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. 2021 Aug 23;187(3):1746–1761. doi: 10.1093/plphys/kiab400

Knock out of transcription factor WRKY53 thickens sclerenchyma cell walls, confers bacterial blight resistance

Wenya Xie 1, Yinggen Ke 1, Jianbo Cao 1, Shiping Wang 1,b, Meng Yuan 1,✉,b
PMCID: PMC8566205  PMID: 34618083

Abstract

Plant cell walls are the first physical barrier against pathogen invasion, and plants thicken the cell wall to strengthen it and restrain pathogen infection. Bacterial blight is a devastating rice (Oryza sativa) disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which typically enters the rice leaf through hydathodes and spreads throughout the plant via the xylem. Xoo interacts with cells surrounding the xylem vessel of a vascular bundle, but whether rice strengthens the sclerenchyma cell walls to stop pathogen proliferation is unclear. Here, we found that a WRKY protein, OsWRKY53, negatively confers resistance to Xoo by strengthening the sclerenchyma cell walls of the vascular bundle. OsMYB63 acts as a transcriptional activator and promotes the expression of three secondary cell wall-related cellulose synthase genes to boost cellulose accumulation, resulting in thickened sclerenchyma cell walls. Both OsWRKY53 and OsMYB63 are abundantly expressed in sclerenchyma cells of leaf vascular bundles. OsWRKY53 functions as a transcriptional repressor and acts genetically upstream of OsMYB63 to suppress its expression. The OsWRKY53-overexpressing and OsMYB63 knockout plants had thinner sclerenchyma cell walls, showing susceptibility to Xoo, while the OsWRKY53 knockout and OsMYB63-overexpressing plants had thicker sclerenchyma cell walls, exhibiting resistance to Xoo. These results suggest that modifying these candidate genes provides a strategy to improve rice resistance to bacterial pathogens.

A rice transcription factor-regulated signal transduction confers sclerenchyma cell wall immunity.

Introduction

Cell walls are necessary for maintaining plant cell structure, and also the first physical barrier against pathogen invasion (Underwood, 2012; Bacete et al., 2018). Strengthened cell walls are an efficient strategy for establishing a defense system, and have been characterized as a feature for plant basal defense. Cell walls are mainly composed of cellulose, hemicelluloses, pectin, and lignin (Keegstra, 2010); of these, cellulose determines the mechanical properties of plant cell walls and affects plant resistance to pathogens (Bacete et al., 2018). A few plant genes promoting cell wall strengthening have been reported as not only improving agronomic performance but also enhancing resistance to different pathogens. Accordingly, integrating the plant cell wall-mediated immunity into crop breeding has been accomplished in several crops, such as by optimizing Xa4 and OsMYB30 in rice, and ZmFBL41 in maize (Zea mays) (Hu et al., 2017; Li et al., 2019, 2020).

WRKY proteins form a large family of plant-specific transcription factors: over 98 genes encode WRKY proteins in rice genomes (Ross et al., 2007). Accumulating evidence indicates that the WRKY proteins play multiple roles in various processes of plant growth and development, and in responses to biotic and abiotic stress, by regulating the expression of target genes via direct binding to the canonical W-box at the target gene promoter. A great number of rice WRKY proteins, such as OsWRKY6, OsWRKY13, OsWRKY28, OsWRKY30, OsWRKY31, OsWRKY42, OsWRKY45, OsWRKY53, OsWRKY67, OsWRKY72, and OsWRKY76, have been characterized as triggering resistance to fungal pathogens, including Magnaporthe oryzae (M. oryzae) and Rhizoctonia solani, bacterial pathogens, including Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola, or viruses (Chujo et al., 2007, 2013; Qiu et al., 2007; Shimono et al., 2007; Zhang et al., 2008; Peng et al., 2012; Yokotani et al., 2013; Cheng et al., 2015; Choi et al., 2015; Liu et al., 2018; Hou et al., 2019). These WRKY proteins activate or suppress the expression of diverse downstream targeting genes to finally moderate plant physiological performance, alter plant phytohormone balance, or modify plant transcriptome network to defend against invasion by different pathogens.

OsWRKY53, a group I WRKY protein, is induced by a fungal cerebroside elicitor and blast fungus M. oryzae. Overexpression of OsWRKY53 results in enhanced resistance to M. oryzae (Chujo et al., 2007). OsWRKY53 also plays a positive role in rice BR signaling. OsWRKY53 overexpressing plants exhibit enlarged leaf angles, dwarfism, and increased grain length and grain width. Conversely, OsWRKY53 knock out plants exhibit decreased leaf angles and also slightly decreased plant height, grain length, and grain width (Tian et al., 2017). OsMPK1, OsMPK3, and OsMPK6 can physically interact with and phosphorylate OsWRKY53, causing the phospho-mimetic form OsWRKY53 to have elevated transactivation activity. Overexpression of phospho-mimetic OsWRKY53 further enhances the basal defence against M. oryzae, more than constitutive overexpression of OsWRKY53 (Chujo et al., 2014; Yoo et al., 2014). Additionally, OsWRKY53 can be phosphorylated by OsMAPK6 and phosphomimetic form of OsWRKY53 is critical for its function in BR response (Tian et al., 2017). Moreover, OsWRKY53 negatively regulates resistance to a chewing herbivore, the striped stem borer (Chilo suppressalis), but positively confers resistance to a piercing–sucking herbivore, the brown planthopper (Nilaparvata lugens) (Hu et al., 2015). The multiple roles of OsWRKY53 are due to either different functions of downstream targeting genes that it regulates or diverse upstream signals to which it responds. However, whether OsWRKY53 plays role in resistance to bacterial pathogens, and even the underlying mechanism, is largely unknown.

