearum infection. ZmPrx5 was localized in the cytoplasm.Compared to control plants,maize plants overexpressing ZmPrx5 showed increased resistance to F. graminearum infection, and ZmPrx5 mutant plants were more susceptible than wild-type plants.Defense-associated pathways including plant-pathogen interactions, phenylalanine metabolism, and benzoxazinoid and flavonoid biosynthesis were suppressed in ZmPrx5 homozygous mutant plants compared with wild-type plants. We suggest that ZmPrx5 positively regulates resistance against stalk rot in maize,likely through defense-oriented transcriptome reprogramming.These results lay a foundation for further research on the roles of Prx5 subfamily proteins in resistance to plant fungal diseases,and provide a potential genetic resource for breeding disease-resistance maize lines.

1. Introduction

As a devastating soilborne disease, maize stalk rot causes plant lodging and other problems,and results in substantial yield losses,reduced grain quality, and difficulty in harvesting [1-3].Fusarium graminearumis the most frequently reported causative agent of maize stalk rot [4,5], which tends to be more common in higheryielding hybrids that produce large ears [6]. The spores ofF.graminearumcan enter maize plants through the roots of seedlings from contaminated soil and can also enter the host stalk directly through mechanically damaged sites. The infection may spread to the upper internodes of the plant, weakening the stalk, which can cause the whole plant to fall over [6-8]. The pathogen also secretes mycotoxins such as deoxynivalenol and zearalenone,which pose threats to human and livestock health [8,9]. Because the pathogen spreads and infects plants via the soil, fungicides are ineffective for controlling maize stalk rot.The use of resistance genes will be the most economical and effective strategy for control of the disease.

Some quantitative trait loci (QTL) for stalk rot resistance have been reported [1,2,7,8,10-13], but only two QTL (qRfg1andqRfg2)have been cloned and identified[14,15].A CCT domain-containing gene,ZmCCT, was identified [14] as the gene responsible for stalk rot resistance associated with theqRfg1locus.ZmAuxRP1,encoding a plastid stroma-localized, auxin-regulated protein, was identified[15] as the gene responsible for stalk rot resistance at theqRfg2locus. The ZmAuxRP1 protein was found to modulate the balance between maize growth and stalk rot resistance. Recently, Ding et al.[16]reported that the oxygenation and subsequent desaturation ofent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yielded predominantlyent-kaurene-associated antibiotics, which are required for stalk rot resistance.Another study [17]indicated thatZmHIR3increases stalk rot resistance by controlling cell death. However, resistance to stalk rot is quantitatively inherited and controlled by multiple genes with additive effects [1,12]. No specific gene conferring immunity to the disease has been identified to date.Because stalk rot causes large-scale damage and resistance to the disease is complex, it is desirable to identify new resistance-associated genes or proteins.

Peroxiredoxin (Prx) is a family of antioxidant enzymes that scavenge reactive oxygen species (ROS) [18,19]. On the basis of their structural and biochemical properties, Prxs can be assigned to six subgroups: Prx1, Prx5, Prx6, Tpx, PrxQ, and AhpE. Only four of these (PrxQ, Prx1, Prx5, and Prx6) are found in plants [20]. The importance of Prx proteins is highlighted by their involvement in multiple cellular processes, including development [21,22], stress tolerance [23], signaling [24], and resistance to abiotic [25,26].Some Prx subfamilies are also involved in disease resistance. For example,the overexpression of a gentian PrxQ homolog in tobacco plants increased their tolerance to fungal diseases [27]. However,whether and how Prxs participate in resistance to maize stalk rot remains unknown.

The objective of this study was to identify proteins potentially associated with stalk rot resistance and to investigate potential response mechanisms using comparative proteomics analysis.Among the up-regulated proteins, ZmPrx5 (B4FN24) involved in antioxidant activity was selected for further study.ZmPrx5overexpressing and mutant plants were used to assess the response of ZmPrx5 in stalk rot resistance. The RNA-seq data obtained fromZmPrx5mutant and wild-type plants were also analyzed.

