Phosphorylation of the transcription factor OsNAC29 by OsMAPK3 activates diterpenoid genes to promote rice immunity

Abstract

Well-conserved mitogen-activated protein kinase (MAPK) cascades are essential for orchestrating of a wide range of cellular processes in plants, including defense responses against pathogen attack. NAC transcription factors (TFs) play important roles in plant immunity, but their targets and how they are regulated remain largely unknown. Here, we identified the TF OsNAC29 as a key component of a MAPK signaling pathway involved in rice (Oryza sativa) disease resistance. OsNAC29 binds directly to CACGTG motifs in the promoters of OsTPS28 and OsCYP71Z2, which are crucial for the biosynthesis of the phytoalexin 5,10-diketo-casbene and consequently rice blast resistance. OsNAC29 positively regulates rice blast resistance by promoting the expression of of OsTPS28 and OsCYP71Z2, and the function of OsNAC29 is genetically dependent on OsCYP71Z2 and OsTPS28. Furthermore, OsNAC29 interacts with OsRACK1A and OsMAPK3/6 to form an immune complex; OsMAPK3 phosphorylates OsNAC29 at Thr304 to prevent its proteasome-mediated degradation and promote its function against rice blast fungus. Phosphorylation of OsNAC29 at Thr304 is induced upon Magnaporthe oryzae infection and chitin treatment. Our data demonstrate the positive role of the OsMAPK3–OsNAC29–OsTPS28/OsCYP71Z2 module in rice blast resistance, providing insights into the molecular regulatory network and fine-tuning of NAC TFs in rice immunity.

OsMAPK3 phosphorylates OsNAC29, preventing its proteasome-mediated degradation and activating transcription of OsTPS28 and OsCYP71Z2 in the rice diterpenoid gene cluster to confer blast resistance.

Introduction

In response to pathogen challenge, plants have evolved 2 elaborate immune systems, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and Dangl 2006; Macho and Zipfel 2014). PTI and ETI are mutually reinforcing and complementary in plant immunity (Yuan et al. 2021). A series of immune responses are produced during the activation of plant immunity, which typically involves activation of mitogen-activated protein kinases (MAPKs), reactive oxygen species (ROS), Ca²⁺ influx, callose deposition, pathogenesis-related (PR) gene expression, and transcriptional reprogramming (Kadota et al. 2014; Saur et al. 2016). Transcriptional reprogramming governed by transcription factors (TFs) and coregulatory proteins is essential for increasing plant resistance by upregulating defense-related gene expression (Tsuda and Katagiri 2010). Accumulating evidence indicates that certain plant TF families, such as basic leucine zipper (bZIP), basic helix–loop helix, WRKY, MYB, and NAM, ATAF, and CUC (NAC) TFs, are involved in controlling the activation of innate immunity (Tsuda and Somssich 2015).

MAPK cascades are critical signaling modules in plant immunity (Kong et al. 2012; Ma et al. 2021; Wang et al. 2023b). In each MAPK cascade, a MAPK is activated via a MAPK kinase (MAPKK)-mediated phosphorylation following phosphorylation of the MAPKK by a MAPK kinase kinase (MAPKKK). Then, activated MAPK phosphorylates various substrate proteins to trigger plant immunity. In Arabidopsis (Arabidopsis thaliana), at least 2 complete MAPK cascades, the MEKK1-MKK1/MKK2-MAPK4 and MAPKKK3/MAPKKK5-MKK4/MKK5-MAPK3/MAPK6 cascades, are well known to be involved in the elicitation of immune responses (Asai et al. 2002; Bi et al. 2018). In rice, studies have indicated that MAPK3 is an important MAPKK involved in both biotic and abiotic stress responses. For example, OsMKK4 activated by chitin phosphorylates OsMAPK3 and OsMAPK6, leading to defense-related gene expression and antimicrobial synthesis (Kishi-Kaboshi et al. 2010). OsMAPK3 blocks OsBHLH002 ubiquitination by OsHOS1 to maintain the stability of OsBHLH002, which can activate OsTPP1 to enhance rice chilling tolerance (Zhang et al. 2017). MAPK3/6 phosphorylate PABP to enhance its binding to the R-motif and promote plant immunity (Wang et al. 2022). OsWRKY31 is phosphorylated by OsMAPK3, while the phosphomimetic OsWRKY31 increases its stability and confers enhanced resistance to Magnaporthe oryzae (Wang et al. 2023b).

The NAM gene in Petunia and the ATAF1 gene together with the CUC gene in Arabidopsis were previously identified as NAC TFs based on their similar and highly conserved N-terminal domain, which was named as the NAC domain (Aida et al. 1997). NAC proteins constitute a large plant-specific TF family that regulates plant development and stress responses (Puranik et al. 2012). NAC TFs have been reported to function in immunity via transcriptional regulation of different sets of target genes in plants. The expression of PR1, PR2, and PR5 is directly activated by the NAC TF NTL to enhance disease resistance in Arabidopsis (Seo et al. 2010). When LrNAC35, an NAC TF from Lilium regale, is ectopically overexpressed in petunia (Petunia hybrida), it specifically regulates the expression of Ph4CL, which is a lignin synthesis gene, to improve plant disease resistance (Sun et al. 2019). Arabidopsis NAC4 functions as a transcription suppressor of the ethylene-responsive element-binding factors (ERFs) ERF11 and ERF13 and inhibits the expression of the targets to trigger a hypersensitive response, which involves cell death induced by pathogenic bacteria (Lee et al. 2017). The tomato NAC protein JA2L induces stomatal immunity by regulating the expression of genes involved in salicylic acid metabolism (Du et al. 2014). In rice, ONAC066 directly binds the promoters of LIP9 and NC4D4 to regulate their expression and modulate the abscisic acid signaling pathway, sugar and amino acid accumulation to enhance rice resistance against M. oryzae and bacterial blight (Liu et al. 2018). The NAC TF ONAC083 negatively regulates rice immunity against M. oryzae by directly activating the transcription of the OsRFPH2-6 gene (Bi et al. 2023). MNAC3, a transcriptional activator, negatively modulates rice immunity against blast and bacterial leaf blight diseases by activating the transcription of immune-negative target genes OsINO80, OsJAZ10, and OsJAZ11 (Wang et al. 2024). OsNAC60 is targeted and regulated by miR164a, and miR164a overexpression and OsNAC60 mutation lead to decreased rice blast resistance (Wang et al. 2018). The NAC TF ONAC096 positively regulates rice blast and bacterial blight resistance by binding to the promoters of OsRap2.6, OsWRKY62, and OsPAL1 (Wang et al. 2021a).

Phytoalexins accumulate in plants invaded by pathogens and are secondary metabolites that exhibit antimicrobial activity against pathogens (Ahuja et al. 2012). 5,10-Diketo-casbene was first reported as a casbene-type diterpene phytoalexin in rice (Inoue et al. 2013) that confers resistance to bacterial blight and rice blast fungus (Zhan et al. 2020; Liang et al. 2021). DGC7 (a rice diterpenoid gene cluster on chromosome 7) is a gene cluster involved in rice immunity and encodes the entire biosynthetic pathway of 5,10-diketo-casbene (Zhan et al. 2020). 5,10-Diketo-casbene is derived by conversion of geranylgeranyl diphosphate (GGPP) to olefin ent-casbene by OsTPS28, followed by further elaboration by OsCYP71Z21 and OsCYP71Z2. DGC7 is crucial for the biosynthesis of 5,10-diketo-casbene (Liang et al. 2021). OsTPS28 knockout plants fail to produce 5,10-diketo-casbene, which decreases disease resistance against M. oryzae. OsCYP71Z2 encodes a C10 enzyme with casbene oxidase activity, and this activity represents an important step in the biosynthesis of medicinal casbene-derived diterpenoids (Li et al. 2013). OsCYP71Z2 is involved in rice immunity, and overexpression of OsCYP71Z2 can enhance bacterial blight (Li et al. 2013). However, how OsCYP71Z2 and OsTPS28 are regulated remains unknown.

Upon pathogen attack, many NAC genes are transcriptionally activated, but how their functions are regulated and how they regulate their targets to increase disease resistance remain largely unknown. In this study, we demonstrated the positive roles of OsNAC29 in rice against M. oryzae. Our findings showed that during M. oryzae infection, OsMAPK3/6-OsNAC29–OsRACK1A interact with each other to form an immune complex, and OsMAPK3 phosphorylates OsNAC29 at T304 to maintain its stability from 26S proteasome-mediated degradation. In addition, OsNAC29 binds the promoter and promotes the expression of OsTPS28 and OsCYP71Z2 to enhance rice blast resistance.

Results

OsNAC29 plays a positive role in rice blast resistance

To identify key factors involved in the interaction between rice and M. oryzae, we performed a transcriptome assay in ZH11 plants inoculated with M. oryzae. Among the differentially expressed genes (DEGs), a TF, termed OsNAC29, was significantly induced at 24 h after M. oryzae infection compared with the control (Supplementary Fig. S1). OsNAC29 has an NAC domain and belongs to the NAC TF family. Phylogenetic analysis revealed that OsNAC29 is a homolog of OsNAC122 and OsNAC131 (Supplementary Fig. S2), which are 2 positive regulators of defense responses against M. oryzae infection (Sun et al. 2013). Then, an RT-qPCR assay was performed and the result also showed that OsNAC29 is induced upon M. oryzae infection in ZH11 (Fig. 1A). To investigate the function of OsNAC29 in M. oryzae infection, Osnac29 mutants and OsNAC29-overexpressing (OsNAC29-OE) plants were constructed in the ZH11 background (Supplementary Fig. S3). When 3-wk-old plants of ZH11, Osnac29 mutants, and OsNAC29-OE plants were inoculated with M. oryzae strain Guy11 by the spray method, the Osnac29 mutants showed more lesions and greater relative fungal biomass, whereas the OsNAC29-OE plants showed fewer lesion number and lower relative fungal biomass than did the wild type (Fig. 1B and Supplementary Fig. S4A). Moreover, OsNAC29-OE plants showed more H₂O₂ accumulation compared with the wild type (Supplementary Fig. S4B). Consistent with these findings, after inoculation with the punch method, the Osnac29 mutants showed longer lesion length and increased fungal growth, whereas the OsNAC29-OE plants showed shorten lesion lengths and decreased fungal growth compared with that of the wild type (Fig. 1C). Moreover, the chitin-induced ROS burst decreased in the Osnac29 mutants and increased in the OsNAC29-OE plants compared with the wild type (Fig. 1D). Thus, the results above suggest that OsNAC29 functions as a positive regulator in rice blast resistance.

