ONAC066, A Stress-Responsive NAC Transcription Activator, Positively Contributes to Rice Immunity Against Magnaprothe oryzae Through Modulating Expression of OsWRKY62 and Three Cytochrome P450 Genes

NAC transcriptional factors constitute a large family in rice and some of them have been demonstrated to play crucial roles in rice immunity. The present study investigated the function and mechanism of ONAC066 in rice immunity. ONAC066 shows transcription activator activity that depends on its C-terminal region in rice cells. ONAC066-OE plants exhibited enhanced resistance while ONAC066-Ri and onac066-1 plants showed attenuated resistance to Magnaporthe oryzae. A total of 81 genes were found to be up-regulated in ONAC066-OE plants, and 26 of them were predicted to be induced by M. oryzae. Four OsWRKY genes, including OsWRKY45 and OsWRKY62, were up-regulated in ONAC066-OE plants but down-regulated in ONAC066-Ri plants. ONAC066 bound to NAC core-binding site in OsWRKY62 promoter and activated OsWRKY62 expression, indicating that OsWRKY62 is a ONAC066 target. A set of cytochrome P450 genes were found to be co-expressed with ONAC066 and 5 of them were up-regulated in ONAC066-OE plants but down-regulated in ONAC066-Ri plants. ONAC066 bound to promoters of cytochrome P450 genes LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 and activated their transcription, indicating that these three cytochrome P450 genes are ONAC066 targets. These results suggest that ONAC066, as a transcription activator, positively contributes to rice immunity through modulating the expression of OsWRKY62 and a set of cytochrome P450 genes to activate defense response.

NAC family is plant-specific and represents one of the largest plant TF families. NAC TFs are characterized by the presence of highly conserved NAC domains at N-terminal, which determine DNA binding activity, and of variable domains at C-terminal, which are responsible for transcription activity (Olsen et al., 2005). Most NAC TFs exhibit binding activities to NAC recognition sequence (NACRS) cis-element with the sequence of CATGT or CACG (Tran et al., 2004), which are frequently present in promoters of certain defense genes. Functional studies using knockout/knockdown mutants and/or overexpression transgenic lines have demonstrated that NAC TFs play significant roles in plant growth, development, and response to biotic and abiotic stress (Puranik et al., 2012;Shao et al., 2015;Kim et al., 2016;Yuan et al., 2019a;Diao et al., 2020;Forlani et al., 2021;Kou et al., 2021).
Genome-wide transcriptome analysis revealed that a set of TF genes including NAC genes is activated by infection of Magnaporthe oryzae, the causal agent of blast disease (Kawahara et al., 2012;Sun et al., 2015). A total of 151 members in rice NAC TF family have been identified (Ooka et al., 2003;Fang et al., 2008;Nuruzzaman et al., 2010) and some of the rice NAC TFs have been shown to be involved in rice immunity, acting as either positive or negative regulators. OsNAC6, belonging to ATAF subfamily (Yuan et al., 2019a), positively regulates resistance to M. grisea and two genes for a cationic peroxidase (LOC_Os01g73200) and a protein containing a conserved DUF26 domain (LOC_Os04g25060) were identified as putative target genes of OsNAC6 (Nakashima et al., 2007). OsNAC111-overexpressing plants showed increased resistance to M. oryzae and constitutively expressed several defense genes, suggesting that OsNAC111, a member of TERN subfamily (Yuan et al., 2019a), positively regulates the promoter activity of a specific set of defense genes including PR2 and PR8 and contributes to rice immunity (Yokotani et al., 2014). Mutation in OsNAC60, whose transcript abundance is regulated by miR164a, and silencing of ONAC122 or ONAC131, coding for NAC TFs belonging to NAP subfamily (Yuan et al., 2019a), increased susceptibility to M. oryzae (Sun et al., 2013;Wang et al., 2018), while overexpression of OsNAC58 increased resistance to Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight disease (Park et al., 2017). ONAC066, which was induced by the blast fungus, positively regulates resistance to M. oryzae and X. oryzae pv. oryzae through modulating of abscisic acid (ABA) signaling pathway . RIM1, belonging to the NTL subfamily (Yuan et al., 2019a), negatively regulates rice immunity to Rice dwarf virus by acting as a host factor that is required for multiplication of the virus in host plants and functions as a molecular link in jasmonic acid signaling (Yoshii et al., 2009(Yoshii et al., , 2010. OsNAC4 was found to be up-regulated during non-host defense response and regulate the occurrence of hypersensitive cell death, accompanied by loss of plasma membrane integrity, nuclear DNA fragmentation and typical morphological changes (Kaneda et al., 2009). Therefore, the NAC TFs play critical roles in different aspects of rice immunity; however, the molecular mechanism for these NAC TFs in rice immunity remains largely unknown.
We previously identified ONAC066 (LOC_Os03g56580) as one of the M. oryzae-responsive NAC TF genes (Sun et al., 2015). ONAC066 is classified into the ONAC022 subgroup of group I in NAC TF family (Ooka et al., 2003) and its Arabidopsis homologous ANAC042/AtJUB1 has been shown to be involved in pathogen-induced defense response (Wu et al., 2012;Shahnejat-Bushehri et al., 2016). ONAC066 has been shown to function as a positive regulator of drought and oxidative stress tolerance as well as disease resistance against M. oryzae and X. oryzae pv. oryzae Yuan et al., 2019b). The present study aimed to elucidate the function of ONAC066 in rice immunity and to identify the putative targets that are regulated by ONAC066. Our data demonstrated that ONAC066 acts as a positive regulator of rice immunity against M. oryzae through modulating expression of OsWRKY62, a WRKY TF that is involved in rice immunity (Fukushima et al., 2016;Liu et al., 2016), and three cytochrome P450 genes.