Xoo causes bacterial blight, the devastating bacterial disease of rice worldwide and severely affects rice yield and quality. Xoo infects rice leaves through hydathodes or wounds, and spreads in the plant through the xylem (Zhang et al., 2019). It has been reported that Xoo could secrete cell wall-degradation enzymes to loosen host plant cell walls for their subsequent proliferation (Cao et al., 2020). Conversely, rice plants employ R genes or defense responsive genes to activate cellulose synthase genes to thicken cell walls of vascular bundles to prevent Xoo colonization (Gu et al., 2005; Fu et al., 2011; Hu et al., 2017). Whether and how WRKY proteins confer resistance to Xoo through strengthening cell walls, especially the sclerenchyma cell walls surrounding the xylem vessel of a vascular bundle, are unclear. Here, we systematically screened rice WRKY genes, which showed differential expression patterns upon Xoo infection, then we focused on and characterized OsWRKY53. We found that OsWRKY53 suppressed the expression of OsMYB63, a transcription activator, and in turn, OsMYB63 activated the secondary cell wall-related cellulose synthase genes to strengthen sclerenchyma cell walls surrounding the xylem vessel of vascular bundle, resulting in the enhanced thickness of cell walls to stop Xoo proliferation.

Results

The response of OsWRKY53 to Xoo

A number of WRKY proteins have been identified as positive or negative regulators involved in rice–pathogen interactions (Viana et al., 2018). To mine more rice WRKY genes involving in response to Xoo, we analyzed an expression microarray dataset (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33411) which represented global transcriptional profiles of R gene-mediated responses to Xoo. Over 93 WRKY genes were systemically integrated and analyzed, of which OsWRKY53 had the highest transcription levels, in both compatible and incompatible rice–Xoo interactions (Supplemental Figure S1). Thus, we focused on OsWRKY53 to investigate its role in response to Xoo. OsWRKY53 has been previously reported to be induced by wounding (Yoo et al., 2014; Hu et al., 2015). The process of Xoo inoculation is accompanied by wounding, and inoculation with H2O can mimic wounding. Therefore, to explore the expression pattern of OsWRKY53 upon Xoo infection, we firstly used the reverse transcription quantitative real-time PCR (RT-qPCR) to assess the expression level of OsWRKY53 in wild-type Zhonghua 11 (ZH11) after inoculation with both Xoo and H2O. RT-qPCR assay showed that the expression level of OsWRKY53 increased at 12 h, but decreased at 24 h after both Xoo infection and H2O-mimiced wounding. However, there was lower expression level of OsWRKY53 at 12 h after Xoo inoculation than H2O control treatment (Figure 1, A). We parallelly carried out H2O-mimicked wounding assay in which the rice leaves were primarily inoculated with H2O, and 12 h later, half of them infected with H2O, the other half infected with Xoo. We found that the accumulation of OsWRKY53 was always lower after inoculation with Xoo than H2O (Figure 1, B). Additionally, we treated rice protoplasts that were transiently transformed with luciferase (LUC) reporter gene under regulation of OsWRKY53 promoter, with Xoo and H2O. Lower LUC activity was observed in protoplasts treated by Xoo than by H2O (Figure 1, C). Taken together, these results suggest that OsWRKY53 is induced by wounding, which is consistent with previous report (Yoo et al., 2014; Hu et al., 2015), and also demonstrate that Xoo infection could suppress OsWRKY53 expression.

Figure 1.

Figure 1

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OsWRKY53 negatively confers resistance to Xoo. A, Transcriptional pattern of OsWRKY53 after Xoo inoculation. hpi, hours post inoculation. B, Relative transcription levels of OsWRKY53 after wounding or Xoo inoculation. C, Activity of OsWRKY53-regulated LUC under wounding and Xoo treatment. D, Relative transcription level of OsWRKY53 and lesion length in OsWRKY53-oe lines. E, The response of oswrky53 mutants to Xoo PXO347. F, Phenotype of OsWRKY53-oe and oswrky53 plants after Xoo infection. G, The response of oswrky53 mutants to different Xoo strains. Plants were inoculated with Xoo at the booting stage. Data represent mean ± sd. n = 3 (A–C), n = 8 (D, E), n = 20–35 (G). Asterisks indicate a significant difference between H2O and Xoo inoculation (A–C), transgenic plants and WT (D, E, G) determined by one-tail Student’s t test at **P < 0.01.