2. Materials and methods

2.1. Plant materials and F. graminearum inoculation

The inbred line Lx9801 was used to investigate the changes in maize in response toF. graminearuminfection. Maize plants were grown under normal conditions (15 h light at 28 °C and 9 h dark at 25°C with 60%humidity)until the 10-leaf stage.F.graminearumspores were produced by incubation for 5 days with shaking(25°C at 150 r min-1)in liquid mung bean broth(40 g mung bean per 1 L H2O).For artificial inoculations,a sterile micropipette tip was used to punch a hole in the stem at the third internode above the soil line,and 20 μL of spore suspension(106mL-1)was injected.Mock inoculations were performed with sterile water instead of spore suspension. The wounds were covered with sterile gauze, and the plants were maintained under normal growth conditions. At each tested time point,the inoculated internodes were cut and split longitudinally for symptom analysis. Three replicates were treated,and the collected stalks were stored at -80 °C for analysis.

2.2.Protein extraction,digestion and tandem mass tag(TMT)labeling

Proteomic analysis was performed using the stalks of Lx9801 at 8 days after inoculation (DAI) with either mock orF. graminearumsuspension. Total protein was extracted from each group as described[28].Protein concentration was measured with BCA Protein Assay Reagent (Promega, Madison, WI, USA). After digestion with trypsin (Promega) [29], peptides were labeled with 6-plex TMT reagents following the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The samples, labeled (Lx9801-Mock1)-126, (Lx9801-Mock2)-127, (Lx9801-Mock3)-128,(Lx9801-F. graminearum1)-129, (Lx9801-F. graminearum2)-130,and (Lx9801-F. graminearum3)-131, were pooled and vacuumdried.

2.3. Peptide fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The AKTA Purifier system (GE Healthcare, Piscataway, NJ, USA)was used to fractionate peptides by strong cation exchange chromatography [30]. The LC-MS/MS analysis was performed on a Q Exactive mass spectrometer coupled with an Easy-nLC instrument(Thermo Fisher Scientific,Waltham,MA,USA)as described[30,31].

2.4. Protein identification and data analyses

The MS raw data files were transformed into the MGF format using Proteome Discoverer 1.4 (Thermo Fisher Scientific). For protein identification and quantification, the UniProt Plant database(UniProt Zeamays)(http://www.uniprot.org)was used.The following search parameters were applied: peptide mass tolerance: ±20 μL L-1, MS/MS tolerance: 0.1 Da, and maximum missed cleavages: 2. Trypsin was used as the cleavage enzyme. The variable modifications were oxidation (M) and TMT 6-plex (Y), and the fixed modifications were carbamidomethyl (C), TMT 6-plex (Nterm), and TMT 6-plex (K). The integration window tolerance was set to 20 μL L-1. The peptide charge state was set to + 2 to+3.A false discovery rate(FDR)≤0.01 was used as the filtering parameter for peptides and proteins. Proteins differentially accumulated betweenF. graminearum- and mock-inoculated samples were those meeting the following criteria: fold change ＞1.5,FDR ＜ 0.05, and unique peptides ≥ 2. The original mass spectrometry-based proteomics data have been deposited in the ProteomeXchange Consortium [32] under the dataset identifier PXD023517.

Differentially accumulated proteins were functionally annotated with Gene Ontology(GO)[33]terms using the Blast2GO program (https://www.blast2go.com/). Metabolic pathways enriched with differentially accumulated proteins were identified using tools of the Kyoto Encyclopedia of Genes and Genomes (KEGG)database [34].

2.5. qRT-PCR analyses

Total RNA was extracted from the collected stalks with a Total RNA kit (Takara, Dalian, China). The Two-Step Prime Script RT Reagent Kit with gDNA Eraser (Takara) was used to synthesize cDNA.Three biological replicates were analyzed.qRT-PCR analyses were performed with a LightCycler 480II Real-Time PCR Detection System (Roche, Basel, Switzerland). The 2-ΔΔCTmethod [35] was used to calculate the relative expression levels with 18SrRNAas the standard.