Plant Materials and Growth Conditions
ONAC066-OE and ONAC066-RNAi transgenic lines in background of rice (Oryza sativa L.) subsp. japonica cv. Zhonghua 11 (ZH11) were identified as previously described (Yuan et al., 2019b). Seeds of T-DNA insertion mutant onac066-1 (1C-13513) in japonica cv. Hwayoung (HY) background were obtained from POSTECH RISD (Rice T-DNA Insertion Sequence Database) (Jeon et al., 2000;An et al., 2003). Homozygous mutants for T-DNA insertion were identified by genomic DNA PCR with gene-specific and T-DNA-specific (2707RB) primers (Supplementary Table S1). Rice plants were grown in a growth room with a cycle of 14 h of light (28 • C) and 10 h of darkness (24 • C), as previously described (Hong et al., 2017).

RNA Isolation and qRT-PCR
Total RNA was extracted using RNA Isolater reagent (Vazyme, Nanjing, China) and treated with genome DNA wiper mix (Vazyme, Nanjing, China) at 42 • C for 2 min to remove the remaining genomic DNA. First-strand cDNA was synthesized with 2 µg purified total RNA using the reverse transcription system (Vazyme, Nanjing, China). qRT-PCR was performed on a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, United States) with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). PCR conditions were as follow: 95 • C for 5 min, then 40 cycles of 95 • C for 10 s, 60 • C for 15 s, and 72 • C for 30 s. Rice 18s rRNA gene was used as an internal control to normalize data (Jain et al., 2006). Quantitative gene expression was analyzed by the 2 − CT method and all primers used were listed in Supplementary Table S1.

Yeast One-Hybrid (Y1H) Assays
Coding region of ONAC066 was fused to GAL4 activation domain in effector vector pGADT7-Rec2 (Clontech, Mountain View, CA, United States). The promoter regions (1,500 bp upstream from ATG) of the genes of interest were amplified and cloned into reporter vector pHis2. pGADT7-Rec2-ONAC066 in combination with a reporter vector harboring the promoter of genes of interest were co-transformed into yeast strain Y187. DNA-protein interactions were verified by growth performance of yeasts grown on medium of SD/-Trp-Leu-His/3-amino-1,2,4triazole (3-AT, a competitive inhibitor of His3 protein) (Clontech, Mountain View, CA, United States). All primers used for vector construction were listed in Supplementary Table S1.

Gene Expression Profiling Analysis
Leave of 3-week-old ONAC066-OE11 and wild type (WT) plants grown in basal nutrient soil mixture under a 16 h light/8 h dark photoperiod at 28 • C were collected and frozen in liquid nitrogen. Total RNA extraction, cDNA synthesis, purification, labeling, and chip hybridization were performed by CapitalBio Technology (Beijing, China) using the Affymetrix Rice GeneChip. Three independent biological replicates were used for each of the genotypes. After log 2 transformation and normalization, genes whose expression showed fold change >2 (up-regulated) or <0.5 (down-regulated) with q-Value (a multiple-test corrected p-Value) (Benjamini and Hochberg, 1995) <5% were considered as differentially expressed genes. GO enrichment analysis of differentially expressed or co-expressed genes were performed using AgriGO website 1 (Tian et al., 2017).

Transient Expression Assays
Promoter regions (1500 bp from ATG codon) of OsWRKY62, LOC_Os02g30110, LOC_Os06g37300 and LOC_Os02g36150 were amplified and cloned into reporter vector pGreenII. pA7-GAL4BD and pA7-GAL4BD::ONAC066 were used as the effectors. Plasmids were extracted using CompactPrep Plasmid Midi Kit (Qiagen, Valencia, CA, United States). Protoplasts were prepared from rice mesophyll cells as previously described (Sun et al., 2012). Briefly, leaf pieces were infiltrated in a vacuum and gently rotated in a cell wall degrading enzyme mixture (0.4 M mannitol, 1.5% cellulose, and 0.3% macerozyme). After 4 h digestion, protoplasts were collected by filtering through 100 mm mesh. For co-transfection assays, 2 µg of the reporter plasmid and 2 µg of the effector plasmid were mixed and introduced into freshly prepared rice protoplasts under 20% PEG solution. After incubated for 12 h at 22 • C in dark, LUC assays were performed using the Dual-Luciferase reporter assay system (Promega, Madison, WI, United States) according to the manufacturer's instructions. The firefly luciferase (LUC) activity was normalized according to the humanized renilla LUC activity in each assay, and the relative ratio was determined by comparing with that of the empty vector. Averages of the relative ratios were calculated from three independent experiments and the primers used for LUC assay are listed in Supplementary Table S1.

Chromatin Immuno-Precipitation (ChIP) and ChIP-PCR
Chromatin immuno-precipitation-PCR assays were performed as described previously (Chung et al., 2018). Briefly, chromatin was isolated from ∼3 g of leaves from 2-week-old ONAC066-OE11 plants. After fragment sonication, the DNA/protein complex was immune-precipitated with ChIP-grade antibody against GFP (Roche, Switzerland). After reverse crosslinking, the immunoprecipitated DNA was extracted with phenol/chloroform. The chromatin samples incubated with preimmune (Pre) serum (GenScript, Nanjing, China) and before immunoprecipitation were used as negative controls and input controls, respectively. PCR was performed using specific primers (Supplementary Table S1) and the products were separated on 1.5% agarose gels and visualized by Goldview staining.