OsWRKY53 negatively regulates rice resistance to Xoo

To investigate the function of OsWRKY53 in bacterial disease resistance, OsWRKY53-overexpressing (OsWRKY53-oe) transgenic plants in which OsWRKY53 was driven by the maize ubiquitin promotor were generated in rice variety ZH11, and 13 independent OsWRKY53-oe plants were obtained. Expression of OsWRKY53 was significantly increased in the 13 transgenic plants. All the OsWRKY53-oe plants exhibited remarkably enhanced susceptibility to Xoo, with lesion length 9.6 ± 0.4 to 14.0 ± 0.7 cm compared with wild-type 7.6 ± 0.5 cm after inoculation with Xoo at the booting stage (Figure 1, D and F). That enhanced susceptibility to Xoo was associated with increased OsWRKY53 expression was further confirmed in two T1 families derived from two independent T0 plants. The correlation coefficient between lesion length and OsWRKY53 transcriptional level was 0.769 and 0.670 for OsWRKY53-oe16 and OsWRKY53-oe25 T1 families, respectively (Supplemental Figure S2). In addition, the OsWRKY53-oe plants had significantly increased susceptibility to different Xoo strains compared with wild-type plants (Supplemental Figure S3, A). Moreover, the Xoo growth in leaves of OsWRKY53-oe plants was approximately 7.6–24.2-fold higher than in wild-type (Supplemental Figure S3, B).

We simultaneously used the CRISPR/Cas9 strategy to knock out the endogenous OsWRKY53 gene in ZH11 to generate oswrky53 mutants. Two 20-nt sequences in the first intron and the second exon of OsWRKY53 gene were selected as target sites for Cas9 cleavage (Supplemental Figure S4, A). Two mutants (designated oswrky53-1 and oswrky53-2) containing large fragment deletion (confirmed through sequencing the target regions after PCR amplification) were selected for further analysis (Supplemental Figure S4, B and C). The two mutants had truncated OsWRKY53 gene sequences, causing partial OsWRKY53 proteins (Supplemental Figure S4, D). After inoculation with Xoo at the booting stage, the two oswrky53 mutants exhibited enhanced resistance to Xoo with lesion length 6.0 ± 1.0 and 6.1 ± 0.9 cm, compared with wild-type 10.6 ± 0.6 cm (Figure 1, E and F). The Xoo growth in leaves of oswrky53 plants was approximately 10–12.5-fold lower than in wild-type (Supplemental Figure S3, B). Additionally, the oswrky53 plants had significantly increased resistance to different Xoo strains from Philippines (PXO61, PXO71, PXO99, PXO341) and China (GD1358, ZHE173) compared with wild-type plants (Figure 1, G). Collectively, these results suggest that OsWRKY53 negatively regulates rice resistance to Xoo.

Apart from the different responses to Xoo, the OsWRKY53-oe and oswrky53 plants also had different physiological performance: OsWRKY53-oe plants had significantly enlarged leaf angles but greatly decreased plant height, while the oswrky53 plants showed erect plant architecture and slightly decreased plant height (Supplemental Figure S5), which were in line with previously published data (Tian et al., 2017). Taken together, these data indicate that OsWRKY53 plays multiple roles not only in growth and development but also in resistance to bacterial pathogen.

Differentially expressed genes regulated by OsWRKY53

To uncover the underlying molecular mechanism for OsWRKY53-mediated resistance to bacterial blight, we analyzed the differentially expressed genes (DEGs) from the microarray dataset of OsWRKY53 overexpressing plants (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48500), which has been deposited on public Gene Expression Omnibus (Chujo et al., 2014). Over 2,487 and 1,556 DEGs were identified in two OsWRKY53 overexpressing lines compared with wild-type, respectively (Supplemental Figure S6, A). After overlapping DEGs, 486 genes were upregulated and 591 genes were downregulated in OsWRKY53 overexpressing plants compared with wild-type (Supplemental Figure S6, B). Gene ontology enrichment analysis showed that the 486 upregulated DEGs in OsWRKY53 overexpressing plants were mainly associated with response to biotic and abiotic stimulus (Supplemental Figure S6, C). We analyzed expression patterns of some representative defense-related genes in OsWRKY53-oe and oswrky53 plants. The RT-qPCR assays showed that a subset of upregulated DEGs, including Chitinase 1, Chitinase 2, Chitinase 3, Chitinase 4, Chitinase 14, Chitinase 15, and PBZ1 genes, had significantly increased expression levels in OsWRKY53-oe plants and decreased transcription accumulation in oswrky53 mutants compared with wild-type (Supplemental Figure S7), which was consistent with that OsWRKY53-oe plants were resistance to fungal blast (Chujo et al., 2007, 2014). Accordingly, the largest subset of 591 downregulated DEGs was associated with cell wall development (Supplemental Figure S6, C), including cellulose synthase-like (CSL) family genes and secondary cell wall-related cellulose synthase (CESA) genes. We simultaneously analyzed expression patterns of these representative CSL and CESA genes in OsWRKY53-oe and oswrky53 plants. Three CSL family genes (CSL2, CSL6, and CSL11) and three CESA genes (OsCesA4, OsCesA7, and OsCesA9) had markedly weakened expression levels in OsWRKY53-oe plants, while dramatically increased transcription contents in oswrky53 mutants (Figure 2, A and Supplemental Figure S7).

Figure 2.