Figure 1.

OsNAC29 plays a positive role in rice immunity. A) The relative expression level of OsNAC29 in ZH11 before and after M. oryzae infection was detected using RT-qPCR, while that in ZH11 treated with water was used as the control. UBQ served as the internal control. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). B) Three-wk-old ZH11, Osnac29-1, and Osnac29-2 plants were inoculated with Guy11 by the spray method. Diseased leaves of all plants were photographed simultaneously at 4 dpi, and different plant samples were labeled with underline, scale bars = 1 cm (left). The relative fungal biomass in the inoculated leaves was measured using RT-qPCR at the DNA level (right). Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). C) Five-wk-old ZH11, Osnac29 mutants, and OsNAC29-OE plants were inoculated with Guy11 by the punch method. Diseased leaves of all plants were photographed simultaneously at 7 dpi, and different plant samples were labeled with underline, scale bars = 1 cm (left). The relative fungal biomass in the inoculated leaves was measured using RT‒qPCR at the DNA level (middle), and the lesion length was measured (right). Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (*P < 0.05, **P < 0.01). D) ROS accumulation dynamics in the ZH11, Osnac29 mutants, and OsNAC29-OE plants after chitin and water (mock) treatments. The error bars represent the Se, n = 16 biological replicates.

OsNAC29 binds to the CACGTG motif and promotes the expression of OsCYP71Z2

To better understand the function of OsNAC29, we examined the expression patterns of OsNAC29 at different developmental stages with an RT-qPCR. OsNAC29 transcripts were highly abundant in mature seeds, geminated seeds, and shoots (Supplementary Fig. S5). Subcellular localization analysis in rice protoplasts revealed that OsNAC29-GFP was localized in the nucleus (Fig. 2A). And a transcriptional activity assay showed that OsNAC29, but not the negative control, has transcriptional activity (Supplementary Fig. S6A). Furthermore, the activation domain was located within amino acids 254 to 346 of its C-terminal region (Fig. 2B). Moreover, we also performed a transcriptional activity assay in rice protoplasts and found that the activation of the LUC (firefly luciferase gene) reporter by OsNAC29 was 10-fold greater than that of the binding domain (BD) vector alone (Fig. 2C). These results suggest that OsNAC29 is a typical TF with transcriptional activity.

Figure 2.

OsNAC29 has transcriptional activity and activates the transcription of OsCYP71Z2 by binding to the CACGTG motif. A) Subcellular localization of OsNAC29-GFP. RFP-fused NLS was used as a nuclear marker, scale bars = 5 μm. BF, bright field. B) Transcriptional activity assay of OsNAC29 in yeast. OsNACO66 was used as a positive control, and BD-Lam was used as a negative control. C) Transcriptional activity assay in rice protoplasts. GAL4-BD was used as the negative control. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). D) Three-wk-old ZH11, Osnac29 mutants, and OsNAC29-OE plants were inoculated with isolated M. oryzae stain Guy11 by spray inoculation. The relative expression level of OsCYP71Z2 in the infected leaves was measured by RT-qPCR. UBQ served as the internal control. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). E) ChIP-qPCR was used to determine the binding of OsNAC29 to the OsCYP71Z2 promoter. P1 and P2 are 2 regions of the OsCYP71Z2 promoter that contain the CACGTG motif, and P3 represents a DNA fragment away from the binding region used as a negative control. The promoter of Ubiquitin was used as an internal control. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). F) EMSAs of recombinant OsNAC29 protein binding to the labeled P1 probe from the OsCYP71Z2 promoter. P1 probe was the mutated probe. The 100-, 200-, and 300-fold excess unlabeled probes were used for competition. Empty-His was used as the control. Arrows indicate the shifted bands and free probes. G) Transcriptional activation of the OsCYP71Z2 promoter by OsNAC29 in rice protoplasts. OsWRKY42 was used as a control. Data are means ± Sd (n = 3 biological replicates), and significant differences were determined by one-way ANOVA (**P < 0.01). EV, empty vector.

To identify the binding motif of OsNAC29, a DNA affinity purification sequencing (DAP-seq) assay was performed, and the results showed that CACGTG was most enriched among the detected motifs (Supplementary Fig. S6B) and the NAC domain of the TF preferentially binds G-BOX (CGTG motif) (Tran et al. 2007), suggesting that OsNAC29 may bind to the CACGTG motif on the promoter of defense-related genes. Then, a transcriptome assay was performed with ZH11 and OsNAC29-OE3 leaves to determine the target genes of OsNAC29. We found 152 upregulated and 29 downregulated genes (with a fold change >2) in OsNAC29-OE3 compared with the wild type (Supplementary Data Set 2). Among the DEGs, OsSPL11, OsPR1a, and OsCYP71Z2 were selected as candidate target genes of OsNAC29 because their promoters contain the CACGTG motif, and they have been reported to play important roles in rice immunity (Liu et al. 2015; Fan et al. 2018; Zhan et al. 2020; Yan et al. 2022).

Then, we used a yeast one-hybrid (Y1H) assay to verify the binding of OsNAC29 to the 3 genes. GAL4 transcriptional activation domain–OsNAC29 (PGAD41–OsNAC29) fusion protein, but not PGAD41 alone, can activate the LacZ reporter gene driven by the OsCYP71Z2 promoter, but not the OsPR1a or OsSPL11 promoter (Supplementary Fig. S6C); the possible reason is that although there are CACGTG motifs on the promoters of OsPR1a and OsSPL11, the flanking sequences of the motif decrease the affinity between the motif and OsNAC29, making it difficult to form a stable interaction. Furthermore, to investigate whether OsCYP71Z2 is the target gene of OsNAC29, RT-qPCR was used to determine the expression of OsCYP71Z2 in Osnac29 mutants, OsNAC29-OE plants, and ZH11 after inoculation with M. oryzae. The results showed that the expression of OsCYP71Z2 was induced upon M. oryzae infection in all infected plants, but was lower in Osnac29 mutants and higher in the OsNAC29-OE plants than in ZH11 (Fig. 2D). The binding of OsNAC29 to the OsCYP71Z2 promoter was subsequently validated via a chromatin immunoprecipitation (ChIP)-qPCR assay in vivo. The P1 and P2 regions containing sequences from −1,186 to −1,191 and −798 to −803 were more highly enriched in the genomic DNA immunoprecipitated by the anti-GFP antibody in the OsNAC29-GFP-OE plants than in the control (Fig. 2E). In addition, electrophoretic mobility shift assays (EMSAs) revealed that the motifs located at P1 and P2 of the OsCYP71Z2 promoter were bound by OsNAC29 (Fig. 2F and Supplementary Fig. S7). To determine the effect of OsNAC29 on OsCYP71Z2 transcription, 35S:OsNAC29 and ProOsCYP71Z2: LUC constructs were cotransfected into rice protoplasts, and the relative LUC activity was significantly increased to more than 100-fold compared with that when ProOsCYP71Z2: LUC cotransfected with EV (vector control); however, OsNAC29 could not activate the promoter of OsWRKY42, which was used as a control (Fig. 2G). The above results demonstrate that OsNAC29 promotes OsCYP71Z2 transcription by binding to the CACGTG motif and that OsCYP71Z2 is the target gene of OsNAC29.

OsCYP71Z2 positively regulates rice resistance to M. oryzae

To investigate the function of OsCYP71Z2 in rice immunity, Oscyp71z2 mutants and OsCYP71Z2-overexpressing (OsCYP71Z2-OE) plants were constructed in ZH11 (Supplementary Fig. S8). Then, the Oscyp71z2 mutants, OsCYP71Z2-OE plants and ZH11 were inoculated with the M. oryzae strain Guy11 by both the punch and spray methods. Compared with ZH11, OsCYP71Z2-OE plants exhibited increased resistance, with decreased lesion length, lesion number, and fungal biomass, whereas the Oscyp71z2 mutants showed decreased resistance, with increased lesion length, lesion numbers, and fungal growth (Fig. 3, A and B). Moreover, chitin-induced ROS burst increased in the OsCYP71Z2-OE plants, but decreased in the Oscyp71z2 mutants compared with the ZH11 plants in response to PAMP treatment (Fig. 3C). In addition, M. oryzae-induced H₂O₂ accumulation and chitin-induced callose deposition increased in the OsCYP71Z2-OE plants (Fig. 3, D and E). Furthermore, neither OsCYP71Z2 nor OsNAC29 is involved in rice development or growth (Supplementary Fig. S9). These results suggest that OsCYP71Z2 positively regulates rice resistance to M. oryzae.

Figure 3.

OsCYP71Z2 positively regulates rice immunity to blast fungus. A) Five-wk-old ZH11, Oscyp71z2 mutants, and OsCYP71Z2-OE plants were inoculated with M. oryzae isolate strain Guy11 by the punch method. Diseased leaves of all plants were photographed simultaneously at 7 dpi, and different plant samples were labeled with underline, scale bars = 1 cm (left). The quantification of fungal biomass in infected leaves was determined using qPCR at the DNA level (middle), and the lesion length was measured (right). Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). B) Three-wk-old ZH11, Oscyp71z2 mutants and OsCYP71Z2-OE plants were inoculated with M. oryzae isolate strain Guy11 by the spray method. Diseased leaves of all plants were photographed simultaneously at 4 dpi, and different plant samples were labeled with underline, scale bars = 1 cm (left). The relative fungal growth in the diseased leaves was determined using qPCR of DNA (right). Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). C) ROS accumulation dynamics in Oscyp71z2 mutants, OsCYP71Z2-OE plants, and ZH11 after chitin and water (mock) treatments. The error bars represent the Se (n = 16 biological replicates). D) Three-wk-old OsCYP71Z2-OE and ZH11 plants were inoculated with Guy11 by the spray method, after which leaves of both OsCYP71Z2-OE and ZH11 plants were cut and stained with DAB simultaneously two days later. Different plant samples were labeled with underline. Scale bars = 1 cm. E) Chitin-induced callose deposition on the 1-wk-old leaves of ZH11 and OsCYP71Z2-OE plants. The rice leaves were treated with 0.6 μM chitin, callose deposition was imaged with a microscope, scale bar = 50 μm, and the amount of callose deposition was quantified with ImageJ. Data are means ± Sd (n = 19 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01).