Identification of Co-expressed Cytochrome P450 Genes
The co-expression data were downloaded from CREP (Collections of Rice Expression Profiling 2 ) and PLANEX (Plant co-expression database 3 ) databases comprising a total of 190 microarray experiments from diverse Chinese cultivated rice varieties and the publicly available GEO data from NCBI, respectively. The permutation test was done to determine the optimal threshold of the Pearson's correlation coefficients (PCCs) (Butte et al., 2000;Carter et al., 2004). Absolute value of PCC>0.75 or 0.55 for CREP and PLANEX database, respectively, were considered as co-expressed genes with ONAC066 (Aoki et al., 2007). Statistical significance for candidate genes in co-expression network construction was further determined by using a student t-test.

RESULTS
ONAC066 Contributes Positively to Rice Immunity Against M. oryzae To further explore the biological function of ONAC066, transgenic rice lines with overexpression of ONAC066 or RNAimediated suppression of ONAC066 were generated and two independent lines for overexpression (ONAC066-OE11 and ONAC066-OE12) and RNAi-mediated suppression (ONAC066-Ri1 and ONAC066-Ri21) were selected for this study (Yuan et al., 2019b). Involvement of ONAC066 in rice immunity was examined by analyzing M. oryzae-caused disease phenotype on leaves of ONAC066-OE and ONAC066-Ri plants using whole plant inoculation and detached leaf inoculation assays. In whole plant inoculation assays, the ONAC066-OE11 and ONAC066-OE12 showed small, scattered lesions while ONAC066-Ri1 and ONAC066-Ri21 plants developed big and dark brown lesions, as compared to WT plants, which displayed typical blast lesions ( Figure 1A). Fungal growth measurement results revealed a 22∼23% of reduction in ONAC066-OE plants and a 263∼396% of increase in ONAC066-Ri plants in comparison to that in WT plants ( Figure 1B). In detached leaf inoculation assays, smaller lesions with a reduction of 37∼44% in lesion length were seen on detached leaves from ONAC066-OE plants while larger lesions with an increase of 28∼53% in lesion length were observed when compared with those in WT plants (Figures 1C,D). Similarly, the ONAC066-OE plants supported less fungal growth, accounting for 18∼33% of that in WT plants, while the ONAC066-Ri plants provided more fungal growth, representing 207∼403% of that in WT plants ( Figure 1E). A mutant line onac066-1, in which T-DNA is inserted in the third exon of ONAC066 gene ( Figure 1F), was identified from RISD (Jeon et al., 2000;An et al., 2003) and homozygous plants were characterized by genotyping ( Figure 1G). Transcript of ONAC066 was undetectable in onac066-1 plants ( Figure 1H), suggesting that onac066-1 is a null mutant. Disease assays indicated that the onac066-1 plants showed more and larger disease lesions and supported a 1.5fold higher level of fungal growth, as compared with WT plants (Figures 1I,J). Taken together, these results suggest that ONAC066 acts as a positive regulator of rice immunity against M. oryzae.

ONAC066 Has Transcriptional Activator Activity in Rice Cells
We previously showed that ONAC066 has the binding ability to NACRS, AtJUB1 binding site (JBS) and JUB-like sequence and is a transcription activator in Y1H assays (Yuan et al., 2019b). The transcription activator activity of ONAC066 was further examined using rice protoplast transient expression system. The effector vectors used for this experiment contain either GAL4-BD alone (GDBD), or ONAC066 (GONAC066), ONAC066-N (GONAC066-N, containing the 1-178 aa N-terminal region), ONAC066-C (GONAC066-C, containing the 179-362 aa C-terminal region), or ONAC022 (GONAC022), closely related to ONAC066 (Hong et al., 2016;Yuan et al., 2019b), fused to GDBD (Figure 2A). In the rice protoplasts co-transfected with the effector and reporter vectors, the LUC activity of the effector GONAC066 or GONAC022 was increased by approximately 1.25-and 2.21-fold of the empty vector GDBD control ( Figure 2B). Furthermore, the LUC activity of the effector GONAC066-N was similar to the empty vector GDBD control while the LUC activity of the effector GONAC066-C showed 1.64-fold higher than that of the empty vector GDBD control ( Figure 2B). These results demonstrate that ONAC066 has transactivation activator activity in rice cells and also indicate that the C-terminus is critical for the transcriptional activator activity of ONAC066.

