Maize CMT030 plants were grown in a greenhouse at 28°C under a 16 h light/8 h dark cycle. The fungal strain was grown at 28°C in potato dextrose agar (PDA) medium for 7 days. Healthy maize seedlings grown for 14 days (3-leaf stage) were treated with a B. maydis spores suspension (10⁵ mL⁻¹) in sterile deionized water. The B. maydis spore suspension was sprayed onto maize leaves, and these were covered with plastic film for 24 h to maintain moisture in the treated areas. For SA treatment, 1 mM SA (Sangon Biotech Co., Ltd., Shanghai, China) was sprayed onto maize seedlings not subject to B. maydis infection. The leaves were harvested for RNA extraction at 0, 12, 24, 48, and 60 h post B. maydis inoculation, and at 0, 1, 6, 12, and 24 h post SA treatment. Untreated plants were harvested at the same time points and used as controls. Six maize plants were collected per treatment at each time point. Three biological replicates were used for all treatments.

The GSDS website (Gene Structure Display Server¹) was used to analyze ZmNBS25 gene structure (Zhang et al., 2013). Alignments between ZmNBS25 and other functional R proteins were performed in MEGA6 (Koichiro et al., 2013), and a phylogenetic tree was constructed by the neighbor-joining method based on whole protein sequences and considering 1,000 bootstrap replicates (Liu et al., 2016). Spatio-temporal expression of ZmNBS25 during maize development was investigated using the microarray data of B73 maize from PLEXdb (Sekhon et al., 2011; Dash et al., 2012). A heat map of the spatio-temporal expression of ZmNBS25 was generated in R/Bioconductor². Promoters of ZmNBS25 were analyzed by RSAT³ (Medina-Rivera et al., 2015).

Total RNA extracted from the leaves of CMT030 seedlings was used to synthesize first-strand cDNA. The full-length cDNA products of ZmNBS25 were obtained using PrimeSTAR Max DNA Polymerase (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) and forward (5′-ATGGCAGAAGCTGTGGTGTT-3′) and reverse (5′-CTATATGCGCAACTCCAGACC-3′) primers, under 35 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 15 s. The products were then cloned and sequenced.

The full-length coding sequence of ZmNBS25 without a termination codon was inserted into the vector pCAMBIA1301 to generate the 35S::ZmNBS25 vector. The 35S::ZmNBS25 and pCAMBIA1301 (control) vectors were transformed into the Agrobacterium tumefaciens strain GV3101. About 100 μL of A. tumefaciens suspensions carrying 35S::ZmNBS25 or control constructs were injected into 4-week N. benthamiana leaves using a small syringe as previously described (Cesari et al., 2014; Zhang et al., 2017). Staining with 1 g L⁻¹ 3′-Diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, United States) and 1 g L⁻¹ lactophenol-trypan blue (Sangon Biotech Co., Ltd., Shanghai, China) was performed as previously described (Hwang and Hwang, 2011; Zhang et al., 2017). N. benthamiana leaves were treated overnight with DAB and the stained leaves were cleared with 95% ethanol. For trypan blue staining, leaves were boiled in the trypan blue solution for 5 min and destained overnight in 2.5 g mL⁻¹ chloral hydrate. The destained leaves were observed under a microscope (Leica DM5000 B, Leica Microsystems Ltd., Heerbrugg, Switzerland). Three biological replicates were used for the experiment.

Electrolyte leakage was determined according to Hwang and Hwang (2011). N. benthamiana leaves were infiltrated with A. tumefaciens GV3101 carrying 35S::ZmNBS25 or control constructs to determine electrolyte leakage. Thirty N. benthamiana leaf disks approximately 1 cm in diameter were punched out with a cork borer, washed in 20 mL double distilled water for 30 min, and then transferred to fresh double distilled water (20 mL). Ion leakage was measured at 0 and 48 h after infiltration. Conductance was measured with a conductivity meter (DDS-11A) and the electrical conductivities of the media were expressed as μS cm⁻¹. Three biological replicates were set for the experiment.