The function of OsNAC29 in rice immunity against M. oryzae genetically depends on OsCYP71Z2 and OsTPS28

A previous study showed that OsCYP71Z2 is a member of the rice diterpenoid gene cluster on chromosome 7 (DGC7). DGC7 is a gene cluster involved in rice immunity (Zhan et al. 2020) and encodes the entire biosynthetic pathway for 5,10-diketo-casbene (Liang et al. 2021). Considerable evidence suggests that 5,10-diketo-casbene is a rice phytoalexin that has antifungal activity against M. oryzae (Zhan et al. 2020). OsTPS28 is another member of DGC7 and plays a positive role in rice resistance against M. oryzae (Liang et al. 2021). Bioinformatics analysis revealed 2 CACGTG motifs in the promoter of OsTPS28. We hypothesized that OsNAC29 may also bind to the promoter of OsTPS28 to promote the transcription and enhance rice blast resistance, though OsTPS28 was not identified in the DAP-seq analyses. Therefore, the expression of OsTPS28 in the Osnac29 mutants and OsNAC29-OE plants before and after M. oryzae inoculation was detected. The results showed that the expression of OsTPS28 was downregulated in the Osnac29 mutants, but upregulated in the OsNAC29-OE plants (Fig. 4A). In vivo ChIP-qPCR assay revealed that the OsTPS28 promoter was most highly enriched at positions P1 and P2 (Fig. 4B). We further confirmed the binding of OsNAC29 to the OsTPS28 promoter using EMSA. The results revealed that OsNAC29 can bind to the P1 and P2 motifs of the promoter of OsTPS28, both of which contain the CACTCG motif (Fig. 4C). Moreover, a transcriptional regulation activity assay was performed to determine the effect of OsNAC29 on OsTPS28 transcriptional activation. 35S:OsNAC29 and ProOsTPS28: LUC constructs were cotransfected into rice protoplasts, and the relative LUC activity was significantly greater to over 100-fold compared with that of ProOsTPS28: LUC was cotransfected with EV (vector control) (Fig. 4D), which was similar to the effect of OsCYP71Z2. These data indicated that OsTPS28 was also a target gene of OsNAC29. To determine whether the increased rice blast resistance of plants overexpressing OsNAC29 depends on OsTPS28 and OsCYP71Z2, Ostps28 Oscyp71z2/OsNAC29-OE3 plants were generated by knocking out OsTPS28 and OsCYP71Z2 in OsNAC29-OE3 plants. After inoculation with M. oryzae, the enhanced resistance mediated by OsNAC29 overexpression was abolished in Ostps28 Oscyp71z2/OsNAC29-OE3 plants (Fig. 4E). These results suggest that the function of OsNAC29 in rice blast resistance is genetically dependent on OsTPS28 and OsCYP71Z2.

Figure 4.

OsNAC29 transcriptionally regulates OsTPS28 and contributes to rice blast resistance depending on OsCYP71Z2 and OsTPS28. A) Relative transcript levels of OsTPS28 in 3-wk-old ZH11, OsNAC29-OE, and Osnac29 plants, which were inoculated with Guy11 by the spray inoculation method, before and after M. oryzae infection were detected by RT-qPCR. UBQ served as the internal control. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). B) ChIP‒qPCR assay was used to determine the binding of OsNAC29 to the OsTPS28 promoter. The Ubiquitin promoter was used as an internal control. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (*P < 0.05, **P < 0.01). C) EMSA of recombinant OsNAC29 binding to the labeled P1 and P2 probes in the OsTPS28 promoter. The 100-, 200-, and 300-fold excess unlabeled probes were used for competition. Empty-His was used as the control. Arrows indicate the shifted bands and free probes. D) The transcriptional activation assay of the OsTPS28 promoter by OsNAC29 in rice protoplasts. Data are means ± Sd (n = 3 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). EV, empty vector. E) Rice blast resistance assay of ZH11, OsNAC29-OE3, and Ostps28 Oscyp71z2/OsNAC29-OE3 plants. Ostps28 Oscyp71z2/OsNAC29-OE3 plants were obtained by knocking out OsCYP71Z2 and OsTPS28 in OsNAC29-OE3 plants. Five-wk-old ZH11, OsNAC29-OE3, and Ostps28 Oscyp71z2/OsNAC29-OE3 plants were inoculated with M. oryzae strain Guy11 by the punch method. Diseased leaves of all plants were photographed simultaneously at 7 dpi (left), and different plant samples were labeled with underline, and the lesion length was measured (middle). The relative fungal biomass was determined using qPCR at the DNA level (right). Data are means ± Sd (n = 3 biological replicates). Scale bars = 1 cm. Significant differences were determined by one-way ANOVA (**P < 0.01).

OsNAC29 forms an immune complex with OsRACK1A and OsMAPK3/6

To further investigate the upstream regulator of OsNAC29, we screened the proteins that interact with OsNAC29 by performing a coimmunoprecipitation (Co-IP)–mass spectrometry (MS) assay (Supplementary Data Set 3) and identified a scaffold protein, OsRACK1A, that interacted with OsNAC29. OsRACK1A plays an important role in the production of ROS and in resistance against rice blast infection (Nakashima et al. 2008). We first examined their interaction by a yeast 2-hybrid (Y2H) assay and found that OsNAC29 interacted with OsRACK1A directly in rice (Supplementary Fig. S10A). A split-luciferase complementation assay (LCA) was further performed in Nicotiana benthamiana leaves, and luminescent signals were detected when OsNAC29-NLUC and OsRACK1A-CLUC were coexpressed (Supplementary Fig. S10B). Furthermore, the Co-IP assay showed that a complex containing OsNAC29-HA and OsRACK1A-GFP formed in rice protoplasts (Supplementary Fig. S10C). Moreover, the OsNAC29-red fluorescent protein (RFP) and OsRACK1A-GFP were colocalized in the nucleus (Supplementary Fig. S10D).

OsRACK1A functions as a scaffold protein in rice innate immunity by interacting with multiple proteins in the Rac1 immune complex (Nakashima et al. 2008). The protease-G protein–RACK1–MAPK cascade module plays an important role in plant immune signaling pathways (Cheng et al. 2015). In rice, OsMAPK3 and OsMAPK6 play important roles in immunity, and they can enhance rice blast resistance by phosphorylating TFs (Wang et al. 2023b), which led us to wonder whether OsMAPK3 and OsMAPK6 interact with OsRACK1A and OsNAC29. To validate this hypothesis, Y2H, Co-IP, and LCI assays were used to determine whether OsMAPK3/OsMAPK6–OsRACK1A–OsNAC29 can interact with each other. Interestingly, they all interacted among OsMAPK3/OsMAPK6–OsRACK1A–OsNAC29, indicating that they together form an immune complex (Fig. 5 and Supplementary Fig. S11). Most importantly, we found that OsMAPK3 physically interacted with OsNAC29 and colocalized in the nucleus (Fig. 5, A and B) and that the protein level of OsNAC29-GFP coimmunoprecipitated with OsMAPK3-HA increased after treatment with chitin or flg22 (Fig. 5E), suggesting that the perception of PAPMs may enhance the interaction between OsNAC29 and OsMAPK3. To further analyze the function of the OsMAPK3/OsMAPK6–OsRACK1A–OsNAC29 module in rice blast resistance, CRISPR/Cas9 technology was used to construct genome-edited mutants in the ZH11 background, including OsMAPK3, OsMAPK6, and OsRACK1A. Unfortunately, only the Osmapk3 mutant was obtained, because knocking out OsMAPK6 or OsRACK1A resulted in homozygous death. Compared with the wild type, Osmapk3 mutants inoculated with M. oryzae displayed increased susceptibility (Supplementary Fig. S12), which is consistent with the findings of a previous study (Wang et al. 2023b). Therefore, we focused on the relationship between OsMAPK3 and OsNAC29.

Figure 5.

OsMAPK3 interacts with OsNAC29. A) Colocalization of OsNAC29-RFP and OsMAPK3-GFP in rice protoplasts, scale bars = 5 μm. RFP, red fluorescence protein; BF, bright field. B) Interaction between OsNAC29 and OsMAPK3 was determined by the Y2H assay. Positively transformed yeast cells were grown on synthetic dextrose minimal media without Leu and Trp plates and further selected on Sd without Leu, Trp, Ade, and His but with x-α-gal (40 μg/mL). Photographs were taken after 3 to 5 d. C) The interaction between OsMAPK3-CLUC and OsNAC29-NLUC was verified by the LCA. OsMAPK3-CLUC and OsNAC29-NLUC were transiently coexpressed in N. benthamiana leaves by co-infiltration. Luminescence was detected by a low-light, cooled, CCD imaging apparatus after 3 d (left), while the recombinant proteins expressed in N. benthamiana leaves were detected using immunoblotting with anti-HA and anti-CLUC antibodies (right). D) Co-IP assay of OsNAC29-GFP and OsMAPK3-HA. The indicated proteins were coexpressed in rice protoplasts. After immunoprecipitation with anti-GFP (α-GFP) beads, precipitated proteins were detected by immunoblotting with an anti-HA (α-HA) antibody. E) Rice protoplasts cotransfected with OsMAPK3-HA and OsNAC29-GFP were treated with or without 100 μg/mL PAMPs for 15 min and subjected to a Co-IP assay. Proteins were precipitated using α-GFP beads. The input proteins and the precipitated proteins were subjected to immunoblotting with α-HA or α-GFP antibodies.