Figure 2

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OsWRKY53 regulates cell wall development. A, Relative transcription levels of three CESA genes in OsWRKY53-oe and oswrky53 mutants. B, Relative transcription levels of three CESA genes in OsWRKY53-oe and oswrky53 mutants upon Xoo infection. C, Cellulose accumulation in leaves of OsWRKY53-oe and oswrky53 mutants. D, Observation of sclerenchyma cells at midrib of the flag leaf of OsWRKY53-oe and oswrky53 mutants via transmission electron microscope. CW, cell wall. XV, xylem vessel. Bars = 2 µm. E, Thickness of sclerenchyma cell walls at midrib of the flag leaf of OsWRKY53-oe and oswrky53 mutants. Data represent mean ± sd. n = 3 (A–C), n = 25 (E). Asterisks indicate a significant difference between transgenic plants and WT determined by one-tail Student’s t test at **P < 0.01 or *P < 0.05.

OsWRKY53 regulates secondary cell wall cellulose synthesis

Since CSL and CESA genes had significantly increased expression accumulation in oswrky53 mutants, and CESA genes have been reported to be involved in response to bacterial Xoo via regulating secondary cell wall cellulose synthesis (Hu et al., 2017). Thus, we speculated that CSL- or CESA-regulated cell wall development contributes to resistance against Xoo in oswrky53 mutants. We firstly assessed the dynamic transcription levels of three representative CESA genes in OsWRKY53-oe and oswrky53 plants upon Xoo infection. The transcription levels of OsCesA4, OsCesA7, and OsCesA9 genes were induced in oswrky53 mutants by Xoo infection, reaching the highest levels at 8 h after bacterial pathogen inoculation, while their expression levels were always inhibited in OsWRKY53-oe plants, compared with that in wild-type (Figure 2, B).

Then, to investigate whether the CESA genes with altered expression affected the cellulose level of plant leaves, we measured the content of cellulose in rice leaves. There was higher cellulose accumulation in leaves of oswrky53 mutants, while lower cellulose levels in leaves of OsWRKY53-oe plants, compared with wild-type (Figure 2, C). We simultaneously visualized the sclerenchyma cell walls in vascular bundle at midrib of flag leaf via transmission electron microscope, and found that the sclerenchyma cell walls were obviously thicker in oswrky53 mutants and thinner in OsWRKY53-oe plants, compared with wild-type (Figure 2, D and E). This indicates that the strengthened sclerenchyma cell wall probably contributes to enhanced resistance to Xoo in oswrky53 plants.

Abundant expression of OsWRKY53 in sclerenchyma cells

That oswrky53 mutants had thicker sclerenchyma cell walls and OsWRKY53-oe plants had thinner sclerenchyma cell walls implies that OsWRKY53 possibly plays roles in cell wall formation or thickening. Therefore, we assessed the spatial expression pattern of OsWRKY53 in vascular bundles of rice leaf. We sampled the midrib of flag leaf for in situ hybridization using OsWRKY53-specific antisense and sense probes, respectively. Strong signals were observed in sclerenchyma cells of vascular bundle, surrounding the xylem vessel, while no signals could be detected in vascular bundles by using OsWRKY53-specific sense probe as a control (Figure 3, A).

Figure 3.

Figure 3

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Spatial expression of OsWRKY53 and OsMYB63 in vascular bundle. A, The in situ hybridization of OsWRKY53 transcript in vascular bundle of rice leaf upon Xoo infection. B, The in situ hybridization of OsMYB63 transcript in vascular bundle of rice leaf upon Xoo infection. S, sclerenchyma cells. P, parenchyma cells. Bars = 25 µm.

To evaluate whether OsWRKY53 could directly bind and regulate the expression of CESA genes since OsWRKY53 acts as a transcriptional repressor (Tian et al., 2017), we firstly carried out electrophoretic mobility shift assay (EMSA). WRKY proteins bind to the canonical W-box with the TTGACC core sequence at the target gene promoter (Rushton et al., 2010). However, there were only between one to three non-canonical W-boxes at the promoters of OsCesA4, OsCesA7, and OsCesA9 (Supplemental Figure S8, A), so we tested these too. The EMSA showed that OsWRKY53 protein specifically bound to the canonical W-box, but did not bind to non-canonical W-boxes (Supplemental Figure S8, B). We further performed chromatin immunoprecipitation (ChIP)-qPCR assay to test whether OsWRKY53 could bind to promoters of OsCesA4, OsCesA7, and OsCesA9 in vivo. We generated constitutively expressed OsWRKY53 fused with GFP tag at its N-terminus in wild-type ZH11. The OsWRKY53-GFP-oe plants had higher mRNA and protein levels and exhibited longer lesion lengths compared with wild-type after inoculation with Xoo at the booting stage (Supplemental Figure S8, C and D). Similar phenotypes of OsWRKY53-oe and OsWRKY53-GFP-oe further supported the negative role of OsWRKY53 in conferring resistance to Xoo. We performed ChIP-qPCR assay to measure the enrichment of OsWRKY53 on the promoters of OsCesA4, OsCesA7, and OsCesA9 genes using anti-GFP antibody in OsWRKY53-GFP-oe plants. However, there were comparable OsWRKY53 enrichments on the three CESA gene promoters in OsWRKY53-GFP-oe and wild-type plants, suggesting that OsWRKY53 could not directly bind to the promoters of OsCesA4, OsCesA7, and OsCesA9 genes (Supplemental Figure S8, E). Taken together, these results indicate that oswrky53 plants exhibited upregulation of three CESA genes (which mediated cellulose accumulation) and thickened sclerenchyma cell walls, which contributed to their enhanced resistance against Xoo. However, the increased expression of three CESA genes was not directly regulated by OsWRKY53.