2.6. Subcellular localization

The full-length coding sequence ofZmPrx5was amplified(Table S1)and cloned into the pCAMBIA1304 vector under the control of theCauliflower mosaic virus(CaMV) 35S promoter to produce CaMV35S::ZmPrx5-GFP. For investigating the subcellular localization of ZmPrx5, the empty CaMV35S::GFP vector was used as the control. The vectors were transfected independently into B73 maize protoplasts as described [36]. The protoplasts were incubated for 18 h at 25°C in the dark,and the GFP signal was visualized and photographed under a LSM710 confocal microscope(Zeiss, Jena, Germany).

2.7. Maize transformation and plant characterization

The full-length coding sequence ofZmPrx5was amplified and cloned into the pCAMBIA3301 vector under the control of the CaMV35S promoter and the NOS terminator. UsingAgrobacteriumtumefaciens-mediated transformation, this vector was introduced into the maize Hi-II (A188 × B73) hybrid [37] using the protocols described [38,39]. Forty positive T0plants were identified using PCR withbargene-specific primers (Table S1). Plants were selfpollinated to produce the T4transgenic generation.

The T4transgenic positive and negative plants, as well as the parents of Hi-II (A188 and B73) were inoculated withF. graminearumspores as described above. Stalks were harvested at 8, 10,14, 16, 18, 20, and 28 DAI. The longitudinal length of brown infected areas was measured as the lesion size at each time using ImageJ software [40]. Three replicates were treated.

2.8. Antibody production and Western blot analysis

A rabbit polyclonal antibody to ZmPrx5 was produced using the standard WB guaranteed polyclonal antibody protocol (http://www.genecreate.com/m-170.html). Briefly, the coding sequence ofZmPrx5was cloned into pET-SUMO (Invitrogen, Carlsbad, CA,USA). The recombinant ZmPrx5-His6 fusion protein was purified fromEscherichia coliusing a Ni-NTA purification system following the supplier’s instructions (Invitrogen). Rabbit polyclonal anti-ZmPrx5 was produced by immunizing rabbits with the purified ZmPrx5-His6 fusion protein.

For Western blot analysis, total proteins were extracted from stalks and a 15 μg aliquot of total protein was separated on 12%SDS-PAGE gel. An electrophoretic transfer system (Bio-Rad, Hercules, CA, USA) was used to transfer the samples to a polyvinylidene difluoride membrane. The membranes were then blocked in 5%w/v skim milk in PBST(10 mmol L-1Tris-HCl,pH 7.5,150 mmol L-1NaCl, 1% Tween-20) for 1 h at room temperature. The membranes were incubated with the primary antibody at 4°C overnight and then with the secondary antibody for another 1 h at room temperature. Protein was visualized by using the Super ECL Detection Reagent (Solarbio, Beijing, China).

2.9. Mutant analysis

ZmPrx5UniformMu (mu1054387 in UFMu-07007) plants were ordered from the Maize Stock Center (http://www.uiuc.edu/ph/www/maize). The UniformMu insertion was confirmed using a modified PCR protocol [41] with a pair ofZmPrx5-specific primers(one upstream and one downstream of the insert site) and a Mu TIR-specific primer(Table S1).The homozygous mutant and corresponding wild-type plants inoculated withF. graminearumwere used to measure resistance levels.

2.10. RNA-Seq analysis

Homozygous mutant and corresponding wild-type plants were inoculated withF. graminearum, and stalks were collected at 20 DAI for RNA-Seq analyses. Three replicates were analyzed. Using 3 μg total RNA from each sample as input material, sequencing libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following the manufacturer’s recommendations. The libraries were sequenced on the Illumina HiSeq (Illumina, San Diego, CA, USA) platform.The raw sequence reads have been deposited in the NCBI Sequence Read Archive under BioProject number PRJNA691732. Clean reads were aligned to theZea maysreference genome (ftp://ftp.ensemblgenomes.org/pub/plants/release-41/fasta/zea_mays/dna) using TopHat v2.0.12 [42]. Then, HTSeq v0.6.1 [43] was used to count the number of reads mapped to each gene, and the fragments per kilobase (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to that gene.The DESeq R package (1.18.0) [44] was used for the differential expression analysis. The resultingP-values were adjusted using Benjamini and Hochberg’s approach [45] to control the FDR. Genes with an adjustedP-value ＜0.05 identified by DESeq were identified as differentially expressed genes (DEGs). The identified DEGs were subjected to KEGG enrichment analyses.