The rice cultivar Zhonghua 11 was selected for ZmNBS25 transformation. Embryogenic calli were obtained using 14-day-old immature embryos. Active embryogenic calli were harvested for transformation with A. tumefaciens GV3101 containing the 35S::ZmNBS25 constructs, which was performed as previously described (Datta et al., 2000). Briefly, embryogenic calli were co-cultivated for 2 days with A. tumefaciens GV3101 in N6 medium (Sigma-Aldrich) containing 200 μM acetosyringone (Sangon Biotech Co., Ltd.). They were then thoroughly washed with sterile water and transferred to fresh medium containing 250 mg L⁻¹ cefotaxime and 50 mg L⁻¹ hygromycin (both Sangon Biotech Co., Ltd.). After several selection cycles, the embryogenic calli were used to propagate plants in regeneration medium. The transgenic plants were transplanted to pots filled with soil. Independent transgenic lines expressing 35S::ZmNBS25 were identified by Southern blot analysis as previously described (Jia et al., 2011; Zhao et al., 2014). Genomic DNA was extracted from the leaves of T0 transgenic lines using CTAB method (cetyl trimethyl ammonium bromide) (Clarke, 2009), and digested by the restriction enzyme EcoRV (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) overnight at 37°C. A fragment of the hygromycin gene, labeled with digoxigenin by a PCR DIG Probe Synthesis Kit (Roche Molecular Diagnostics, Pleasanton, CA, United States) was used as the hybridization probe.

Arabidopsis plants were transformed with the floral dip method (Clough and Bent, 1998) using A. tumefaciens GV3101 carrying the 35S::ZmNBS25 construct or the control vector. Transformed Arabidopsis seeds were sown onto Murashige and Skoog (MS; Sigma-Aldrich) agar plates containing 20 mg mL⁻¹ hygromycin (Roche Molecular Diagnostics) to screen for positive transformants, which were then transplanted for further growth. Genomic DNA was extracted from the leaves of transgenic Arabidopsis lines using the CTAB (cetyl trimethyl ammonium bromide) method (Clarke, 2009). The DNA extracted from each transgenic Arabidopsis plant was used as template to determine positive transgene integration by PCR. The 2× Taq Master Mix (Dye Plus; Vazyme Biotech Co., Ltd., Nanjing City, PRC) was used for PCR amplification under the following profile: 95°C for 10 s, 32 cycles of 55°C for 30 s, and 72°C for 30 s. Seeds from positive transformants were harvested, subjected to hygromycin screening, and the process was repeated until only seeds capable of growing in hygromycin medium remained. The homozygous T3 generation was selected for subsequent experiments.

In Arabidopsis and rice, SA was extracted and quantified according to a previously described method (Meuwly and Metraux, 1993; Cho et al., 2013). Briefly, leaves were flash-frozen in liquid nitrogen and ground to a very fine powder. The SA extracted from 0.5 g Arabidopsis leaf powder was determined by high-performance liquid chromatography (HPLC) (Agilent 1100 series with a C18 column, Agilent Technologies, Santa Clara, CA, United States), using SA from Dikma (Beijing, China) as the internal standard. The SA extracted from 0.3 g rice leaf powder was also determined by HPLC (Agilent 1200 series with a C18 column, Agilent Technologies) using SA from Sigma-Aldrich as the internal standard.

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was grown for 2 days at 28°C on King’s B (KB) medium (tryptone 20 g L⁻¹, glycerol 10 mg L⁻¹, K₂HPO₄ 1.5 g L⁻¹, MgSO₄ 1.5 g L⁻¹, and agar 15 g L⁻¹). After this period, a single Pst DC3000 colony was used to inoculate 5 mL KB medium and left to proliferate for 1 day at 28°C. One milliliter of the culture was used to inoculate 100 mL KB and left to grow for 1 day. The bacterial suspension was centrifuged at 3,000 rpm for 15 min and the pellet was homogenized in a resuspension solution of 0.01% Silwet L-77 and 10 mM MgSO₄. The final optical density at 600 nm (OD₆₀₀) was adjusted to 1.0 and Arabidopsis presenting 14 to 18 rosette leaves were sprayed with the bacterial suspension [10⁵ colony forming units (CFU) mL⁻¹] and control were sprayed with resuspension solution without Pst DC3000 (mock). Plants were then covered with plastic film for 3 days to retain moisture in the treated area. Evaluation of the disease index (DI) was performed 7 days post inoculation (dpi) as follows: DI (%) = [Σ(ni × vi)/(V × N)/100], where vi = disease rating; ni = number of plants with that disease rating; V = highest disease rating; and N = total number of observed plants (Niu et al., 2011). Three biological replicates were set and each replicate contained nine Arabidopsis plants. Cell death was measured by Evans blue staining as previously described (Xie et al., 2017).