OsWRKY53 directly binds and suppresses expression of OsMYB63

R2R3-MYB transcription factors have been reported as potentially regulating expression of CESA genes (Huang et al., 2015; Noda et al., 2015). We hypothesized that OsWRKY53 directly repressed expression of MYB genes, and in turn, the MYB proteins activated transcription of CESA genes. To validate the hypothesis, we mined the putative MYB genes from DEGs that were regulated by OsWRKY53. Three MYB genes, OsMYB61, OsMYB63, and OsMYB103, were identified; OsMYB61 and OsMYB63 showed significantly weakened expression in OsWRKY53-oe plants, but only OsMYB63 had markedly increased transcription level in oswrky53 mutants (Supplemental Figure S9, A). Additionally, we examined the expression pattern of OsMYB63 in OsWRKY53-oe and oswrky53 plants upon Xoo infection, with the results that the transcription levels of OsMYB63 were induced in oswrky53 mutants but suppressed in OsWRKY53-oe plants after Xoo infection, compared with that in wild-type (Supplemental Figure S9, B). The expression patterns between OsMYB63 and CESA genes were similar, but were opposite to that of OsWRKY53, partially supporting our hypothesis.

A canonical W-box with TTGACC sequence was identified at the OsMYB63 promoter (Figure 4, A). Firstly, we used EMSA to determine whether OsWRKY53 could directly bind to the canonical W-box at OsMYB63 promoter in vitro. The EMSA showed that OsWRKY53 protein specifically bound to the intact W-box, but did not bind to mutated W-box with TTAAGCC sequence (Figure 4, A). Then, we performed ChIP-qPCR assay on the binding of OsWRKY53 to OsMYB63 promoter in vivo, in OsWRKY53 plants constitutively expressing OsWRKY53-GFP-oe. OsWRKY53 binding enriched at the W-box in the OsMYB63 promoter but not other positions, suggesting OsWRKY53 could bind to OsMYB63 promoter in vivo (Supplemental Figure S8, F). We parallelly carried out ChIP-qPCR assay in OsWRKY53-complementation (OsWRKY53-com) plants where OsWRKY53 fused with GFP regulated by the OsWRKY53 native promoter was transformed into oswrky53-1 mutant. The generated OsWRKY53-com plants had similar lesion length and Xoo population as wild-type, indicating that OsWRKY53-GFP fusion protein functionally complemented the loss-of-function of oswrky53 mutant (Supplemental Figure S10). The following ChIP-qPCR assay again verified that OsWRKY53 binding was enriched at the W-box in the OsMYB63 promoter (Figure 4, B). Lastly, we analyzed OsWRKY53 action on OsMYB63 expression in a transient expression assay with the LUC reporter driven by ∼2 kb putative promoter region of OsMYB63 (POsMYB63:LUC) in rice protoplast. The LUC activity of POsMYB63:LUC was repressed by OsWRKY53 (Figure 4, C).

Figure 4.

Figure 4

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OsWRKY53 binds and suppresses expression of OsMYB63. A, DNA binding activity assay of OsWRKY53 by EMSA. The blue capital letters indicate the intact W-box, the red capital letters represent the mutated W-box. B, Binding assay of OsWRKY53 to the promoter of OsMYB63 by ChIP-qPCR in OsWRKY53-Com plants using the anti-GFP antibody. C, Activity assay of OsWRKY53 in regulating OsMYB63 expression. B and C, data represent mean (three replicates) ± sd. Asterisks indicate a significant difference between control and effector determined by one-tail Student’s t test at **P < 0.01.

We simultaneously detected the spatial expression pattern of OsMYB63 in vascular bundle of rice leaf to test whether both OsMYB63 and OsWRKY53 were expressed in the same or similar cells. We sampled the midrib of the flag leaf for in situ hybridization using OsMYB63-specific antisense and sense probes, respectively. The signals were mainly observed in sclerenchyma cells of vascular bundle when using OsMYB63-specific antisense probe, and no signals could be detected in vascular bundles by using OsMYB63-specific sense probe as a control (Figure 3, B). The in situ hybridization assay indicates that both OsMYB63 and OsWRKY53 are expressed in the same sclerenchyma cells of vascular bundles. Taken together, these results indicate that OsWRKY53 directly binds to the OsMYB63 promoter both in vitro and in vivo, and suppresses OsMYB63 expression.