To validate DEGs, 15 representative genes were subjected to qRT-PCR analysis. Spearman’s rank correlation coefficients between qRT-PCR and RNA-Seq expression values were computed.

2.11. Statistical analysis

One-way ANOVA with Tukey’spost hoctest were performed to separate means at a significance level ofP＜0.05.

3. Results

3.1. Identification of differentially accumulated proteins in maize in response to F. Graminearum infection

To identify proteins related to resistance toF. graminearumin maize, we used a quantitative proteomics analysis. The maize inbred line Lx9801 was inoculated withF.graminearumspore suspensions or a mock solution. At 8 DAI,F. graminearum-inoculated maize plants showed severe stalk rot symptoms(Fig.1A),whereas mock-inoculated plants remained healthy (Fig. 1B). LC-MS/MS yielded 24,738 spectra corresponding to 6814 proteins (Fig. 1C;Tables S2, S3), of which 763 were differentially accumulated, with 436 up-and 327 down-accumulated in response toF.graminearuminfection (Fig. 1D; Table S4).

3.2. Functional classification and biological pathways of differentially accumulated proteins

The KEGG pathways most enriched in differentially accumulated proteins were metabolic pathways,biosynthesis of secondary metabolites,and carbon metabolism(Figs.2A,S1).The GO subcategories enriched in differentially accumulated proteins were single-organism metabolic process and oxidation-reduction process(Fig.2B).Thus,a wide range of cellular processes may function in maize response toF. graminearuminfection. In the molecular function category, the subcategory most enriched in differentially accumulated proteins was oxidoreductase activity (Fig. 2C), indicating that ROS metabolism functions in stalk rot resistance.Among these up-accumulated proteins,ZmPrx5(B4FN24)involved in antioxidant activity was selected for further study.

3.3. Expression pattern and subcellular location of ZmPrx5

We identified expression patterns ofZmPrx5by qRT-PCR. We detected transcripts ofZmPrx5in multiple maize tissues,including root, stalk, leaf, ear, and reproductive tissues (Fig. 3A). We also measured the expression ofZmPrx5in Lx9801 after inoculation withF. graminearum, and found that it was significantly upregulated byF. graminearuminfection (Fig. 3B). Western blotting showed that the protein levels of ZmPrx5 were up-accumulated afterF. graminearuminfection (Fig. 3C). We also measured the expression ofZmPrx5in other maize inbred lines, including A188 and B73.The expression ofZmPrx5in all of these lines was strongly up-regulated byF. graminearuminfection (Fig. 3D, E), in a pattern similar to that observed in Lx9801. These results suggest that ZmPrx5 may function in maize response toF. graminearuminfection.

Because protein localization is closely associated with its function, we performed subcellular localization analyses. When CaMV35S::ZmPrx5-GFP was transiently expressed in maize protoplasts,ZmPrx5 was localized in the cytoplasm(Fig.3F),like its protein homologs in other plants [46-48].

Fig.1. Comparative proteomic analysis.Phenotype of the maize inbred line Lx9801 at 8 days after inoculation with Fusarium graminearum(A)or mock(B)solution.Scale bars,1 cm. (C) TMT labeling and the LC-MS/MS workflow for the identification of differentially accumulated proteins in the maize inbred line Lx9801 after inoculation with F.graminearum or mock solution. Three biological replicates were analyzed. (D) Distribution of differentially accumulated proteins.