OsMAPK3 enhances rice blast resistance by stabilizing OsNAC29

A previous study showed that OsMAPK3 inhibits the OsHOS1–OsbHLH002 interaction and prevents ubiquitination-mediated degradation of OsbHLH002 during chilling stress (Zhang et al. 2017), and OsMAPK3 can phosphorylate and stabilize OsWRKY31 to promote resistance (Wang et al. 2023b). Thus, we hypothesize that OsMAPK3 may stabilize OsNAC29. To verify this hypothesis, we performed a time-course degradation assay in rice protoplasts. Rice protoplasts were treated with the protein synthesis inhibitor cycloheximide (CHX) to block OsNAC29-HA synthesis after transfection, and the OsNAC29-HA levels were determined by immunoblotting at different time points. A 26S proteasome inhibitor (MG132) was added to determine whether OsNAC29 is degraded via the ubiquitin‒proteasome system. The results showed that the degradation of OsNAC29 in rice protoplasts was obviously blocked by addition of MG132 after 2 h (Fig. 6A). The abundance of the OsNAC29 protein in the OsNAC29-OE3 plants was subsequently examined, and the results showed that the abundance of OsNAC29-GFP was low in the OsNAC29-OE3 plants. However, the abundance of OsNAC29-GFP markedly increased after inoculation with M. oryzae (Fig. 6B). Moreover, we compared OsNAC29-GFP degradation in WT protoplasts cotransformed with OsMAPK3-HA, OsRACK1A-FLAG, or 3AH-YFP. Compared with those of OsRACK1A-FLAG, the degradation rate of OsNAC29 obviously decreased at the tested time points when cotransformed with OsMAPK3 (Supplementary Fig. S13). In addition, we measured the accumulation of OsNAC29-HA in the presence of MG132 or OsMAPK3-YFP in rice protoplasts, and GFP was used as a control. The results showed that OsMAPK3 prevented the degradation of OsNAC29 by the 26S ubiquitin‒proteasome system (Fig. 6C). Furthermore, cell-free degradation assays revealed that in vitro purified OsNAC29-His was more stable when it was incubated with the total extract from the wild-type seedlings than when it was incubated with the Osmapk3 extract (Fig. 6D). Similarly, transiently expressed OsNAC29-HA in the wild-type protoplasts was more stable than that in the Osmapk3 protoplasts in the presence of the protein synthesis inhibitor CHX (Fig. 6E). The results above indicate that OsNAC29 is degraded via the ubiquitin proteasome system, which can be prevented by OsMAPK3.

Figure 6.

OsMAPK3 regulates OsNAC29 stability to enhance resistance to rice blast. A) Time-course degradation of OsNAC29-HA in ZH11 rice protoplasts. Cotransfected rice protoplasts were treated with CHX (250 μM) to block protein synthesis, and OsNAC29-HA levels were monitored by immunoblotting with HA antibody. The 26S proteasome inhibitor MG132 (40 μM) was added to determine whether OsNAC29-HA is degraded via the 26S proteasome pathway. The GFP was used as an internal control. B) Total proteins were extracted from OsNAC29-OE3 plants before and after Guy11 inoculation by the spray method and then subjected to IP assays with anti-GFP beads and immunoblotting with anti-GFP antibody. C) Degradation of OsNAC29-HA in the presence of OsMAPK3 or MG132 (40 μM) in rice protoplasts. The GFP was used as an internal control. D) Purified OsNAC29-His protein was incubated with the total extracts from ZH11 and the Osmapk3 mutant at room temperature for 0, 15, 30, 45, and 90 min, respectively. The level of the OsNAC29-His protein was analyzed by immunoblotting with an anti-His antibody. E) OsNAC29-HA was transiently expressed in the ZH11 and Osmapk3 rice protoplasts, which were subsequently treated with CHX (250 μM) and/or MG132 (40 μM). GFP was transiently coexpressed as an internal control. F) The protein levels of OsNAC29-GFP in OsNAC29-GFP/ZH11 plants and OsNAC29-GFP/Osmapk3 plants were detected by immunoblotting with anti-GFP antibodies. The transcription level of OsNAC29 was analyzed by RT-qPCR. Data are means ± Sd (n = 3 biological replicates). The relative protein levels were analyzed by ImageJ software. Significant differences were determined by one-way ANOVA (**P < 0.01). NS, no significant. G) Three-wk-old seedlings of Osmapk3, OsNAC29-GFP/ZH11, OsNAC29-GFP/Osmapk3, and ZH11 were inoculated with Guy11 by the punch method. Diseased leaves of all plants were photographed simultaneously at 7 dpi (top) and different plant samples were labeled with underline, and the lesion length was measured (middle). The relative fungal biomass was determined using RT‒qPCR at the DNA level (bottom). Data are means ± Sd (n = 10 biological replicates). Scale bars = 1 cm. Significant differences were determined by one-way ANOVA (**P < 0.01). To better detect OsNAC29-GFP proteins with immunoblotting, standard ECL substrate chemiluminescence solution and short exposure time were used for image B, while high-sensitivity ECL substrate chemiluminescence solution and long exposure time were used for image F.

To elucidate the biological significance of the OsNAC29–OsMAPK3 interaction in rice blast resistance, OsNAC29-GFP/ZH11 and OsNAC29-GFP/Osmapk3 plants were separated from F2 plants by crossing OsNAC29-OE plants (OsNAC29-GFP) with Osmapk3 mutants. Then, OsNAC29-GFP levels in OsNAC29-GFP/ZH11 plants and OsNAC29-GFP/Osmapk3 plants were detected by immunoblotting with GFP antibodies. We found that the protein level of OsNAC29-GFP was lower in OsNAC29-GFP/Osmapk3 plants than in OsNAC29-GFP/ZH11 plants, while there was no difference in the transcription level of OsNAC29 (Fig. 6F), indicating that OsNAC29-GFP was more unstable when OsMAPK3 was absent, suggesting that OsMAPK3 maintained the stability of OsNAC29 both in vivo and in vitro. To determine whether OsNAC29-mediated resistance is dependent on OsMAPK3, we inoculated OsNAC29-GFP/ZH11, OsNAC29-GFP/Osmapk3, Osmapk3, and wild-type plants with Guy11 and found that OsNAC29-GFP/Osmapk3 was more susceptible than OsNAC29-GFP/ZH11 plants to rice blast (Fig. 6G), indicating that OsNAC29 regulates disease resistance to M. oryzae partly in an OsMAPK3-dependent manner.

OsMAPK3 stabilizes OsNAC29 by phosphorylating OsNAC29 at Thr304

OsMAPK3 can interact with OsNAC29 directly and stabilize OsNAC29 in planta, and OsMAPK3 commonly functions by phosphorylating its targets (Wang et al. 2023a, 2023b; Zhang et al. 2024), which prompted us to investigate whether OsNAC29 is a phosphorylated substrate of OsMAPK3. Previous studies have shown that OsMAPK3 has autophosphorylation activity in vitro (Zhang et al. 2017; Wang et al. 2023b). We therefore performed an in vitro kinase assay by incubating purified recombinant OsNAC29-His protein with OsMAPK3-GST or without OsMAPK3-GST, followed by MS analysis of OsNAC29-His to identify the phosphorylation site. The threonine 304 residue (Thr304) of OsNAC29 was identified as the site phosphorylated by OsMAPK3 (Fig. 7A). To examine the function of the phosphorylation site of OsNAC29, we separately substituted the phosphorylated amino acid residue for aspartic acid (OsNAC29^T304D) to mimic constitutive phosphorylation and alanine acid (OsNAC29^T304A), which is structurally very similar to T and blocks phosphorylation at the site. The phosphorylation of OsNAC29-His by OsMAPK3-GST was detected in the presence of γ-³²P ATP, while mutation of T304 decreased the phosphorylation of OsNAC29 by OsMAPK3 (Fig. 7B). An in vivo phosphorylation assay was performed using an anti-phosphoserine/threonine (anti-pSpT) antibody that specifically recognizes the phosphorylated S or T residue. Consistently, we observed a strong T304 phosphorylation signal when OsNAC29-GFP was coexpressed with OsMAPK3-FLAG in N. benthamiana leaves, whereas no signal was detected in the negative control (Fig. 7C).

Figure 7.

OsMAPK3 stabilizes OsNAC29 by phosphorylating OsNAC29 at Thr304. A) Identification of the OsNAC29 residue phosphorylated by OsMAPK3 via MS analysis. In vitro kinase assays were performed with recombinant OsMAPK3-GST and OsNAC29-His, while the single OsNAC29 protein was used as a control. Annotated spectra for the phosphorylated peptide of OsNAC29 are indicated by an arrow indicating the phosphorylation site. B) In vitro phosphorylation assay was used to determine whether OsNAC29-His was phosphorylated by OsMAPK3-GST. Protein phosphorylation was detected by autoradiography (upper panel), and protein loading is indicated by Coomassie Brilliant Blue (CBB) staining (lower panel). C) In vivo phosphorylation assay was used to determine whether OsNAC29-GFP can be phosphorylated by OsMAPK3-FLAG at Thr304 in plants. Total proteins were extracted from N. benthamiana leaves coinfiltrated with the indicated construct combinations and subjected to IP with anti-GFP beads, followed by immunoblotting with the anti-pSpT antibody. D) In vitro phosphorylation of OsNAC29 by OsMAPK3 at Thr304 was detected by immunoblotting with an antibody specifically recognizing phosphorylated Thr304 of OsNAC29 (anti-pT304). E) In vivo phosphorylation of OsNAC29 by OsMAPK3 at Thr304 was detected by immunoblotting with antibody specifically recognizing phosphorylated Thr304 of OsNAC29. F) ATPase activity of the purified proteins in B was detected. Data are means ± Sd (n = 5 biological replicates). Significant differences were determined by one-way ANOVA (**P < 0.01). G) The stability of OsNAC29-FLAG and OsNAC29^T304A-FLAG expressed in Osmapk3 rice protoplasts after CHX treatment for 2 h was determined using immunoblotting with an anti-FLAG antibody, while GFP was transiently coexpressed as an internal reference. H) The purified proteins of OsNAC29-His and OsNAC29^T304A-His were incubated with the total extract from the ZH11 plant at room temperature for 0, 30, or 60 min and then subjected to immunoblotting, with an anti-His antibody.