To evaluate the resistance of transgenic rice to disease, rice overexpressing ZmNBS25 (ZmNBS25-OE) and control rice (EV) were inoculated with Rhizoctonia solani at the seedling stage using the leaf sheath inoculation method. Rhizoctonia solani cultured on PDA plates was cut into equal size pieces and then inoculated in rice leaf sheath (ZmNBS25-OE transgenic and control plant). Plants were then covered with plastic film to maintain moisture. Disease phenotype was obtained at 14 days post R. solani inoculation.

Total RNA was isolated with TRIzol (Thermo Fisher Scientific, Waltham, MA, United States) from 6 maize plants, 12 Arabidopsis plants, and 6 rice plants subject to each treatment including maize treatment with SA, B. maydis and control, Arabidopsis treatment with Pst. DC3000 and control, rice treatment with Rhizoctonia solani and control. Three biological repeats were performed for each treatment. DNase (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) was used to eliminate genomic DNA contamination. A reverse transcription kit (Roche Molecular Systems, Inc., Pleasanton, CA, United States) was used to synthesize first-strand cDNA from 1 μg total RNA from each sample. Quantitative real-time polymerase chain reactions (qRT-PCR) were run on an Applied Biosystems 7300 system (Applied Biosystems, Foster City, CA, United States) using the primers listed in Supplementary Table S1. The relative expression level of genes investigated in this study was calculated with the formula 2^−ΔΔCt, ΔΔCt = (CT_gene - CT_actin)_treat - (CT_gene - CT_actin)_control (Zhang et al., 2017).

The data were analyzed by Student’s t-test to evaluate differences between control and treated samples. Statistical significance was set at ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. Each assay contained three independent replicates.

Our previous study has identified 109 NBS encoding genes in maize (Cheng et al., 2012), including ZmNBS25. The protein encoded by this gene clustered with the Arabidopsis disease resistance protein AtRPM1 (Cheng et al., 2012). The full-length cDNA sequence of ZmNBS25 (GRMZM2G050959) is 3077 bp long, containing a 2736 bp coding sequence (Supplementary Figure S1). The phylogenetic analysis, based on whole protein sequences, showed that ZmNBS25 clustered with HvSL8 of Hordeum vulgare, ZmMRPR1 of Z. mays, SiRPM1-like of Setaria italica, and OsYR5 of O. sativa (Figure 1A and Supplementary Table S2). Whole-protein sequence identities between ZmNBS25 and HvSL8, ZmMRPR1, SiRPM1-like, OsYR5 were 45.84, 45.54, 47.46, and 46.92%, respectively. Protein sequence motif analysis showed that ZmNBS25 has conserved NBS motifs, including P-Loop, GLPL, kinase-2, MHD motif, and a C-terminal LRR domain (Bertioli et al., 2003; Cheng et al., 2012; Cesari et al., 2014) (Figure 1B).

Expression patterns of ZmNBS25 in various maize organs and different developmental stages were investigated by using the microarray data. We found that ZmNBS25 expressed highly in root and stem, but relatively low in other tissues, such as leaf and cob (Figure 2A). We further examined the spatial expression patterns of ZmNBS25 in seven tissues (root, stem, leaf, tassel, silk, husk, and cob) of maize by qRT-PCR and found that, similar to the microarray analysis, ZmNBS25 expression was high in root and stem, and low in leaf and cob (Figure 2B).