ONAC066 Activates the Expression of Defense Genes
To explore the molecular mechanism of ONAC066 in rice immunity and identify candidates for the ONAC066 target genes, gene expression profiling was performed and compared between ONAC066-OE11 and WT plants. After data processing, a total of 81 (ONAC066-OE11/WT fold changes >2 and q-Value < 5%) and 28 (ONAC066-OE11/WT fold changes <0.5 . Photographs and leaf samples were taken at 5 days post inoculation. Relative fungal growth was quantified by genomic qRT-RCR analyzing of the M. oryzae MoPot2 and rice OsUbi genes and shown as ratios of MoPot2/OsUbi. Experiments were repeated at least three times with similar results, and results from one representative experiment are shown in panels (A,C,I). Data presented in panels (B,D,E,J) are the means ± SD from three independent experiments and **/* indicated significant difference at p < 0.01 and p < 0.05 levels, respectively, by Student's t-test, in comparison to WT. and q-Value < 5%) genes were found to be up-and downregulated in ONAC066-OE11 plants grown under normal condition ( Table 1 and Supplementary Table S2). The upregulated and down-regulated genes in ONAC066-OE plants were clustered into 29 and four major categories, respectively ( Figure 3A and Supplementary Table S3). The 7 main categories for up-regulated genes in ONAC066-OE plants belong to biological processes, cellular components, and molecular functions ( Figure 3A). Notably, three genes for calmodulinrelated proteins, LOC_Os03g59770, LOC_Os12g36910, and LOC_Os01g04280, were identified as ONAC066-up-regulated, and LOC_Os01g04280 shows 43.3% identity to Arabidopsis SARD1, which plays a critical role in salicylic acid (SA)-mediated defense signaling . LOC_Os02g50460 (OsPUB40) and LOC_Os03g13740 (OsPUB41) have 43.5 and 43.8% of identity, respectively, to tomato CMPG1, which is required for efficient activation of defense mechanisms in tomato (González-Lamothe et al., 2006). Among the up-regulated genes belonging to the DNA binding category, four WRKY TFs, including OsWRKY45 (Shimono et al., 2007(Shimono et al., , 2012Cheng et al., 2015), OsWRKY62 (Peng et al., 2008;Fukushima et al., 2016;Liu et al., 2016), and OsWRKY76 (Yokotani et al., 2013;Liu et al., 2016), which have been shown to be involved in rice immunity, were identified (Table 1). In addition, three defense genes encoding for PR1, PR2 (β-1,3-glucanase), and PR8 (chitinase) were also up-regulated in ONAC066-OE plants ( Table 1).
Publicly available microarray data GSE7256 (Ribot et al., 2008), which was generated from 2-week-old cv. Nipponbare plants infected with M. grisea virulent isolate FR13, was used to examine whether the up-regulated genes in ONAC066-OE11 plants are also responsive to M. oryzae. Among the 81 upregulated genes in ONAC066-OE plants, 26 genes (32% of the up-regulated genes), were up-regulated in rice plants at 3 and/or 4 dpi after infection by M. oryzae isolate FR13 (Table 1). It is thus likely that overexpression of ONAC066 activates the expression of a set of genes that are responsive to M. oryzae.
To verify the expression patterns of the up-regulated genes in ONAC066-OE plants, we further compared the expression levels of 12 genes (9 ONAC066-up-regulated genes and 3 previously identified defense genes) among WT, ONAC066-OE and ONAC066-Ri plants by qRT-PCR. The expression levels of the ONAC066-up-regulated genes OsPR1, OsPR2, OsPR8, OsWRKY45, OsWRKY62, OsWRKY79, Myb (LOC_Os03g04760), POD (LOC_Os04g59200), and OsLOX7 (LOC_Os08g39840) as well as of two well-known defense genes OsPR1b and OsPAL1 (Inui et al., 1997;Agrawal et al., 2001) were significantly up-regulated in ONAC066-OE plants but markedly downregulated in ONAC066-Ri plants ( Figure 3B). Expression of another well-known defense gene OsPBZ1 (Midoh and Iwata, 1996) was significantly down-regulated in ONAC066-Ri plants but not changed in ONAC066-OE plants ( Figure 3B). In the transcriptomic analyses, no expression data for OsPR1b were obtained and OsPBZ1 and OsPAL1 were not considered as differentially expressed genes as their expression was upregulated by ∼1 fold with q-Value > 5%. The difference in expression change of OsPAL1 in ONAC066-OE plants, as revealed by qRT-PCR and transcriptomic analyses, may be due to different techniques used. Collectively, these data indicate that ONAC066 plays a role in activation of defense signaling and response in rice immunity.