To confirm that Thr304 in OsNAC29 is an important phosphorylation residue mediated by OsMAPK3, a site-specific antibody against OsNAC29 (OsNAC29^pT304) was developed. We confirmed the phosphorylation of OsNAC29 at T304 by OsMAPK3 in vitro and in vivo with the anti-OsNAC29^pT304 antibody (Fig. 7, D and E). Moreover, we further verified the T304 site by measuring ATP consumption. OsMAPK3 catalyzes the transfer of the phosphoric group on ATP to the hydroxyl group on OsNAC29 and OsNAC29^T304A in an in vitro kinase assay, which converts ATP to ADP. After the phosphorylation reaction, we measured the ATPase activity. The results showed that the ATP surplus in the kinase assay containing OsMAPK3 with OsNAC29 was significantly less than that of OsMAPK3 alone, while there was no significant difference between OsMAPK3 with OsNAC29^T304A and OsMAPK3 alone (Fig. 7F). Thus, the above results demonstrate that OsNAC29 is phosphorylated at Thr304 by OsMAPK3 both in vitro and in vivo.

Our results showed that OsMAPK3 can stabilize OsNAC29 and phosphorylate OsNAC29 at Thr304, which prompted us to test whether OsMAPK3 stabilizes OsNAC29 by phosphorylating Thr304. To confirm this hypothesis, the phosphomimetic mutants OsNAC29^T304D-FLAG and OsNAC29-FLAG were cotransiently expressed in ZH11 and Osmapk3 protoplasts. The results showed that OsNAC29-FLAG is more easily to be degraded in Osmapk3 cells than in ZH11 cells. However, OsNAC29^T304D-FLAG was more stable than OsNAC29-FLAG when expressed in Osmapk3 cells (Fig. 7G). In addition, an in vivo degradation assay was performed in ZH11 rice protoplasts. As shown in Supplementary Fig. S14A, the degradation rate of OsNAC29^T304A was faster than that of OsNAC29^T304D and OsNAC29. Furthermore, the purified OsNAC29-His fusion protein was more stable than OsNAC29^T304A-His when incubated with the total extract from ZH11 leaves (Fig. 7H). The purified OsNAC29-His protein was less stable than OsNAC29^T304D-His when incubated with the total extract from Osmapk3 leaves (Supplementary Fig. S14B), suggesting that OsMAPK3 stabilizes OsNAC29 by phosphorylating T304.

In addition, to assess whether phosphorylation of OsNAC29 is necessary for regulating the expression of OsCYP71Z2 and OsTPS28, we examined the transcriptional activation of OsNAC29, OsNAC29^T304A, and OsNAC29^T304D on OsCYP71Z2 and OsTPS28, as well as the expression levels of OsCYP71Z2 and OsTPS28 in FLAG-OsNAC29-OE/Osnac29-1, FLAG-OsNAC29^T304A-OE/Osnac29-1, and FLAG-OsNAC29^T304D-OE/Osnac29-1 plants. The results revealed that OsNAC29^T304D had higher transcription activation on OsCYP71Z2 and OsTPS28 than did OsNAC29^T304A, and the expression levels of OsCYP71Z2 and OsTPS28 were higher in FLAG-OsNAC29^T304D-OE/Osnac29-1 plants than in FLAG-OsNAC29^T304A-OE/Osnac29-1 and FLAG-OsNAC29-OE/Osnac29-1 plants (Supplementary Fig. S15), indicating that phosphorylation of OsNAC29 by OsMAPK3 is necessary for regulating the expression of OsCYP71Z2 and OsTPS28.

The phosphomimic OsNAC29 confers enhanced resistance to M. oryzae

To determine the function of Thr304 in OsNAC29-mediated rice immunity, we investigated whether pathogens or patterns could induce OsNAC29 phosphorylation at Thr304. First, we detected OsNAC29 phosphorylation at Thr304 by transfecting the OsNAC29-HA construct into ZH11 and Osmapk3 protoplasts after treatment with chitin for 20 min. OsNAC29 was phosphorylated at Thr304 in ZH11 but not in Osmapk3 protoplasts (Fig. 8A), indicating that OsMAPK3 is required for the phosphorylation of OsNAC29 at Thr304 in rice. Furthermore, we found that phosphorylation at Thr304 of OsNAC29 gradually increased after chitin treatment in ZH11 protoplasts (Fig. 8B). Importantly, when the OsNAC29-OE3 plants were inoculated with M. oryzae, the OsNAC29-GFP in the samples was immunoprecipitated with an anti-GFP antibody and then subjected to an immunoblotting assay with the anti-pT304 antibody. The results showed increased phosphorylation at Thr304 of OsNAC29 after M. oryzae infection (Fig. 8C), suggesting that the phosphorylation of OsNAC29 at Thr304 is involved in its function in rice blast resistance.

Figure 8.

Phosphorylation of OsNAC29 at Thr304 by OsMAPK3 contributes to rice resistance against M. oryzae.A) OsNAC29-HA was transiently transfected into ZH11 and Osmapk3 protoplasts. Following treatment with 1 μM chitin for 20 min, phosphorylation at T304 of OsNAC29 was analyzed by immunoblotting with the anti-pT304 antibody. B) OsNAC29-FLAG was transiently expressed for 12 h in rice protoplasts. The transformed protoplasts were treated with chitin for the indicated times and then collected for protein extraction. Phosphorylation at Thr304 of OsNAC29 was detected with an anti-pT304 antibody. C) Three-wk-old plants of OsNAC29-OE3 were inoculated with M. oryzae strain Guy11 by the spray method. Leaves were harvested at the indicated times and then subjected to immunoblot analyses with anti-GFP and anti-pT304 antibodies. D) Relative transcript levels of OsNAC29 in ZH11, Osnac29-1, and transgenic lines overexpressing FLAG-OsNAC29 (#1 and #4), FLAG-OsNAC29^T304A (#4 and #7), and FLAG-OsNAC29^T304D (#3 and #7) in the Osnac29-1 mutant were detected using RT-qPCR. UBQ was used as the internal control. Data are means ± Sd (n = 3 biological replicates). Scale bars = 0.5 cm. Significant differences were determined by one-way ANOVA (**P < 0.01) (left). Five-wk-old ZH11, Osnac29-1, FLAG-OsNAC29/Osnac29-1, FLAG-OsNAC29^T304A/Osnac29-1, and FLAG-OsNAC29^T304D/Osnac29-1 plants were inoculated with M. oryzae strain Guy11 by the punch method. Images were taken 7 dpi (right). E) Different transgenic plants and ZH11 plants were inoculated with M. oryzae by the punch method. Lesion length was measured at 7 dpi. Data are means ± Sd (n = 10 biological replicates). Significant differences were determined by one-way ANOVA (*P < 0.05, **P < 0.01). F) Five-wk-old ZH11, Osnac29-1, FLAG-OsNAC29/Osnac29-1, OsNAC29^T304A/Osnac29-1, and OsNAC29^T304D/Osnac29-1 plants were inoculated with M. oryzae strain Guy11. Leaves were cut, stained with DAB, and photographed after decoloring. Scale bars = 1 cm.

To verify the contribution of phosphorylation at Thr304 of OsNAC29 to rice resistance against M. oryzae, we constructed transgenic lines overexpressing OsNAC29, OsNAC29^T304D, or OsNAC29^T304A with a FLAG tag in the Osnac29-1 mutant (FLAG-OsNAC29-OE/Osnac29-1, FLAG-OsNAC29^T304A-OE/Osnac29-1, and FLAG-OsNAC29^T304D-OE/Osnac29-1). The relative expression level of OsNAC29 was greater in the selected transgenic lines than in the wild type (Fig. 8D). After inoculation with M. oryzae by the punch method, the resistance of the FLAG-OsNAC29^T304D-OE/Osnac29-1 transgenic plants to M. oryzae was significantly greater than that of the FLAG-OsNAC29-OE/Osnac29-1 and wild-type plants (Fig. 8E), indicating complementation of the Osnac29-1 phenotype by the expression of FLAG-OsNAC29^T304D. In contrast, the enhanced susceptibility of Osnac29-1 to M. oryzae could not be suppressed by the expression of FLAG-OsNAC29^T304A. In addition, the DAB staining assay showed that H₂O₂ accumulation, indicated by brown spots, was lower in the FLAG-OsNAC29^T304A-OE/Osnac29-1 plants compared than in the FLAG-OsNAC29^T304D-OE/Osnac29-1 and FLAG-OsNAC29-OE/Osnac29-1 plants (Fig. 8F). Taken together, the above results suggested that the phosphorylation of OsNAC29 at Thr304 is essential for its function in rice blast resistance.

Discussion

OsNAC29 is a NAC TF that positively regulates plant immunity

Plants trigger a series of immune responses upon pathogen attack, including transcriptional reprogramming, indicating the crucial roles of transcriptional regulators in plant immunity (Jones and Dangl 2006; Lu et al. 2022). NAC TFs constitute the largest family of plant-specific TFs and play critical roles in plant immunity (Olsen et al. 2005; Lee et al. 2017; Bi et al. 2023). Recently, OsNAC083 was reported to negatively regulate rice immunity against M. oryzae by directly activating the transcription of OsPFPH2-6 through the ACGCAA element in its promoter (Bi et al. 2023). JA2 and JA2L, 2 closely related NAC TFs in tomato, differentially regulate stomatal closure and reopening during pathogen attack (Du et al. 2014). At present, 151 NAC genes have been identified in rice (Nuruzzaman et al. 2010). Ten ONAC TFs are known to act as either positive or negative regulators in rice immunity against different pathogens (Bi et al. 2023). However, immunity-related NAC TFs in rice remain largely unknown. In this study, we found that the expression of OsNAC29, an NAC family member, was upregulated during M. oryzae infection (Supplementary Figs. S1 and S2). Two CRISPR/Cas9 alleles of Osnac29 displayed increased susceptibility, while OsNAC29-overexpressing plants showed enhanced resistance to M. oryzae (Fig. 1). Our results revealed an immunity-related NAC TF, OsNAC29, in rice, and OsNAC29 functions as a positive regulator in the rice immune response against blast fungus. OsNAC29 plays a crucial role in rice fungal diseases, such as rice blast caused by M. oryzae. However, it is unknown whether OsNAC29 also plays important roles in rice bacterial diseases. Research on the function of OsNAC29 in rice bacterial diseases, such as rice blight, is highly valuable.