To test whether ZmNBS25 is responsible for disease resistance in maize, we sprayed either a spore suspension of B. maydis or SA on leaves to mimic the natural disease environment. After inoculating maize CMT030 with B. maydis, the expression level of ZmNBS25 was first reduced at 12 h post inoculation (hpi), followed by a significantly increased at 24, 48, and 60 hpi (Figure 3A). On the other hand, after SA treatment, the expression level of ZmNBS25 was consistently increased from 1 to 24 hpi when compared to control samples (Figure 3B).

To determine whether ZmNBS25 is involved in disease resistance in other crops, we cloned ZmNBS25 from maize CMT030 and generated an overexpression vector of ZmNBS25 using the pCAMBIA1301 vector with a CaMV35S promoter. After transforming the plasmid in A. tumefaciens GV3101, this was infiltrated into N. benthamiana leaves to verify whether transient ZmNBS25 overexpression could cause HR cell death. Large amounts of H₂O₂ accumulation were observed in N. benthamiana leaves after ZmNBS25 transient overexpression for 48 h by DAB staining (Figure 4A and Supplementary Figure S2). Trypan blue staining and electrical conductivity are two well established indicators of electrolyte leakage and correlate with the severity of visual damage (Zhang et al., 2016). As shown in Figure 4B, ion conductivity significantly increased in plants overexpressing ZmNBS25 compared with plants expressing pCAMBIA1301. Furthermore, trypan blue staining was darker in the leaves of ZmNBS25-OE plants than in the leaves of pCAMBIA1301 transgenic plants (Figure 4A and Supplementary Figure S2). These results suggested that the transient overexpression of ZmNBS25 in tobacco leaves induced HR and H₂O₂ accumulation, acting as a defense response to stress.

To investigate whether ZmNBS25 has a universal role in disease resistance, we cloned ZmNBS25 into a pCAMBIA1301 binary vector and generated ZmNBS25-OE transgenic Arabidopsis plants (Supplementary Figure S3). As shown in the HPLC chromatogram, total SA levels were drastically increased in ZmNBS25-OE plants (Peak area of OE1 was 460.4 g⁻¹ and peak area of OE2 was 486.2 g⁻¹) compared with non-transgenic Col-0 plants (Peak area was 75.5 g⁻¹) without any treatment (Figure 5A). We then examined the effect of ZmNBS25 overexpression after Pst DC3000 inoculation. Two transgenic Arabidopsis lines and Col-0 were sprayed with a virulent Pst DC3000 suspension (10⁵ CFU mL⁻¹). Seven days after inoculation, the ZmNBS25-OE lines showed significant resistance to Pst DC3000 (Figures 5B–D). Evans blue staining of infected Arabidopsis plants showed that cell death was more evident in Col-0 plants than in ZmNBS25-OE plants (Figure 5B). The disease severity of ZmNBS25-OE lines was 23.66% on average, while that of Col-0 plants was 45.58% (Figure 5C). The density of Pst DC3000 on ZmNBS25-OE plants was significantly lower than in Col-0 plants at 7 dpi (Figure 5D). Moreover, the density of Pst DC3000 grown on the ZmNBS25-OE1 plants were significant lower than that on the ZmNBS25-OE2 plants at 7 dpi (P = 0.0114) (Figure 5D). This might be caused by the higher ZmNBS25 expression in ZmNBS25-OE1 Arabidopsis plants than in ZmNBS25-OE2 plants (Supplementary Figure S3). These results strongly suggest that ZmNBS25 plays important roles in disease resistance.

To examine if the enhanced resistance to Pst DC3000 is related to changes in defense responsive genes, we measured the expression levels of some defense related genes in Col-0 and ZmNBS25-OE plants upon Pst DC3000 infection. Particularly, we examined the relative expression levels of genes involved in pathogen resistance (PR) and on the SA-dependent defense signaling pathway, including AtEDS1 (Enhanced Disease Susceptibility 1) (Falk et al., 1999), AtNDR1 (Non-race Specific Disease Resistance 1) (Day et al., 2006), AtTAO1 (Target of AvrB Operation) (Eitas et al., 2008), AtRPS5 (Resistance to P. syringae protein 5) (Warren et al., 1998), AtPR1, and AtPR5 in Col-0 and ZmNBS25-OE plants. Except for AtRPS5 and AtPR5, defense genes showed higher expression levels in ZmNBS25-OE plants than in Col-0 plants without Pst DC3000 infection. On the other hand, the expression levels of AtEDS1, AtNDR1, AtTAO1, AtPR1, and AtPR5 in Col-0 plants were significantly induced by Pst DC3000 (Figure 5E and Supplementary Tables S3, S4). Similar inductions were observed in Pst DC3000-infected ZmNBS25-OE plants compared with control ZmNBS25-OE plants, except AtPR5. When compared with Pst DC3000-infected Col-0 plants, the expression levels of AtEDS1, AtTAO1, and AtPR1 were significantly increased in the Pst DC3000-infected ZmNBS25-OE plants (Figure 5E and Supplementary Tables S3, S4). These results indicate that ZmNBS25 overexpression enhanced disease resistance of transgenic Arabidopsis against Pst DC3000.