Fig.2. Functional annotation of differentially accumulated proteins in maize in response to F.graminearum infection.(A)Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of differentially accumulated proteins.Gene Ontology(GO)enrichment analysis of differentially accumulated proteins.Subcategories enriched in differentially accumulated proteins in biological process (B) and molecular function (C) categories.

Fig. 3. Expression patterns of ZmPrx5 and subcellular localization of ZmPrx5. (A) Transcript levels of ZmPrx5 in tissues of B73 as determined by qRT-PCR. (B) Relative expression levels of ZmPrx5 in maize inbred line Lx9801 at several days after inoculation(DAI)as determined by qRT-PCR.The expression levels of ZmPrx5 were normalized against that of 18S rRNA. (C) Protein accumulation levels of ZmPrx5 in Lx9801 infected with F. graminearum inoculation at several DAIs. β-Actin served as loading control.Relative expression levels of ZmPrx5 in maize inbred lines A188(D)and B73(E)at several DAIs as determined by qRT-PCR.The expression levels of ZmPrx5 were normalized against that of 18S rRNA. Values are means ± standard errors of means of three independent experiments. (F) Subcellular localization of GFP and ZmPrx5-GFP in maize protoplasts. Scale bars, 20 μm.

3.4. Overexpression of ZmPrx5 increased maize resistance to stalk rot caused by F. graminearum

Because the gene transcription and protein accumulation of ZmPrx5 were both significantly induced uponF. graminearuminfection, we further investigated whether increasingZmPrx5expression could increase maize resistance to stalk rot.To test this possibility, we generated transgenic maize plants overexpressingZmPrx5(CaMV35S::ZmPrx5) (Fig. S2). The results of qRT-PCR(Fig. 4A) and Western blot (Fig. 4B) analyses confirmed higher ZmPrx5 transcript and protein accumulation levels in T4transgenic positive lines(2073 and 2074)than in negative lines(2073sib and 2074sib).

To test the resistance of these transgenic positive and negative lines, we inoculatedF. graminearumspores into the third internodes of plants and then recorded stalk rot symptoms. Between 8 and 28 DAI, brown lesions caused by the infection were detected in both transgenic positive and negative lines (Fig. 5A). However,the lesions were significantly smaller in theZmPrx5transgenic positive lines than in the negative lines (Fig. 5B). Because the transgenic plants were developed from the Hi-II line (A188 × B73),we also investigated the phenotypes of the parents, A188 and B73, in response toF. graminearuminfection. The results showed that both A188 and B73 were susceptible to maize stalk rot(Fig. S3), indicating that the resistance of the transgenic positive lines was due to the overexpression ofZmPrx5. Thus, overexpression ofZmPrx5promoted resistance to maize stalk rot caused byF. graminearuminfection.

3.5.Increased susceptibility of ZmPrx5 mutant to stalk rot caused by F.graminearum

To further confirm the positive role of ZmPrx5 in maize resistance to stalk rot, a maize transposon insertion mutant ofZmPrx5was selected and selfed. Homozygous plants were identified by PCR analysis of genomic DNA (Fig. 6A). The expression levels ofZmPrx5were significantly lower in mutant than in wild-type plants(Fig.6B).Western blotting experiments confirmed that the ZmPrx5 protein band was present in wild-type plants but not inZmPrx5mutant plants (Fig. 6C).

The susceptibility of theZmPrx5mutant to stalk rot was then investigated. After inoculation withF. graminearumspores, the split internodes at the inoculation sites turned brown. This occurred slowly in wild-type plants but more rapidly in mutant plants(Fig.6D).Lesions were significantly larger inZmPrx5mutant than in wild-type plants(Fig.6E),indicating that the infection was faster,and more severe, in mutant than in wild-type plants.These results further support the positive role of ZmPrx5 in maize stalk rot resistance.

Fig. 4. Confirmation of transgenic maize plants. (A) Relative expression levels of ZmPrx5 in transgenic positive (2073 and 2074) and transgenic negative (2073sib and 2074sib) lines. The expression levels of ZmPrx5 were normalized against that of 18S rRNA. Values are means ± standard errors of means of three independent experiments.Different letters indicate significant differences(P ＜0.05),as determined by one-way ANOVA.(B)Western blot analysis of ZmPrx5 protein accumulation levels in transgenic positive and transgenic negative lines.β-Actin served as loading control.Numbers above gel lanes represent the ratio of band intensity between the ZmPrx5 band and β-Actin band for each lane.