ONAC066 Directly Activates Expression of OsWRKY62
The up-regulation pattern of OsWRKY45, OsWRKY62, OsWRKY76, and OsWRKY79 in ONAC066-OE plants led to examine whether these WRKY genes were ONAC066 targets. The binding ability of ONAC066 to the promoters of these  a Averages of fold changes from three independent biological samples were shown. b q-Value (%) shows the significance analysis based on three independent biological replicates. The genes with a cutoff q-Value < 5% and fold changes >2 were defined as differentially expressed genes. c Fold changes (M. oryzae FR13-infected/mock control) were retrieved from microarray dataset GSE7256 (Ribot et al., 2008), and genes with >2-fold changes were considered as up-regulated genes (shaded data).
WRKY genes was examined by Y1H assays. For this purpose, the 1500 bp promoter regions from start codons of these WRKY genes were amplified and cloned into pHis2 vectors ( Figure 4A) and were then co-transformed with vector Rec2-ONAC066 into yeast strain Y187. Yeast co-transformed with Rec2-ONAC066 and pHis2-pWRKY62 grew normally on 3-AT containing medium, while yeast co-transformed with empty Rec2 and pHis2 vectors did not grow well at a high concentration of 3-AT ( Figure 4B), suggesting that ONAC066 could directly bind to the promoter of OsWRKY62. However, growth performance of yeasts co-transformed with Rec2-ONAC066 and pHis2-pWRKY45, pHis2-pWRKY76, or pHis2-pWRKY79 was indistinguishable to yeast co-transformed with empty Rec2 and pHis2 vectors on 3-AT containing medium (Supplementary Figure S1), indicating that ONAC066 did not bind to the promoters of OsWRKY45, OsWRKY76, and OsWRKY79. The binding site of ONAC066 in OsWRKY62 promoter was further mapped by ChIP-PCR. Four NAC core-binding sites (CACG) (Tran et al., 2004) were identified within the 1500 bp promoter region of the OsWRKY62 gene and four probe regions were chosen for ChIP-PCR assays ( Figure 4C). No PCR amplicon was detected in the four probe regions when pre-immune serum was used in IP ( Figure 4D). A clear amplicon was observed in P4 probe region and no amplicon was detected in P1, P2, and P3 probe regions, when anti-GFP antibody was used in IP ( Figure 4D), demonstrating that ONAC066 binds to P4 region, which contains three NAC core-binding sites (Figure 4C), in the OsWRKY62 promoter.
The ability of ONAC066 to drive the transcription of OsWRKY62 was further explored by using the rice protoplast transient expression system with the reporter vector harboring OsWRKY62 promoter, and the effector vector expressing ONAC066 (Figure 4E). In rice protoplasts co-transfected with the effector vector ONAC066 and reporter vector pOsWRKY62, the LUC activity was significantly higher than that in rice protoplasts co-transfected with the empty ChIP-PCR analysis of ONAC066 binding to OsWRKY62 promoter. ChIP of ONAC066-GFP transgenic line using GFP antibody (α-GFP) or pre-immune (Pre) serum was performed and precipitated DNA fragments were subject to PCR analysis using OsWRKY62 promoter primers. 10% of chromatin amount before IP was used as positive controls (input) and IP sample with pre-immune serum was used as a negative control. (E,F) Activation of OsWRKY62 promoter by ONAC066. (E) Diagrams of the effector and reporter constructs. A 1500 bp from start codon was used as the OsWRKY62 promoter. (F) Luciferase activity in protoplasts co-transfected with the reporter and different combinations of effectors was determined. Experiments were repeated at least three times with similar results, and results from one representative experiment are shown in panels (B,D). Data presented in panel (F) are the means ± SD from three independent experiments and ** indicated significant difference at p < 0.01 by Student's t-test, in comparison to empty vector. effector vector and reporter vector pOsWRKY62 ( Figure 4F). These results indicate that ONAC066 transcriptionally drives the OsWRKY62 promoter. Taken together, it is clear that ONAC066 can bind to the NAC core-binding sites in OsWRKY62 promoter and thus drives the expression of OsWRKY62.

ONAC066 Modulates Expression of Three P450 Genes
To further characterize putative ONAC066 targets, ONAC066 was used as a guide gene to identify its co-expressed genes using expression profiling data from 190 datasets in CREP and NCBI GEO-based PLANEX databases. As results, 86 and 114 genes with PCC values greater than 0.75 or 0.55 for CREP and PLANES databases, respectively, were identified as tightly co-expressed genes with ONAC066 (Supplementary  Tables S4, S5). Enriched GO annotations were particularly clustered into categories of biological process and molecular functions, which are tightly associated with catalytic activity, binding, electron carrier activity, transcription regulator and transporter activity (Figure 5A).
A total of 15 co-expressed genes were simultaneously identified in CREP and PLANEX databases ( Table 2), among which 5 belong to the cytochrome P450 gene family. In fact, 14 cytochrome P450 genes were found to be co-expressed with ONAC066 in CREP and PLANEX databases and five of them were also induced by M. oryzae FR13 (Table 3). To verify the coexpression of these cytochrome P450 genes with ONAC066, we compared their expression patterns of 5 cytochrome P450 genes among WT, ONAC066-OE and ONAC066-Ri plants by qRT-PCR. The expression levels of these 5 cytochrome P450 genes, including LOC_Os01g41820 (CYP72A33), LOC_Os02g30110 (CYP81L6), LOC_Os03g25480 (CYP709B2), LOC_Os06g37300 (CYP701A8/OsKOL4), and LOC_Os02g36150 (CYP71Z6), were significantly up-regulated in ONAC066-OE plants but markedly down-regulated in ONAC066-Ri plants (Figure 5B), further confirming the co-expression feature of these cytochrome P450 genes with ONAC066. However, the expression patterns of the remaining co-expressed cytochrome P450 genes in Data represented in panels (B,C) are the means ± SD from three independent experiments and **/* indicated significant difference at p < 0.01 and p < 0.05 levels, respectively, by Student's t-test, in comparison to WT. Figure S2). Moreover, DPF, encoding a basic helix-loop-helix transcription factor acting as a master transcription factor in biosynthesis of diterpenoid phytoalexins (Yamamura et al., 2015), was also significantly up-regulated in ONAC066-OE plants but markedly down-regulated in ONAC066-Ri plants (Figure 5C).