OsNAC29 was involved in rice blast resistance by transcriptional regulation of OsCYP71Z2 and OsTPS28

OsNAC122 and OsNAC132, 2 genes most homologous to OsNAC29 in rice, are localized to the nucleus and exhibit transcriptional activation activities (Sun et al. 2013). Similarly, we showed that OsNAC29 is located in the nucleus (Fig. 2A) and has transcriptional activity in yeast (Fig. 2, B and C and Supplementary Fig. S6A), indicating that OsNAC29 may function as a transcriptional activator or repressor. Based on the analyses of comminated RNA-seq with DAP-seq data, as well as EMSA and ChIP‒qPCR data, we demonstrated that OsNAC29 can bind to the CACGTG motif in the promoters of OsCYP71Z2 and OsTPS28 and promote their expression (Figs. 2, D–G and 4, A–D). OsCYP71Z2 and OsTPS28 are 2 members of the DGC7 cluster. DGC7 (a rice diterpenoid gene cluster on chromosome 7) is a gene cluster involved in rice immunity that encodes the diterpenoid biosynthesis pathway to 5,10-diketo-casbene, which leads to enhanced rice resistance (Liang et al. 2021). 5,10-Diketo-casbene is derived from GGPP to olefin ent-casbene by TPS28, followed by further elaboration combined with CYP71Z21 and CYP71Z2. Moreover, it has been reported that OsTPS28 knockout lines no longer produce casbane-type diterpenoids and exhibit impaired resistance to the rice fungal blast pathogen M. oryzae (Okada et al. 2007). OsCYP71Z2 plays an important role in bacterial blight resistance by regulating diterpenoid phytoalexin biosynthesis and H₂O₂ generation (Li et al. 2013; Zhan et al. 2020), and phytoalexins accumulate in plants invaded by pathogens and are secondary metabolites that exhibit antimicrobial activity against pathogens (Ahuja et al. 2012). However, the function of OsCYP71Z2 in the resistance of rice to blast is currently unknown. Our study revealed that following M. oryzae invasion, the OsCYP71Z2-overexpressing lines exhibited enhanced resistance to M. oryzae. In contrast, Oscyp71z2 displayed increased susceptibility (Fig. 3), indicating that OsCYP71Z2 is also a positive regulator, which is consistent with the role of OsNAC29 in rice resistance against blast. Importantly, our results showed that the OsCYP71Z2 and OsTPS28 knockout lines in the OsNAC29-OE3 background exhibited increased susceptibility compared with those in the OsNAC29-OE3 and WT backgrounds (Fig. 4E), indicating that OsNAC29 regulates disease resistance to M. oryzae in an OsCYP71Z2- and OsTPS28-dependent manner.

OsTPS28 and OsCYP71Z2 are 2 key genes associated with 5,10-diketo-casbene synthesis (Zhan et al. 2020; Liang et al. 2021). OsCYP71Z2 has been reported to be involved in biosynthesis of momilactone A and phytocassane B biosynthesis to regulate rice bacterial blight resistance (Li et al. 2013). Therefore, it is worth determining the content of phytoalexins, such as 5,10-diketo-casbene, momilactone A, and phytocassane B, in Osnac29-1 and OsNAC29-OE3 plants before and after M. oryzae infection in the future.

Interestingly, ROS burst was also affected in OsNAC29 and OsCYP71Z2 knockout mutants and overexpressing plants. OsNAC29 and OsCYP71Z2 may directly or indirectly affect the expression of ROS burst-related genes. How OsNAC29 and OsCYP71Z2 affect the ROS burst remains to be investigated in the future.

In addition, OsPR1a and OsSPL11 were expressed differently in ZH11 and OsNAC29-OE plants after M. oryzae infection and their promoters contain the CACGTG motif, indicating that they may be the target genes of OsNAC29. Although the Y1H assay could not detect the activation of OsANC29 on OsPR1a and OsSPL11, more experiments need to be performed to verify whether OsPR1a, OsSPL11, and even other genes are the targets of OsNAC29.

OsMAPK3-mediated phosphorylation is crucial for OsNAC29 stability

Accumulating evidence indicates that MAPK cascades are critical signaling modules in plant immunity (Kong et al. 2012; Ma et al. 2021; Wang et al. 2023b). The MAPKKK3/MAPKKK5-MKK4/MKK5-MAPK3/MAPK6 cascades are well known to be involved in the elicitation of immune responses (Asai et al. 2002; Bi et al. 2018). Stimulation of pattern recognition receptors results in the activation of MAPK3, MAPK4, MAPK6, and MAPK11, which are hallmarks of the immune response (Furlan et al. 2017). MAPK3- and MAPK6-mediated phosphorylation has been implicated in regulating the turnover of various downstream substrates, including TFs, involved in stress responses (Zhang et al. 2024). For instance, phosphorylation of ERF6 by MAPK3/6 increases ERF6 protein stability in vivo in response to Botrytis cinerea infection (Meng et al. 2013). OsMKK10-2 activated by biotic stimuli phosphorylates OsMAPK3/6. This leads to the phosphorylation of OsWRKY31 to promote immunity; thus, the OsWRKY31 protein is controlled by phosphorylation and ubiquitination (Wang et al. 2023b). In this study, OsNAC29, a substrate for OsMAPK3, was shown to positively regulate immunity, and the phosphorylation of OsNAC29 by OsMAPK3 attenuated 26S proteasome-mediated degradation, resulting in a significant increase in the protein levels (Fig. 6, A–E). Further analyses showed that the protein level of OsNAC29-GFP as well as its ability to mediate rice blast resistance deceased markedly in the Osmapk3 mutant (Fig. 6, F and G), suggesting that OsNAC29 regulates disease resistance to M. oryzae partly in an OsMAPK3-dependent manner. Our study showed that OsNAC29 was degraded by the 26S proteasome and that OsMAPK3 stabilized OsNAC29 by phosphorylation (Fig. 7). Identifying the E3 of OsNAC29 would provide more insights into the mechanism by which OsMAPK3–OsNAC29–E3 interacts with and regulates rice resistance against M. oryzae.

Phosphorylation at Thr304 of OsNAC29 is required for its function in blast resistance

Our study showed that the degradation of OsNAC29 is mediated by the 26S proteasome (Fig. 6A). Interestingly, the level of the OsNAC29 protein in the OsNAC29-overexpressing plants was very low without inoculation (Fig. 6B). However, it rapidly increased after M. oryzae infection (Fig. 6B). Furthermore, we found that OsMAPK3 influences OsNAC29 protein accumulation to enhance rice resistance against M. oryzae by phosphorylating OsNAC29 at the T304 site (Fig. 7). The phosphomimetic OsNAC29^T304D protein was more stable than the wild-type and OsNAC29^T304A proteins (Fig. 7, G and H). Similarly, in vivo phosphorylation of OsNAC29 at T304 was indeed enhanced by M. oryzae inoculation and chitin treatment (Fig. 8, A–C). Consistently, the resistance of the FLAG-OsNAC29^T304D-OE/Osnac29-1 transgenic lines to M. oryzae was significantly greater than that of the FLAG-OsNAC29-OE/Osnac29-1 and the wild-type plants (Fig. 8, D–F), indicating that the phosphorylation of OsNAC29 at Thr304 is essential for its function in rice defense against M. oryzae. It remains worth determining whether the phosphorylation of OsNAC29 at Thr304 influences its ability to bind DNA. In addition, OsNAC29^T304A is still phosphorylated by OsMAPK3-GST (Fig. 7B), indicating that OsMAPK3 has other phosphorylation sites on OsNAC29 in addition to Thr304, which is worth to be detected in the future.

OsNAC29 forms an immune complex with OsRACK1A and OsMAPK3/6

It is well known that the protease-G protein–RACK1–MAPK cascade modules play important roles in plant immune signaling pathways in Arabidopsis (Cheng et al. 2015). However, their relationship in rice is not very clear. Here, through an interaction assay, we showed that OsMAPK3/OsMAPK6, OsRACK1A, and OsNAC29 all interacted with each other, indicating that they together form a complex (Fig. 5 and Supplementary Figs. S10 and S11). It has been reported that OsRACK1A, OsMAPK3, and OsMAPK6 play important roles in rice immunity (Nakashima et al. 2008; Wang et al. 2023b). Similarly, the susceptibility of the OsMAPK3 knockout mutant increased compared with that of the wild type (Supplementary Fig. S12). These findings further suggested that OsNAC29 forms an immune complex with OsRACK1A and OsMAPK3/6 to regulate rice resistance against M. oryzae.

Taken together, our study revealed the important role of the OsMAPK3–OsNAC29–OsCYP71Z2/OsTPS28 module in the regulation of rice immunity. When rice is subjected to blast fungus infection, the MAPK cascade is activated, and subsequently, activated OsMAPK3 phosphorylates OsNAC29 at Thr304 to prevent its degradation and enhance its transcriptional activity. Finally, OsNAC29 directly activates OsCYP71Z2 and OsTPS28 transcription to increase the ROS burst, H₂O₂ accumulation and possibly phytoalexin biosynthesis, thereby enhancing rice resistance to blast fungus. In addition, OsRACK1A may function as a scaffold protein to recruit MAPK cascade members and OsNAC29 to form an immune complex (Fig. 9).

Figure 9.

The working model of the OsMAPK3–OsNAC29–OsTPS28/OsCYP71Z2 module in rice immunity. Upon M. oryzae infection, the MAPK cascade was activated. Then activated OsMAPK3 phosphorylates OsNAC29 at Thr304 and stabilizes OsNAC29 from 26S proteasome-mediated degradation. OsNAC29 increases rice blast resistance by directly binding to the promoters of OsCYP71Z2 and OsTPS28 and activating their transcription. Moreover, OsRACK1A may function as a scaffold protein to recruit MAPKs and OsNAC29 to form an immune complex.