To determine whether ZmNBS25 overexpression confers disease resistance in other heterologous plant systems, we generated ZmNBS25-OE transgenic rice lines. Southern blot analysis showed three separate transfer (T)-DNA insertions events with only one copy each (1, 5, and 7; Supplementary Figure S4); thus, we selected two independent transgenic lines, L5 and L7, for subsequent experiments. Plant resistant against biotrophic pathogens is usually regulated by the SA-dependent pathway (Felton and Korth, 2000), so we first measured the SA content in ZmNBS25-OE and control plants. HPLC chromatograms showed that ZmNBS25-OE lines accumulated more SA (peak area 309.489 g⁻¹) than control transgenic plants (peak area 74.749 g⁻¹) without pathogen inoculation (Figure 6A). To further determine the disease resistance capacity of ZmNBS25 overexpression, we inoculated R. solani, which can cause rice sheath blight, at rice leaf sheath. ZmNBS25-OE transgenic rice lines developed fewer and smaller disease lesions than control transgenic plants at 14 dpi (Figure 6B). We found that two typical defense related genes, OsPAL06 and OsPXa5, were significantly upregulated in ZmNBS25-OE lines after R. solani infection (Figure 6C).

To investigate the effect of overexpressing the ZmNBS25 gene on yield-related traits, we compared seeds’ size and 1000-grain weight between wild type (WT) and ZmNBS25-OE rice lines (Supplementary Figure S5). No phenotypic differences, including seed length and width, were observed between WT and ZmNBS25-OE rice lines (Supplementary Figures S5A,C,D). Except for ZmNBS25-OE line L7, no significant reductions in 1000-grain weight were observed in ZmNBS25-OE lines (L1, L5) (Supplementary Figure S5B). In addition, we observed similar results in Arabidopsis, where seeds showed no phenotypic differences between WT and ZmNBS25-OE lines (Supplementary Figure S6).

Nucleotide-binding site-leucine-rich repeat proteins play important roles in pathogen recognition and defense response signal transduction (Ameline-Torregrosa et al., 2008; Gao et al., 2010). Some NBS-LRRs that confer resistance to microbial pathogens and certain environmental stressors have been cloned from higher plants (Liu et al., 2007). In the present study, we cloned and systematically characterized a novel NBS-LRR-encoding gene, ZmNBS25, in maize. We found that ZmNBS25 could be induced by B. maydis inoculation and SA treatment. Overexpression of ZmNBS25 provides enhanced disease resistance in transgenic rice and Arabidopsis. This is similar to the function of ZmRXO1, which is a maize NBS-LRR gene involved in resistance to diverse pathogens, including rice blast (Zhao et al., 2004, 2005). Our phylogenetic analysis based on whole-protein sequences also showed that ZmNBS25 is closely related to ZmRXO1. In addition, ZmNBS25 has typical NBS-ARC, P-Loop, GLPL, kinase-2, MHD, and other conserved motifs similar to S-L8, MRPR1, RPM1-like, and YR5 proteins, suggesting that ZmNBS25 can participate in maize pathogen interactions or defense responses.