ONAC066-OE and ONAC066-Ri plants were similar to WT (Supplementary
The co-expression feature of the cytochrome P450 genes with ONAC066 raised the possibility that they were ONAC066 targets. The binding ability of ONAC066 to promoters of the co-expressed cytochrome P450 genes was therefore examined by Y1H assays. For this purpose, the 1500 bp promoter regions from start codons of these cytochrome P450 genes were cloned into pHis2 vectors and were then co-transformed with vector Rec2-ONAC066 into yeast strain Y187. Yeast co-transformed with Rec2-ONAC066 and pHis2-pLOC_Os02g30110 or pHis2-pLOC_Os06g37300 or pHis2-pLOC_Os02g36150 grew well on SD/-Trp/-Leu/-His medium containing 100 mM 3-AT, while yeasts co-transformed with Rec2-ONAC066 and pHis2-pLOC_Os01g41820 or pHis2-pLOC_Os03g25480 did not grow on the same medium ( Figure 6A). These results indicate that ONAC066 could bind to the promoters of LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 to activate the reporter gene expression in yeasts.
The binding site of ONAC0066 in the promoters of LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 was further mapped by ChIP-PCR. One, three and five NAC core-binding sites (CACG) (Tran et al., 2004) were identified within the 1500 bp promoter regions of LOC_Os02g30110, LOC_Os06g37300 and LOC_Os02g36150, respectively, and four probe regions for each promoter of the genes were chosen a Pearson's correlation coefficients value of the co-expressed genes identified from CREP and PLANEX databases, respectively. b Fold changes (M. oryzae FR13-infected/mock control) were retrieved from microarray dataset GSE7256 (Ribot et al., 2008), and genes with >1.5-fold changes were considered as up-regulated genes (shaded data).  (Ribot et al., 2008), and genes with >1.5-fold changes were considered as up-regulated genes (shaded data). c Data from http://crep.ncpgr.cn/crep-cgi/home.pl. d CYP genes in bold were found in two databases. e Data from http://planex.plantbioinformatics.org.
for ChIP-PCR assays ( Figure 6B). No PCR amplicon was detected in the four probe regions for each promoter of the genes tested when pre-immune serum was used in IP ( Figure 6C). Amplicons were observed in P5 and P6 probe regions in LOC_Os02g30110 promoter, P12 probe region in LOC_Os06g37300 promoter and P13 and P14 probe regions in LOC_Os02g36150 promoter, when anti-GFP antibody was used in IP ( Figure 6C). Notably, a clear amplicon was detected in P5 probe region of LOC_Os02g30110 promoter, which does not contain a NAC core-binding site ( Figure 6C). Together, these ChIP-PCR analysis of ONAC066 binding to promoters of the selected cytochrome P450 genes. ChIP of ONAC066-GFP transgenic line using GFP antibody (α-GFP) or pre-immune (Pre) serum was performed and precipitated DNA fragments were subject to PCR analysis using different primers for promoters of the selected cytochrome P450 genes. 10% of chromatin amount before IP was used as positive controls (input) and IP sample with pre-immune serum was used as a negative control. (D) Activation of promoters of the selected cytochrome P450 genes by ONAC066. Fragments of 1500 bp from start codon were used as promoters of the selected cytochrome P450 genes. Luciferase activity in protoplasts co-transfected with the reporter and different combinations of effectors was determined. Experiments were repeated at least three times with similar results, and results from one representative experiment are shown in panels (A,C). Data presented in panel (D) are the means ± SD from three independent experiments and ** indicated significant difference at p < 0.01 by Student's t-test, in comparison to empty vector.
results suggest that ONAC066 binds to the LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 promoters. The ability of ONAC066 to drive the transcription of LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 genes was further explored by using the rice protoplast transient expression system with the effector vector expressing ONAC066 and the reporter vector harboring the promoters of LOC_Os02g30110, LOC_Os06g37300, or LOC_Os02g36150. In rice protoplasts co-transfected with the effector vector ONAC066 and reporter vector pOs02g30110, pOs06g37300, or pOs02g36150, the LUC activity was significantly higher than that in rice protoplasts co-transfected with the empty effector vector and reporter vectors (Figure 6D). These results indicate that ONAC066 transcriptionally drives the promoters of the LOC_Os02g30110, LOC_Os06g37300 and LOC_Os02g36150 genes. Collectively, it is clear that ONAC066 can bind to the promoters of LOC_Os02g30110, LOC_Os06g37300 and LOC_Os02g36150 and thus drives their expression.