OsMYB63 positively regulates resistance to Xoo

To explore the role of OsMYB63 in rice–Xoo interactions, OsMYB63-overexpressing (OsMYB63-FLAG-oe) transgenic plants in which OsMYB63 fused with FLAG was driven by the maize ubiquitin promotor were generated in rice variety ZH11. Expressions of OsMYB63 were significantly increased in the 13 OsMYB63-FLAG-oe plants. All the OsMYB63-FLAG-oe plants exhibited enhanced resistance to Xoo after inoculation with Xoo at the booting stage (Figure 5, A). That enhanced resistance to Xoo was associated with increased OsMYB63 expression was further confirmed in two T1 and T2 families derived from two independent T0 plants. The two T1 families showed enhanced resistance to Xoo with lesion length 5.6 ± 0.6 to 8.0 ± 0.7 cm, and 6.7 ± 0.4 to 7.8 ± 0.7 cm, compared with wild-type 10.3 ± 0.7cm (Supplemental Figure S11). The correlation coefficient between lesion length and OsMYB63 transcriptional level was −0.832 and −0.884 for OsMYB63-FLAG-oe15 and OsMYB63-FLAG-oe31 T1 families, respectively (Supplemental Figure S11, A). The derived OsMYB63-FLAG-oe15-2 and OsMYB63-FLAG-oe31-11 T2 families had also lower lesion length than wild-type (Supplemental Figure S11, B). Correspondingly, the Xoo growth in leaves of OsMYB63-FLAG-oe plants was lower than in wild-type at different days post infection (Figure 5, D).

Figure 5.

Figure 5

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OsMYB63 positively regulates resistance to Xoo. A, Relative transcription level of OsMYB63 and lesion length in OsMYB63-FLAG-oe lines. B, The response of osmyb63 mutants to Xoo PXO347. C, Phenotype of OsMYB63-FLAG-oe and osmyb63 plants after Xoo infection. D, Growth of Xoo in leaves of OsMYB63-FLAG-oe and osmyb63 plants. Plants were inoculated with Xoo at the booting stage. A, B, and D, data represent mean ± sd. n = 3 (A, D), n = 30 (B). Asterisks indicate significant difference between transgenic plants and WT determined by one-tail Student’s t test at *P < 0.05 or **P < 0.01.

Simultaneously, osmyb63 mutants were generated via the CRISPR/Cas9 strategy in ZH11 background. Two 20-nt sequences in the first exon and first intron of OsMYB63 gene were selected as target sites for Cas9 cleavage (Supplemental Figure S12, A). Two mutants (designated osmyb63-1 and osmyb63-2) containing large fragment deletions (confirmed through sequencing the target regions after PCR amplification) were selected for further analysis (Supplemental Figure S12, B and C). These two mutants had truncated OsMYB63 gene sequences, causing a premature stop codon (Supplemental Figure S12, D). After inoculation with Xoo at the booting stage, the two osmyb63 mutants exhibited enhanced susceptibility to Xoo with lesion length 13.1 ± 0.5 and 13.5 ± 1.0 cm compared with wild-type 10.1 ± 0.6 cm (Figure 5, B and C). The Xoo growth in leaves of osmyb63 mutants was higher than in wild-type (Figure 5, D). We further transformed OsMYB63 fused with FLAG regulated by the native promoter of OsMYB63 into osmyb63-1 mutant. The generated OsMYB63-com plants had similar lesion length and Xoo growth as wild-type, indicating that OsMYB63-FLAG fusion protein complemented the loss-of-function of osmyb63 mutant (Supplemental Figure S13). Collectively, these results suggest that OsMYB63 positively regulates rice resistance to Xoo.

OsMYB63 specifically binds and promotes expression of CESA genes

OsMYB63 has been reported binding to OsCesA7 promoter at the two distinct MYB-binding sites, AC-II and SMRE3 (Noda et al., 2015). Both AC-II and SMRE3 are also distributed at the promoters of OsCesA4 and OsCesA9 (Supplemental Figure S14, A). To investigate whether OsMYB63 similarly bound to the promoters of OsCesA4 and OsCesA9 like to OsCesA7, we performed EMSA. A specific band was observed after OsMYB63 incubation with AC-II probe, while no band could be obtained when OsMYB63 incubation with mutated AC-II probe (Figure 6, A). Similarly, OsMYB63 protein could also bind to the intact SMRE3, but not the mutated SMRE3 of the promoters of OsCesA4 and OsCesA9 (Supplemental Figure S14, B).

Figure 6.

Figure 6

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OsMYB63 binds and promotes expression of three CESA genes. A, DNA binding activity assay of OsMYB63 by EMSA. The blue capital letters indicate the intact AC-II, the red capital letters represent the mutated AC-II. B, Binding assay of OsMYB63 to the promoter of three CESA genes by ChIP-qPCR in OsMYB63-FLAG-oe plants using the anti-FLAG antibody. C, Activity assay of OsMYB63 in regulating OsCesA4, OsCesA7, and OsCesA9 expression. B and C, Data represent mean ± sd. n = 3 (B and C). Asterisks indicate a significant difference between control and effector determined by one-tail Student’s t test at **P < 0.01.