3.6. ZmPrx5-induced transcriptome reprogramming in maize in response to F. Graminearum infection

To elucidate the potential mechanism underlying ZmPrx5-mediated resistance to stalk rot in maize,we performed a comparative transcriptome analysis ofZmPrx5mutant and wild-type plants infected withF. graminearumat 20 DAI. The differences in the expression levels of each gene between the mutant and wildtype plants infected withF.graminearumwere measured.We used stringent values of FDR ＜0.05 and fold change ＞2 as thresholds to identify genes showing differences in expression levels. Of 3884 genes identified as DEGs, 1795 were up-regulated and 2089 down-regulated in mutant plants relative to wild-type plants(Fig. 7A; Table S5). To validate the results obtained by RNA-Seq analysis,qRT-PCR was performed to measure the expression levels of 15 DEGs identified by RNA-Seq. The expression levels of the 15 genes were generally consistent (R2= 0.9329) with the RNA-Seq data in the mutant and wild-type plants (Fig. 7B).

To elucidate the pathways involved inZmPrx5-mediated maize resistance toF. graminearuminfection, a KEGG analysis was performed. The pathways enriched with down-regulated DEGs in the mutant line were those involved in defense response,including plant-pathogen interactions, phenylalanine metabolism, benzoxazinoid biosynthesis, and flavone and flavonol biosynthesis(Fig. 7C; Table S6). Thus, defense-oriented transcriptome reprogramming was suppressed in the absence ofZmPrx5. The expression patterns of five defense-associated genes were further investigated in transgenic positive and negative lines at 20 DAI.All five genes were up-regulated in transgenic positive lines relative to negative lines (Fig. 7D). Taken together,these findings suggest a link betweenZmPrx5-mediated stalk rot resistance and defense-oriented transcriptome reprogramming.

Fig. 7. Transcriptomic analyses of ZmPrx5 mutant and wild-type plants infected with F. graminearum. (A) Numbers of down- and up-regulated genes between ZmPrx5 homozygous mutant and corresponding wild-type plants after F. graminearum inoculation. (B) Validation of expression patterns of 15 selected genes by qRT-PCR. Fold changes in expression levels in ZmPrx5 mutant relative to wild-type plants were transformed to a log2 scale.Linear regression of log2 fold changes between mutant and wildtype plants measured by qRT-PCR (y-axis) and by RNA-Seq (x-axis). Coefficient of determination (R2) = 0.9329. Each point represents one gene. Values are means of three biological replicates. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of down-regulated DEGs in the ZmPrx5 homozygous mutant and corresponding wild-type plants. (D) Expression patterns of five defense-associated genes in transgenic positive (2073 and 2074) and transgenic negative (2073sib and 2074sib)lines at 20 days after inoculation(DAI).The expression levels of each gene were normalized against that of 18S rRNA.Values are means±standard errors of means of three independent experiments. Different letters indicate significant differences (P ＜0.05), as determined by one-way ANOVA.

4. Discussion

Comparative proteomics analysis is an attractive way to identify resistance- or defense-associated proteins, and to elucidate the mechanisms involved in plant-pathogen interactions [49-51].Based on comparative proteomics analysis,our previous study revealed thatZmREM1.3plays important roles in southern corn rust resistance[28].In this study,comparative proteomics analysis showed that differentially accumulated proteins were involved in the oxidation-reduction process and oxidoreductase activity, indicating that ROS metabolism functions in stalk rot resistance.A previous study [52] showed that antioxidant proteins are directly involved in defense against ROS. Overexpression of an antioxidant protein in tomato increased the capacity of the ROS scavenging system, resulting in resistance to an oomycete pathogen. Antioxidant proteins also function in cell wall modifications by catalyzing lignification, implying that plant cell-wall structures are changed around the sites of pathogen entry. This was observed to occur around the sites ofF. oxysporumingress, and established physical barriers to pathogen invasion [53,54]. In the present study,comparative proteomics, qRT-PCR, and Western blotting analyses confirmed that the antioxidant protein ZmPrx5 was induced byF.graminearuminfection. These results suggest that ZmPrx5 plays a role in the resistance of maize toF. graminearum.