DISCUSSION
The present study further investigated the function and explored the molecular mechanism of ONAC066 in rice immunity. ONAC066 belongs to ONAC022 subgroup (Ooka et al., 2003) and its Arabidopsis homologous ANAC042 was found to suppress defense responses to a bacterial pathogen (Shahnejat-Bushehri et al., 2016). ONAC066 transcriptionally responded to M. oryzae infection and abiotic stress factors Yuan et al., 2019b) and thus is a stress-responsive NAC TF that can respond to multiple abiotic and biotic factors in rice. A recent study revealed that ONAC066 positively regulates resistance to blast and bacterial blight diseases through ABA-dependent signaling pathway . In this study, ONAC066-OE plants exhibited enhanced resistance while ONAC066-Ri and onac066-1 plants showed attenuated resistance to M. oryzae (Figure 1), clearly demonstrating that ONAC066 plays a positive role in rice immunity against M. oryzae. Furthermore, we previously demonstrated that ONAC066 functions as a positive regulator of drought and oxidative stress response in rice (Yuan et al., 2019b). Collectively, these findings enable ONAC066 as a promising factor to be used in creating novel rice germplasm with improved abiotic stress tolerance and disease resistance.
ONAC066 is a nucleus localized TF with an ability to bind to a canonical NAC recognition sequence (Tran et al., 2004) and a newly identified cis-element JUB1 binding site (Wu et al., 2012) in yeasts Yuan et al., 2019b). ONAC066 has transcription activator activity that depends on its C-terminal in yeast (Yuan et al., 2019b). This is further demonstrated in the present study that ONAC066 has transcription activator activity in rice cells (Figure 2). Therefore, ONAC066 acts as a transcription activator and exerts its biological function through activating downstream target genes. Notably, the C-terminal region of ONAC066 was capable of initiating the transcription in rice cells (Figure 2), which is similar to the previous observations on ONAC022 and OsNAP, whose C-terminal regions showed abilities to activate the transcription in yeast (Liang et al., 2014;Hong et al., 2016). Generally, the N-terminals of NAC proteins recognize their cis-element sequences while the C-terminals is responsible to initiate the transcription of the downstream target genes (Olsen et al., 2005). Whether the C-terminal of ONAC066 affect or abolish its interactions with the cis-elements in OsWRKY62 promoter is unknown. However, our previous study showed that the full ONAC022 with its C-terminal could bind to the NACRS element in electrophoretic mobility shift assays (Hong et al., 2016), implying that the C-terminals may not affect the interactions of NAC proteins with the cis-elements in promoters of their target genes. On the other hand, ONAC066 was found to bind to probe regions harboring NAC core-binding sites in the promoters of OsWRKY62, LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 genes (Figures 4, 6). However, ONAC066 also bound to a probe region without a NAC core-binding site in LOC_Os02g30110 promoter (Figure 6), suggesting that ONAC066 can bind to other unknown cis-elements in the promoters of its target genes. This is partially supported by our previous observation that ONAC066 not only bound to NACRS and JBS cis-elements in Y1H assays but also bound to a JBS-like cis-element in OsDREB2A promoter in ChIP-PCR and EMSA assays (Yuan et al., 2019b). Although the interaction of ONAC066 with OsWRKY62 promoter was validated by different experiments in the present study, including Y1H, ChIP-PCR and rice protoplast transient expression assays, the detailed ciselement(s) in OsWRKY62 promoter for ONAC066 need to be further identified.
Because ONAC066 has transcription activator activity in rice cells (Figure 2), we thus sought for genes that were up-regulated in ONAC066-OE plants by gene expression profiling analyses between ONAC066-OE11 and WT plants. Surprisingly, only 81 up-regulated genes were identified in ONAC066-OE plants ( Table 1). The number of the up-regulated genes in ONAC066-OE plants is comparable to the numbers of co-expressed genes identified in two different databases (Supplementary  Tables S4, S5). The up-regulated expression of genes for PRs, WRKYs and homologues of immunity-related factors (SARD1 and CMPG1) demonstrates the activation of defense response in ONAC066-OE plants. Importantly, 26 of these up-regulated genes were predicted to be induced by M. oryzae infection ( Table 1), suggesting that ONAC066 may modulate a group of genes that are responsive to M. oryzae. Defense and signaling genes were significantly up-regulated in ONAC066-OE plants but downregulated in ONAC066-Ri plants (Figure 3), indicating that the expression of these defense and signaling genes depends largely on ONAC066 function. This is partially similar to OsNAC111, which positively contributes to disease resistance by regulating the expression of a specific set of defense genes in disease response (Yokotani et al., 2014). Unlike OsNAC111 whose suppression did not affect rice immunity, ONAC066-Ri plants showed increased susceptibility to M. oryzae infection (Figure 1), implying that ONAC066 is required for basal immunity against M. oryzae.
Although the immunity-related functions of rice NAC TFs have been well documented (Nakashima et al., 2007;Kaneda et al., 2009;Yoshii et al., 2009Yoshii et al., , 2010Sun et al., 2013;Yokotani et al., 2014;Park et al., 2017;Liu et al., 2018;Wang et al., 2018), the molecular mechanisms of these NAC TFs to regulate rice immunity are largely unknown yet. ONAC066 was previously found to directly bind to the promoter of OsNCED4 to modulate its expression . OsNCED4 is one of the 9-cisepoxycarotenoid dioxygenases acting as a rate-limiting enzyme for abscisic acid biosynthesis (Huang et al., , 2019Hwang et al., 2018). Considering that ONAC066 is a transcription activator (Figure 2), the binding of ONAC066 to OsNCED4 promoter should drive the expression of OsNCED4, which in turn leads to ABA biosynthesis. However, the expression level of OsNCED4 was significantly down-regulated and ABA level was also markedly reduced in ONAC066-overexpressing plants . Therefore, whether OsNCED4 is a true target of ONAC066 and whether ABA signaling is involved in ONAC066-mediated regulation of rice immunity need to be further examined. On the other hand, SARD1 has been demonstrated to play a critical role in SA signaling through regulating pathogen-induced biosynthesis of SA in Arabidopsis (Zhang et al., 2010;. The up-regulated expression of LOC_Os01g04280, which shows 43.3% of identity to Arabidopsis SARD1, in ONAC066-OE plants raises a