Our results indicated that ZmNBS25 was expressed at relatively low levels in uninfected maize leaves; however, it was significantly upregulated at 24 h post B. maydis inoculation, indicating that ZmNBS25 might be associated with B. maydis resistance in maize. Similar expression patterns have been observed in other plants NBS-LRR genes, such as: AhRRS5, a R. solanacearum resistance gene in peanut (Zhang et al., 2017); Xa1, a bacterial resistance gene from rice (Yoshimura et al., 1998); and SacMi, a M. incognita resistance gene (Zhou et al., 2018). In addition, the expression level of ZmNBS25 was significantly upregulated by SA treatment. SA is a well-known signaling molecule involved in defense against stress (Divi et al., 2010). It accumulates in plant tissues challenged by pathogen infections, and it can induce SAR and increase the expression of certain PR genes, which generally increase resistance to a wide range of diseases (Gaffney et al., 1993; Yang et al., 2013; Novakova et al., 2014). The expression of disease resistance genes such as RCY1 and NPR1 in Arabidopsis partially depends upon SA signaling (Cao et al., 1997; Takken and Joosten, 2000). Given the similar expression pattern of ZmNBS25 upon SA treatment and B. maydis induction, the enhanced defense response to B. maydis in ZmNBS25-OE lines is likely associated with SA signaling. Moreover, we also found that binding elements such as WBOXATNPR1, WRKY71OS, BIHD1OS, and GCCCORE could participate in plant disease resistance and were enriched in the ZmNBS25 promoter (Supplementary Figure S7). For example, WBOXATNPR1 recognizes the WRKY DNA binding proteins induced by SA (Eulgem et al., 2000), and WRKY71OS interacts with certain WRKY family members that play vital roles in plant disease resistance (Eulgem et al., 2000; Shimono et al., 2012). Together, these results suggest that ZmNBS25 plays a vital role in the defense response to diseases.

Activation of NBS-LRR proteins triggers programmed cell death and reactive oxygen species (ROS) production in plants (Andersson et al., 2006; Qi and Innes, 2013). Indeed, we found that ZmNBS25 might positively regulate SA-dependent cell death and disease resistance during pathogen infection. Transient overexpression of ZmNBS25 in N. benthamiana induced a HR and caused cell death and H₂O₂ accumulation without pathogenic infection. Therefore, ZmNBS25 could be involved in ROS signaling pathways for disease resistance. An R gene that can be integrated into a heterologous cereal crop will significantly improve disease resistance strategies for that species. An earlier study on the ZmRXO1 gene in rice showed that it is possible to transfer a NBS-LRR-type R gene to a distantly related cereal species (Zhao et al., 2005). Studies have demonstrated that SA is involved in defense responses mediated by plant NBS-LRR R proteins (Roberts et al., 2013). For instance, the expression of ADR1-L2D848V in Arabidopsis caused increased disease resistance and constitutively high SA levels (Roberts et al., 2013). In our study, ZmNBS25 overexpression in transgenic Arabidopsis and rice produced more SA than in control plants and enhanced transgenic plants resistance to disease, indicating that ZmNBS25 might participate in disease responses by inducing SA accumulation.

Without Pst DC3000 infection, the expression of defense genes AtEDS1, AtNDR1, AtTAO1, and AtPR1 was higher in ZmNBS25-OE plants than in Col-0 plants, indicating that ZmNBS25 overexpression affected the expression of defense-related genes and resulted in enhanced resistance to disease. In Pst DC3000 infected ZmNBS25-OE transgenic Arabidopsis plants, disease severity was reduced, and ZmNBS25-OE also affected the expression of some defense genes after Pst DC3000 inoculation as defense genes were significantly upregulated in ZmNBS25-OE plants after Pst DC3000 infection. Moreover, ZmNBS25-OE rice plants showed similar changes of defense-related genes after R. solani infection. These data indicated that ZmNBS25 overexpression could promote the activation of defense response in Arabidopsis and rice plants infected with Pst DC3000 and R. solani, respectively. Taken together, our results suggest that ZmNBS25 can function as a positive regulator to prime defense response upon pathogen infection.

The results in our current study strongly suggest that ZmNBS25 can function as a disease resistance gene across different species, being a valuable candidate for engineering resistance in breeding programs. Given the common difficulties of gene editing in maize, a cost-effective and high-efficient transformation method is desired in the future to further evaluate the disease resistance function of ZmNBS25 in maize by manipulating its gene expression.