Materials and methods

Plant materials and fungal strain growth conditions

The rice plants in this study were grown in a greenhouse with 16 h of light (600 to 800 µmol/m²/s) and 8 h of darkness at a temperature of 28 °C or in the paddy field of Fujian Agriculture and Forestry University and Science and Technology Backyard of Fujian Pucheng Rice Seed Industry in Fuzhou, Fujian Province. The rice plants used here were all of the (Oryza sativa) cultivar ZH11 background. The generation of OsNAC29, OsCYP71Z2, and OsMAPK3 knockout mutants was conducted via the CRISPR/Cas9 system. The sgRNAs of Osnac29, Oscyp71z2, Osmapk3, and Ostps28 Oscyp71z2/OsNAC29-OE3 are shown in Supplementary Data Set 1. For the generation of rice OsNAC29-OE and OsCYP71Z2-OE overexpression plants, their full-length cDNAs were cloned from wild-type (ZH11) cDNA by PCR using specific primers (Supplementary Data Set 1) and then cloned and inserted into the binary vector PCAMBIA2300 with a GFP tag, respectively. For transgenic plants overexpressing OsNAC29, OsNAC29^T304A, and OsNAC29^T304D, their respective coding sequences were cloned and inserted into the BGV007 vector fused with sequences encoding a FLAG tag driven by the 35S promoter. These constructs were subsequently genetically transformed into the Osnac29-1 mutant. The Ostps28 Oscyp71z2/OsNAC29-OE3 plant was constructed by knocking out OsTPS28 and OsCYP71Z2 in the OsNAC29-OE3 plant via the CRISPR/Cas9 system.

The M. oryzae strain Guy11 in this study was cultured at 28 °C on rice bran medium in the dark for approximately half a month and then exposed to light for sporulation for 1 wk after the surface mycoflora on the rice bran medium was removed.

Transcriptome analysis to identify genes induced by M. oryzae infection

Three-wk-old ZH11 leaves were collected at 24 h after spraying with M. oryzae infection, while leaves sprayed with H₂O were used as the control. Then, the samples were subjected to RNA sequence analysis. The preparation and sequencing of the libraries were performed by the Beijing Allwegene Technology Company Limited (Beijing, China). DEGs were determined in the R environment using the DESeq2 package (version 1.30.1). Genes with an adjusted P-value <0.05 found by DESeq2 and a cutoff of log2 (fold change) ≥1 or ≤−1 were defined as differentially expressed.

Protein expression in rice protoplasts and N. benthamiana

For protein expression assays in rice protoplasts, plasmids were constructed and then extracted with a Plasmid Maxi kit. ZH11 seedlings were cultured in the dark for ∼2 wk. Rice protoplasts were prepared from the ZH11 seedlings. The plasmids were subsequently transformed into rice protoplasts via polyethylene 4000 glycol/calcium-mediated transformation. After transformation for 14 h, the proteins were extracted with buffer (50 mm Tris–HCl, 150 mm KCl, 1 mm EDTA, 1 mm DTT, 1× protease inhibitor cocktail) or observed using a confocal microscope (Zeiss LSM880). For expression in N. benthamiana, constructed plasmids were transformed into Agrobacterium strain GV3101 and infiltrated into N. benthamiana using a needleless syringe. After 3 d, the N. benthamiana leaves were detached to detect proteins or detected with a confocal microscope. For GFP, the laser wavelength is 488 nm, intensity is 1% to 2%, collection bandwidth is 493 to 574 nm, and detector gain is 540 to 620. For RFP, the laser wavelength is 561 nm, intensity is 1% to 2%, collection bandwidth is 578 to 696 nm, and detector gain is 510 to 630.

RNA extraction and RT-qPCR

RNA extraction and RT-qPCR were performed as described previously with minor modification (Li et al. 2024). Rice leaves were cut and ground in liquid nitrogen. Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions, and first-strand cDNA was subsequently synthesized by M-MLV reverse transcriptase (Promega) with 1 μg of total RNA from each sample. RT-qPCR was performed using SYBR Green mix (QuantiNova SYBR Green PCR Kit, QIAGEN) with a Bio-Rad C1000TM Thermal Cycler (Bio-Rad). The rice UBQ gene (LOC_Os03g13170) was used as an internal control for gene expression. The primers used to detect the expression of the genes in this study are listed in Supplementary Data Set 1.

Rice disease assay

For spray inoculation, 3-wk-old plants were sprayed with Guy11. The spore concentration was adjusted to 20 × 10⁴ spores/mL in 0.02% Tween-20 solution. Then, the sprayed plants were placed into a chamber at 28 °C with a 12-h light/dark photoperiod. For punch inoculation, the leaves of 5-wk-old plants were lightly wounded with a mouse ear punch, and 10 µL of spore suspension was added to the wound following a method described previously (Lu et al. 2022). For the fungal biomass assay, the punch-inoculated or spray-inoculated leaves were measured using the DNA amounts of M. oryzae Mopot2 against rice ubiquitin DNA amounts by qPCR (Wang et al. 2021b). For the callose deposition assay, chitin-induced callose deposition was performed with 7-day-old seedlings using previously described method (Niu et al. 2022).

The ROS detection assay was performed using a previously described method with minor modifications (Tian et al. 2020). Briefly, leaf discs from ∼10-day-old seedlings were placed in 96-well plates with 100 μL of deionized water overnight in the dark. To detect ROS production, the water was replaced with 100 μL of 20 mm luminal, 5 mg/mL peroxidase, and PAMPs (0.4 mm chitin). ROS production was recorded with a Promage GloMAX 20/20 luminometer.

For the DAB staining assay following the method of Liu et al. (2017), rice leaves were cut 2 d postinoculation (dpi) and were infiltrated with DAB (3,3′-diaminobenzidine) staining solution, which was dissolved in water. Leaves were vacuum-infiltrated with DAB solution for 30 min and incubated overnight. The leaves were destained with 100% ethanol and boiled. The destained leaves were then scanned with a photo scanner.

Transactivation assay in rice protoplasts and yeast

To examine the transcriptional activity of OsNAC29 in rice protoplasts using the GAL4/UAS-based system, the coding sequence of OsNAC29 was cloned and inserted into the 35S-GAL4BD plasmid to generate the GAL4BD-OsNAC29 vector. GAL4BD-OsNAC29 and GAL4BD (empty vector) were transformed into rice protoplasts. The transcriptional activity of OsNAC29 was detected using a previously described method (Bart et al. 2006). To determine the transcriptional activity of OsNAC29 in yeast, the cDNA of OsNAC29 was inserted into the pGBKT7 vector and then transformed into Y2H yeast cells. The transformed cells were applied to the selection medium following the method of Tang et al. (2021).

Yeast two-hybrid and Y1H assays

For the yeast two-hybrid (Y2H) assay, the cDNAs of OsNAC29, OsMAPK3, and OsRACK1A were amplified by PCR using gene-specific primers (Supplementary Data Set 1) and cloned and inserted into pGADT7 or pGBKT7 as indicated. Different combinations transformed into using a previously described method (Park et al. 2012). For a Y1H assay to delineate the DNA sequences to which OsNAC29 binds, the coding sequences of OsNAC29 were cloned and inserted into the pB42AD vector, and the promoters were cloned and inserted into pLacZi as indicated. Different combinations transformed into yeast strain EGY48 using the same method as the Y2H. Yeast cell growth was monitored on plates containing SD/-Trp-Ura selective media supplemented with X-gal.

Luciferase activity assay in rice protoplasts

A luciferase activity assay in rice protoplasts was performed as described previously (Lu et al. 2022). The promoter of OsCYP71Z2 or OsTPS28 (1,500-bp upstream of ATG) was cloned and inserted into the pGreenII 0800-LUC plasmid. The firefly luciferase (LUC) gene, which is expressed under the control of the maize ubiquitin promoter, was used as a reporter to monitor protoplast viability. The coding sequence of OsNAC29, OsNAC29^T304A, and OsNAC29^T304D were cloned and inserted into the pGreenII 62-SK plasmid as an effector. The constructed plasmids were subsequently cotransformed into rice protoplasts. The protein from protoplast samples was harvested, and 1× lysis buffer provided in the Luciferase Assay Report Kit (Promega) was added. Luciferase activity was detected with a Promage GloMAX 20/20 luminometer.

Chromatin immunoprecipitation-qPCR

ChIP assays were performed as described previously (Haring et al. 2007). In brief, the leaves of plants overexpressing OsNAC29 were fixed in 1% formaldehyde and subsequently quenched with glycine under vacuum. The cells were then lysed, and the chromatin was isolated and sheared by sonication. The sheared DNA fragments were precipitated using Protein A Agarose (Thermo Fisher Scientific, 20333). Immunoprecipitation was carried out using a specific antibody (anti-FLAG, Abmart, ab205606 or anti-GFP, Invitrogen, A11122) against the protein. After washing and elution, cross-links were reversed by heating, and DNA was purified. The purified DNA was subsequently subjected to RT-qPCR analysis to examine the relative enrichment of the promoter fragment using the primers (Supplementary Data Set 1).

Electrophoretic mobility shift assay

For EMSA, the cDNA of OsNAC29 was cloned and inserted into the PET32a vector. The fusion proteins were overexpressed and produced in an Escherichia coli strain. Oligonucleotides were synthesized and labeled with a Cy5 end-label as probes, and nonlabeled oligonucleotides (100-, 200-, and 300-fold excesses of labeled probes) were used as competitor probes. The sequences are provided in Supplementary Data Set 1. For the binding reactions, nuclear extracts or purified proteins were incubated with the labeled DNA probes in a binding buffer containing 10 mm Tris–HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, 5% glycerol, and 1 µg of poly (dI-dC) as a nonspecific competitor. The reaction mixtures were incubated at room temperature for 30 min. After protein‒DNA-binding reactions, electrophoretic separation and band visualization were carried out using methods described previously (Liu et al. 2014).

Luciferase complementation assay

The coding sequences of OsNAC29, OsMAPK3, and OsRACK1A were subsequently cloned and inserted into the nLUC or cLUC vector. The LCA was performed in N. benthamiana leaves as previously described (Zhou et al. 2018). Three days postinfiltration, 1 mM d-luciferin was sprayed onto the leaves, and the LUC activity was measured using an LB985 NightSHADE plant imaging system (Berthold, Germany).