To confirm OsMYB63 could bind to the promoters of OsCesA4, OsCesA7, and OsCesA9 in vivo, we carried out ChIP-qPCR assay in OsMYB63-FLAG-oe plants, where the OsMYB63-FLAG-oe plants had accumulated OsMYB63-FLAG fusion protein (Supplemental Figure S14, C). The ChIP-qPCR assay showed that OsMYB63 binding was enriched at the AC-II and SMRE3 sites in the OsCesA4, OsCesA7, and OsCesA9 promoters, suggesting OsMYB63 can directly bind to OsCesA4, OsCesA7, and OsCesA9 promoters in vivo (Figure 6, B and Supplemental Figure S14, D). Likewise, the enrichment of OsMYB63 on AC-II and SMRE3 sites in the OsCesA4, OsCesA7, and OsCesA9 promoters were obtained in OsMYB63-com plants (Supplemental Figure S14, E and F).

Additionally, we analyzed OsMYB63 action on expression of these three CESA genes in a transient expression assay with the LUC reporter driven by ∼1 kb putative promoter regions of OsCesA4 (POsCesA4:LUC), OsCesA7 (POsCesA7:LUC), and OsCesA9 (POsCesA9:LUC) by cotransformation of the relevant constructs in rice protoplast. The LUC activity of POsCesA4:LUC, POsCesA7:LUC, and POsCesA9:LUC was significantly activated by OsMYB63 (Figure 6, C). Taken together, these results indicate that OsMYB63 binds to the OsCesA4, OsCesA7, and OsCesA9 promoters both in vitro and in vivo, and promotes their expression.

OsMYB63 promotes sclerenchyma cell wall development

We assessed the expression of secondary cell wall-related CESA genes in OsMYB63-FLAG-oe and osmyb63 plants by RT-qPCR. There were obviously higher transcription levels of OsCesA4, OsCesA7, and OsCesA9 in OsMYB63-FLAG-oe plants, but markedly lower expression of them in osmyb63 mutants (Figure 7, A and B). We also measured the cellulose content in OsMYB63-FLAG-oe and osmyb63 plants. Consistent with the expression patterns of OsCesA4, OsCesA7, and OsCesA9, and abundant expression of OsMYB63 in sclerenchyma cells by in situ hybridization assay (Figure 3, B), the OsMYB63-FLAG-oe plants had significantly increased cellulose content and the osmyb63 mutants showed lower cellulose levels (Figure 7, C). The sclerenchyma cell walls in vascular bundles of flag leaves in OsMYB63-FLAG-oe and osmyb63 plants were also significantly thicker and thinner than that in wild-type plants, respectively (Figure 7, D and E). The results together suggest that OsMYB63 promotes sclerenchyma cell wall development.

Figure 7.

Figure 7

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OsMYB63 promotes cell wall development. A, Relative transcription levels of CESA genes in OsMYB63-FLAG-oe plants. B, Relative transcription levels of CESA genes in osmyb63 plants. C, Cellulose accumulation in leaves of OsMYB63-FLAG-oe and osmyb63 plants. D, Observation of sclerenchyma cell walls from the flag leaf of OsMYB63-FLAG-oe and osmyb63 plants via transmission electron microscope. CW, cell wall. Bars = 2 µm. E, Thickness of sclerenchyma cell walls from the flag leaf of OsMYB63-FLAG-oe and osmyb63 plants. Data represent mean ± sd. n = 3 (A–C), n = 30 (E). Asterisks indicate a significant difference between transgenic plants and WT determined by one-tail Student’s t test at **P < 0.01.

OsWRKY53 genetically acts upstream of OsMYB63

Since OsWRKY53 could bind and suppress the expression of OsMYB63, to genetically examine whether OsWRKY53 acted upstream of OsMYB63 in response to Xoo infection, we crossed OsWRKY53-oe plants with OsMYB63-FLAG-oe plants and isolated OsWRKY53-oe/OsMYB63-FLAG-oe homozygotes from the F2 population. The OsWRKY53-oe/OsMYB63-FLAG-oe plants had substantially taller plant height than OsWRKY53-oe plants, and slightly shorter plant height than OsMYB63-FLAG-oe plants or wild-type (Figure 8, A). After inoculating with Xoo at the booting stage, the OsWRKY53-oe/OsMYB63-FLAG-oe plants had comparable lesion length as OsMYB63-FLAG-oe plants, exhibiting increased resistance to Xoo, compared with wild-type, and the OsWRKY53-oe plants exhibited the longest lesion length among all the plants (Figure 8, B and Supplemental Figure S15, A–C). The transcription level of OsMYB63 was decreased in OsWRKY53-oe/OsMYB63-FLAG-oe plants compared with that in OsMYB63-FLAG-oe plants (Supplemental Figure S15, D). Accordingly, the expression of OsCesA4, OsCesA7, and OsCesA9 was slightly weakened in OsWRKY53-oe/OsMYB63-FLAG-oe plants compared with that in OsMYB63-FLAG-oe plants, while significantly higher than that in wild-type (Supplemental Figure S15, E). In line with higher transcriptional levels of three CESA genes, the OsWRKY53-oe/OsMYB63-FLAG-oe plants had increased cellulose content (Figure 8, C), and enhanced thickness of sclerenchyma cell walls of vascular bundles (Figure 8, D and E). Collectively, these results suggest that OsWRKY53 acts upstream of OsMYB63, and regulates rice response to Xoo by direct binding to the promoter of OsMYB63 to suppress its transcription.

Figure 8.