possibility that ONAC066 may regulate rice immunity through SA signaling via modulating LOC_Os01g04280 expression. In the present study, immunity-related WRKY genes including OsWRKY45 and OsWRKY62 (Shimono et al., 2007(Shimono et al., , 2012Peng et al., 2008;Yokotani et al., 2013;Cheng et al., 2015;Fukushima et al., 2016;Liu et al., 2016) were significantly up-regulated in ONAC066-OE plants but down-regulated in ONAC066-Ri plants (Figure 3 and Table 1). Y1H assays revealed ONAC066 could bind the OsWRKY62 promoter but not the promoters of OsWRKY45, OsWRKY76 and OsWRKY79 ( Figure 4A and Supplementary Figure S1). ChIP-PCR and rice protoplast transient expression assays demonstrated that ONAC066 bound to the NAC core-binding site in OsWRKY62 promoter and activated OsWRKY62 expression (Figure 4). It is thus likely that OsWRKY62 is a target of ONAC066 and ONAC066 modulates the expression of OsWRKY62 through direct binding to NAC core-binding site in OsWRKY62 promoter. Among these four WRKY genes up-regulated in ONAC066-OE plants, only OsWRKY62 was identified as one of the ONAC066 targets (Figure 4). The portion between verified ONAC066 binding and up-regulated expression of the genes in ONAC066-OE plants was 25%, which is similar to values reported for other plant TFs such as Arabidopsis WRKY33 . The altered expression of OsWRKY45 and OsWRKY76 in ONAC066-OE and ONAC066-Ri plants may be affected indirectly by the ONAC066-activated defense signaling pathway. OsWRKY45, a central component of the SA signaling pathway, is a positive regulator of rice immunity (Shimono et al., 2007(Shimono et al., , 2012Goto et al., 2015Goto et al., , 2016. However, inconsistent conclusions regarding the function of OsWRKY62 in rice immunity were previously reported; for example, OsWRKY62 and OsWRKY76 are negative regulators of rice immunity against blast and bacterial leaf blight diseases (Peng et al., 2008(Peng et al., , 2010Liu et al., 2016;Liang et al., 2017) while OsWRKY62 was reported to play a positive role in rice immunity (Fukushima et al., 2016). OsWRKY62 can form homodimers and heterodimers with OsWRKY45, and the OsWRKY45-OsWRKY62 heterodimer acts as a strong activator while the OsWRKY62 homodimer acts as a repressor of rice immunity (Fukushima et al., 2016). Simultaneous up-regulation of OsWRKY45 and OsWRKY62 in ONAC066-OE plants implies the formation of the OsWRKY45-OsWRKY62 heterodimers, which leads to the activation of defense response.
Due to limited number of up-regulated genes was identified in ONAC066-OE plants, we tried to mine putative target genes of ONAC066 through co-expression analysis, which has been successfully used to establish the transcriptional regulatory network and identify target genes for given TFs (Fang et al., 2015;Smita et al., 2015). Hundreds of functional genes were found to be co-expressed with ONAC066 through two different online tools CREP and PLANEX (Supplementary Tables S4, S5). Among 15 co-expressed genes simultaneously identified in two different databases, 5 encode cytochrome P450s (Table 3). Cytochromes P450s constitute one of the largest families of enzymatic proteins with diverse functions from critical structural components to key signaling molecules (Mizutani and Ohta, 2010;Nelson and Werck-Reichhart, 2011;Pandian et al., 2020). Rice genome harbors 326 genes coding for 355 cytochrome P450s (Wei and Chen, 2018). Some of rice cytochrome P450s are involved in biosynthesis of diterpenoid phytoalexins and therefore play roles in resistance against fungal and bacterial pathogens (Wang Q. et al., 2011;Li et al., 2013Li et al., , 2015Maeda et al., 2019;Shen et al., 2019;Kitaoka et al., 2021;Liang et al., 2021). In our gene expression profiling and co-expression analysis, a set of cytochrome P450 genes were identified to be co-expressed with ONAC066 ( Table 3). The co-expression feature of 5 cytochrome P450 genes was confirmed by their up-regulated expressions in ONAC066-OE plants and down-regulated expressions in ONAC066-Ri plants (Figure 5). ONAC066 directly bound to the promoters of 3 co-expressed cytochrome P450 genes, LOC_Os02g30110, LOC_Os06g37300 and LOC_Os02g36150, and activated their transcription (Figure 6), demonstrating that the LOC_Os02g30110, LOC_Os06g37300, and LOC_Os02g36150 cytochrome P450 genes are ONAC066 targets. The fact that DPF, a positive regulator of biosynthesis of diterpenoid phytoalexins (Yamamura et al., 2015), was up-regulated in ONAC066-OE plants but down-regulated in ONAC066-Ri plants ( Figure 5) imply that ONAC066 may play a role in regulating biosynthesis of diterpenoid phytoalexins in rice. Another, among the co-expressed cytochrome P450 genes, OsCAld5H1 (LOC_Os10g36848) has been shown to be involved in regulating the structure of cell wall lignin (Takeda et al., 2017(Takeda et al., , 2019, which is related to rice immunity against M. oryzae and X. oryzae pv. oryzae Li et al., 2020;Onohata and Gomi, 2020;Shamsunnaher et al., 2020). It is likely that ONAC066 contributes positively to rice immunity through direct regulation of a set of cytochrome P450s with distinct functions in biosynthesis of diterpenoid phytoalexins, cell wall lignin and other secondary metabolisms. However, the involvement of these co-expressed cytochrome P450s in rice immunity needs further investigation.

CONCLUSION
In the present study, we further demonstrated that ONAC066 is a positive regulator of rice immunity against M. oryzae and identified OsWRKY62 and three cytochrome P450 genes as ONAC066 targets. Therefore, ONAC066 contributes positively to rice immunity through regulating OsWRKY62 expression to activate defense response and/or activating a set of cytochrome P450 genes to promote the biosynthesis of defense compounds including phytoalexins. Together with our previous study on the function of ONAC066 in abiotic stress response (Yuan et al., 2019b), we concluded that ONAC066 acts as a positive regulator of abiotic and biotic stress response in rice and thus can be used in creating novel rice germplasm with improved abiotic stress tolerance and disease resistance.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
XY, DL, and FS conceived the project, designed the experiments, analyzed the data, and drafted the manuscript. XY generated all material used in this study (cloning, vector, transformations, and transgenic plants). XY, HW, YB, YY, YG, XX, and JW performed the experiments and collected the data. All authors commented on the manuscript.
Supplementary Figure S2 | Expression patterns of identified co-expressed cytochrome P450 genes in ONAC066-OE and ONAC066-Ri plants.
Supplementary Table S1 | List of primers used in this study.