Co-IP assay

Co-IP assays were performed as previously described (Wang et al. 2019). In brief, protoplasts isolated from WT plants were transfected with the desired plasmids and incubated overnight. Expressed proteins were extracted by the buffer (50 mm Tris–HCl, 150 mm KCl, 1 mm EDTA, 1 mm DTT, 1× protease inhibitor cocktail). 80 μL each supernatant was stored as an input sample, and the remaining supernatant was incubated with GFP-Trap or HA-Trap magnetic beads for 90 min. Following incubation, the beads were washed 4 or 5 times with PBS and boiled at 95 °C for 10 min. Immunoblotting was subsequently conducted with the anti-GFP (TransGen, HT801)/anti-HA (Abmart, 26D11)/anti-FLAG (Sigma-Aldrich, F1804) antibodies.

In vitro and in vivo phosphorylation assays

In vitro phosphorylation analysis was performed as previously reported (Ma et al. 2017). Proteins were incubated in kinase reaction buffer (50 mm pH 7.5, Tris–HCl, 10 mm CaCl₂, 10 mm MgCl₂, 10 mm MnCl₂, 1 mm DTT, 0.1 mm ATP with or without 5 μ Ci of γ-³²P ATP) for 30 min at room temperature. The reactions were stopped by the addition of SDS-loading buffer after 30 min. After phosphorylation, one half of the protein products were then exposed to Fuji X-ray film or subjected to immunoblot analysis using an anti-pSpT antibody (9477; Cell Signaling Technology, USA) or phosphosite-specific antibody (anti-pT304). The other half was stained with Coomassie brilliant blue.

To detect the phosphorylation of OsNAC29 by OsMAPK3 in planta, combinations of the OsNAC29-GFP + OsMAPK3-HA, OsNAC29-GFP + 3HA, and OsNAC29^T304A-GFP + OsMAPK3-HA constructs were transformed into rice protoplasts or expressed in N. benthamiana leaves. Total proteins from each sample were extracted and immunoprecipitated with 40 μL of anti-HA agarose beads. Immunoblotting was then performed with anti-pT304/anti-pSpT/anti-HA/anti-GFP antibodies.

ATPase activity

The ATPase activity assay was performed using a previously described method with slight modifications (Geng et al. 2022). In brief, to assess the phosphorylation of OsNAC29 by OsMAPK3, the proteins were incubated in kinase reaction buffer (50 mm Tris–HCl (pH 7.5), 10 mm CaCl₂, 10 mm MgCl₂, 10 mm MnCl₂, 1 mm DTT, and 1 mm ATP) at room temperature for 30 min. Then, 10 μL of the reaction mixture was added to 96-well plates with 10 μL of the luminescent kinase assay kit (S0158S, Beyotime, China) for 10 min. The ATPase activity was detected by a Promage GloMAX 20/20 luminometer, and the process was repeated 3 times.

Cell-free protein degradation assay

A cell-free protein degradation assay was carried out as described previously (Fu et al. 2012). In brief, recombinant OsNAC29-His/OsNAC29^T304A-His/OsNAC29^T304D-His was expressed in E. coli BL21 cells and purified. Total proteins were extracted from 3-wk-old Osmapk3 mutants and the corresponding WT (ZH11) plants with degradation buffer. Then, the same amount of OsNAC29-His protein was mixed with the total proteins and incubated at room temperature. Samples were taken at the indicated times for protein blotting.

Measurements of the half-life assay

The coding sequences of OsNAC29, OsNAC29^T304A and OsNAC29^T304D were cloned and inserted into the PYBA-1143 (with a HA tag) vector, respectively. The cDNAs of OsMAPK3 and OsRACK1A were, respectively, cloned into the pCAMBA1300 (with a FLAG tag) vector. The half-life assay of the fusion protein OsNAC29-HA was carried out as described previously (Yoon and Kieber 2013). Briefly, the indicated proteins were coexpressed in rice protoplasts, and the protoplasts were harvested after they were cultured at 26 °C for 16 h. CHX (250 μM) was added to the suspended protoplasts, and the protein was extracted at different time points for the blotting assay. The protein concentration was measured using the ImageJ software.

Phosphosite-specific antibody (anti-pT304) generation

The anti-pT304 antibody was custom-made by Abmart (Shanghai, China) using the phosphopeptide: C-AGGGH(pT)PAKR as an antigen. According to the antigen design sequence, peptides are synthesized, and quality control of peptide synthesis is conducted using HPLC-MS to ensure peptide purity >90%. Then, the rabbits were immunized with the phosphopeptide. Finally, the antibody quality was tested using ELISA.

DAP-seq assay

DAP-seq assay was carried out as described previously (O’Malley et al. 2016). In brief, OsNAC29 is expressed in vitro and tagged with an epitope for affinity purification. Genomic DNA of ZH11 is extracted and fragmented to generate a library of DNA sequences. The expressed OsNAC29 is incubated with the fragmented genomic DNA, allowing it to bind to its specific target sites. The OsNAC29 protein–DNA complexes are isolated using the epitope tag, which allows for the specific purification of DNA bound by OsNAC29. The purified DNA is then sequenced, and the data are analyzed to identify the binding sites of the TF across the genome.

Phylogenetic analysis

For phylogenetic analyses of OsNAC29, the full-length OsNAC29 protein sequence was used as query sequence for BLAST in the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) nonredundant protein sequence database, and some homologous proteins were identified. Then, these protein sequences were aligned with ClustalW, and a phylogenetic tree was constructed using MEGA7. See Supplementary Files 1 and 2.

Transcriptome analysis to identify DEGs in the OsNAC29-OE plants

Leaves of 3-wk-old OsNAC29-OE plants and ZH11 were collected and subjected to transcriptome analysis. Briefly, mRNA was extracted from each sample, and sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and sequenced on an Illumina Novaseq 6000 platform by the Beijing Allwegene Technology Company Limited (Beijing, China). All clean reads were mapped to the O. sativa L. Nipponbare reference genome by STAR. Then, HTSeq was used to count the numbers of reads mapped to each gene. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. The differential expression analysis was performed in the R environment with the DESeq2 package. Genes with an adjusted P-value < 0.05 found by DESeq2 and a cutoff of log2 (fold change) ≥1 or ≤−1 were considered differentially expressed.

Statistical analyses

All the data were analyzed by one-way ANOVA and are provided in Supplementary Data Set 4.

Accession numbers

Gene information in this article can be found in Rice Genome Annotation Project database (http://rice.uga.edu/) with the following accession numbers: OsNAC29 (LOC_Os07g48450), OsMAPK3 (LOC_Os03g17700), OsMAPK6 (LOC_Os06g06090), OsRACK1A (LOC_Os01g49290), OsCYP71Z2 (LOC_Os07g11739), OsTPS28 (LOC_Os07g11790), OsWRKY45 (LOC_Os05g25770), OsWRKY42 (LOC_Os02g26430), OsSPL11 (LOC_Os12g38210), OsPR8 (LOC_Os10g28080), OsPR1a (LOC_Os07g03710), UBQ (LOC_Os03g13170). The MoPot2 accession number can be found in the NCBI as Z33638.

Author contributions

S.L. and D.T. conceived and designed the research. L.L., J.F., J.Z., N.X., Z.D., Y.L., and X.W. carried out most of the experiments. L.L. and S.L. analyzed the data. L.L., S.L., and D.T. wrote the manuscript. All the authors have read and approved the final manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Transcriptome analysis revealed that OsNAC29 is upregulated after M. oryzae infection.

Supplementary Figure S2. Phylogenetic analysis of OsNAC29.

Supplementary Figure S3. Identification of Osnac29 mutants and OsNAC29-OE plants.

Supplementary Figure S4. OsNAC29 plays a positive role in rice immunity.

Supplementary Figure S5. Expression pattern of OsNAC29 in different tissues.

Supplementary Figure S6. Transcriptional activity and DAP-seq analysis of OsNAC29.

Supplementary Figure S7. EMSA of recombinant OsNAC29 protein binding to the labeled P2 probe from the OsCYP71Z2 promoter.

Supplementary Figure S8. Identification of Oscyp71z2 mutants and OsCYP71Z2-OE plants.

Supplementary Figure S9. Phenotypes of the Oscyp71z2 and Osnac29 mutants and the OsCYP71Z2-OE and OsNAC29-OE plants.

Supplementary Figure S10. OsNAC29 interacts with OsRACK1A.

Supplementary Figure S11. OsMAPK3/OsMAPK6-OsRACK1A-OsNAC29 interact with each other.

Supplementary Figure S12. Compared with the wild type, the Osmapk3 mutant displayed increased susceptibility.

Supplementary Figure S13. OsMAPK3 enhanced OsNAC29 stability.

Supplementary Figure S14. OsMAPK3-mediated OsNAC29 phosphorylation positively regulates OsNAC29 stability.

Supplementary Figure S15. Phosphorylated OsNAC29 at Thr304 enhances its transcriptional activity on OsCYP71Z2 and OsTPS28.

Supplementary Data Set 1. Sequences of the primers and sgRNA targets used in this study.

Supplementary Data Set 2. Differentially expressed genes (DEGs) in OsNAC29-OE3 and ZH11.

Supplementary Data Set 3. List of Ip-MS genes of OsNAC29.

Supplementary Data Set 4. ANOVA results.

Supplementary File 1. Alignment used for phylogenetic analysis.

Supplementary File 2. Phylogenetic tree in Newick format.

Funding

This work was supported by the National Key Research and Development Program of China (2022YFF1001500).

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• NTL Gramene: AT1G11570

• NTL Araport: AT1G11570

• PR5 Gramene: AT1G75040

• PR5 Araport: AT1G75040

• ATAF1 Gramene: AT1G01720

• ATAF1 Araport: AT1G01720

• NAM Gramene: AT1G52880

• NAM Araport: AT1G52880

• ERF11 Gramene: Mp7g17020.1

• ERF11 Araport: Mp7g17020.1

• ERF13 Gramene: Mp6g08690.1

• ERF13 Araport: Mp6g08690.1

• PR1 Gramene: At2g14610

• PR1 Araport: At2g14610

• ATP CHEBI: CHEBI:15422

• EMSA Gramene: electrophoretic mobility shift assay

• EMSA Araport: electrophoretic mobility shift assay

• EDTA Gramene: Ethylene Diamine Tetraacetic Acid

• EDTA Araport: Ethylene Diamine Tetraacetic Acid

• ELISA Gramene: enzyme linked immunosorbent assay

• ELISA Araport: enzyme linked immunosorbent assay