Prxs are thiol peroxidases that have multiple functions in antioxidant defense and redox signaling networks [55]. Previous studies[56-58]have shown that Prxs act to protect plants against oxidative and abiotic stress. Overexpression of aPrxin potato increased tolerance to methyl viologen-mediated oxidative stress and high temperature [59]. APrxfromTamarix hispidaincreased plants’ tolerance to salt stress by increasing the expression and activity of antioxidant enzymes and increasing ROS scavenging capacity [60]. Some Prx subfamilies are also involved in disease resistance. PrxQ was shown [27] to participate in plant resistance to a fungal pathogen. In our study,ZmPrx5transgenic positive plants showed greater resistance toF. graminearuminfection than transgenic negative plants,whileZmPrx5mutant plants were more susceptible than wild-type plants, showing thatZmPrx5increases maize resistance to stalk rot. Differences resistance performance between transgenic and mutant plants may be due to the differing genetic backgrounds. Taken together, these results confirm that a Prx5 subfamily protein participates in resistance to fungal disease.

RNA-Seq analyses of theZmPrx5mutant and wild-type afterF.graminearuminfection revealed DEGs acting in pathways associated with disease resistance, including mitogen-activated protein kinases, heat shock proteins, pathogenesis-related (PR) proteins,and hormone signaling proteins [61-63]. Plants often produce PR proteins and hormones as a defense strategy against pathogen invasion[4,64-67].Defense-associated secondary metabolite pathways including benzoxazinoid and flavonoid biosynthesis were enriched in down-regulated DEGs in the mutant. Benzoxazinoids are secondary metabolites that are effective in defense and innate immune responses against pests and diseases[15,68,69].In a previous study[70], maize wall-associated kinase genes conferred resistance to northern corn leaf blight that was correlated with reduced benzoxazinoid content.Flavonoids have diverse functions,including roles in defense responses against pathogens and ROS scavenging[71,72]. In another study [73], activation of flavonoid biosynthesis under phosphate deficiency increased resistance of cotton plants to the pathogenVerticillium dahliae. Taken together, these results suggest a link betweenZmPrx5-mediated stalk rot resistance and defense-oriented transcriptome reprogramming.

5. Conclusions

A comparative proteomics approach was used to identify candidate proteins associated with stalk rot resistance. On the basis of those results and the results of qRT-PCR and Western blot analyses,an up-accumulated antioxidant protein ZmPrx5 was selected for further study. Analyses of transgenic and mutant plants showed thatZmPrx5promotes maize stalk rot resistance. Transcriptomic analysis suggested a link betweenZmPrx5-mediated stalk rot resistance and defense-oriented transcriptome reprogramming. These findings lay a foundation for further studies of the roles of plant Prx5 subfamily proteins in resistance to fungal diseases, and provide a potential genetic resource for breeding disease-resistant maize lines.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Liuji Wu:Experimental design, Funding acquisition, Writing -review and editing.Shunxi Wang, Wencheng Liu, Zan Chen, Jinghua Zhang:Sample processing,Investigation,Validation,Writing- original draft.Xingmeng Jia, Mingyue Gou, Xueyan Chen,Yuqian Zhang,Hehuan Li,Yanhui Chen:Investigation and Validation. All authors read and approved the manuscript.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (U1804113, 31872872 and 31671675), the National Key Research and Development Program of China(2016YFD0102000),the Open Project Funding of the State Key Laboratory of Crop Stress Adaptation and Improvement, the 111 Project#D16014, and Shandong Provincial Natural Science Foundation (ZR2015CM034 and ZR2016CM30).

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.12.001.