Figure 8

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OsWRKY53 genetically acts upstream of OsMYB63. A, The phenotype of OsWRKY53-oe, OsMYB63-FLAG-oe, and OsWRKY53-oe/OsMYB63-FLAG-oe plants at the booting stage. B, The response of OsWRKY53-oe, OsMYB63-FLAG-oe, and OsWRKY53-oe/OsMYB63-FLAG-oe plants to Xoo PXO347. C, Cellulose accumulation in leaves of OsWRKY53-oe/OsMYB63-FLAG-oe plants. D, Observation of sclerenchyma cell walls from the flag leaf of OsWRKY53-oe/OsMYB63-FLAG-oe plants via transmission electron microscope. Bars = 2 µm. E, Thickness of sclerenchyma cell walls from the flag leaf of OsWRKY53-oe/OsMYB63-FLAG-oe plants. Data represent mean ± sd. n = 20–35 (B), n = 3 (C), n = 30 (E). Asterisks indicate a significant difference between transgenic plants and WT determined by one-tail Student’s t test at **P < 0.01.

Discussion

Plants can strengthen the outer cell walls of leaf, stem, or root to provide the first physical barrier against pathogen invasion (Underwood, 2012; Bacete et al., 2018). Here, we found that rice could strengthen the inner cell walls of sclerenchyma cells surrounding the xylem vessel of vascular bundle, restraining Xoo from multiplying and proliferating, through regulation mediated sequentially by two transcription factors.

OsWRKY53 functions as a transcriptional repressor conferring resistance to a bacterial pathogen

WRKYs act as either transcriptional activators or transcriptional repressors in the process of defense response signal transduction or regulatory network (Rushton et al., 2010; Viana et al., 2018). Some rice WRKYs, including OsWRKY6, OsWRKY30, OsWRKY31, OsWRKY45, and OsWRKY67, act as transcriptional activators conferring resistance to fungal blast (Shimono et al., 2007; Zhang et al., 2008; Peng et al., 2012; Choi et al., 2015; Liu et al., 2018). OsWRKY72, OsWRKY76, and OsWRKY28 function as transcriptional repressors against bacterial blight or fungal blast (Chujo et al., 2013; Yokotani et al., 2013; Hou et al., 2019). Our previously reported OsWRKY13 and OsWRKY42 act as transcriptional repressors downregulating a set of genes, while OsWRKY45 functions as a transcriptional activator upregulating a series of genes, mediating fungal blast resistance (Cheng et al., 2015). Occasionally, a WRKY protein can serve as both transcriptional activator and transcriptional repressor triggering resistance to different pathogens. OsWRKY13 is an activator of the SA-dependent pathway and a repressor of JA-dependent pathway, conferring resistance to bacterial blight and fungal blast, respectively (Qiu et al., 2007).

OsWRKY53 has been previously identified as a positive regulator where it acts as a transcriptional activator by activating several Chitinase and PR genes to trigger resistance to fungal blast (Chujo et al., 2007, 2014). However, here, our biochemical assays and genetic results verified that OsWRKY53 served as a transcriptional repressor conferring resistance to bacterial blight (Figure 1), by directly repressing related genes such as MYB transcription factor OsMYB63. We found OsWRKY53 could directly bind to the canonical W-box of OsMYB63 promoter via EMSA and ChIP-qPCR assay, and suppress the expression of OsMYB63 through transient expression assay (Figure 4). Additionally, the expression level of OsMYB63 was significantly decreased in OsWRKY53 overexpressing plants, but obviously increased in oswrky53 knockout mutants. Owing to the inverse action model, OsWRKY53 and OsMYB63 mediated opposite immunity to Xoo, in that knocking out OsWRKY53 and overexpressing OsMYB63 improved rice resistance to Xoo. Apart from involvement in defense response, OsWRKY53 is also reported as participating in resistance to herbivore by suppressing a subset of WRKYs and JA-related genes (Hu et al., 2015). Thus, OsWRKY53 acts as a transcriptional activator or a repressor in diverse signal transduction pathways or regulatory networks that depend on different plant developmental or physiological processes and biotic or abiotic stress responses that OsWRKY53 participates in.

OsMYB63 acts as a transcriptional activator triggering resistance to a bacterial pathogen

Some members of transcription factor families, like WRKY, ERF, NAC, bHLH, and MADS, have been largely referenced as involved in plant resistance to diverse pathogens (Baillo et al., 2019; Li et al., 2019), while only a few MYB transcription factors have been reported as regulating immunity. Arabidopsis MYB46 functions as a negative regulator mediating resistance to fungal pathogen (Ramirez et al., 2011). Rice OsMYBS1, OsMYB30, OsMYB55, and OsMYB110 serve as transcriptional activators enhancing resistance to fungal blast or bacterial blight (Li et al., 2017, 2020; Kishi-Kaboshi et al., 2018). OsMYB-R1 plays a positive role in conferring resistance to fungal sheath blight (Tiwari et al., 2020). Here, we characterized OsMYB63, which acted as a transcriptional activator by directly binding to the conserved AC-II and SMRE3 sites at promoters of three CESA genes to upregulate their expression, triggering positive resistance to bacterial blight (Figure 6). OsMYB63 could directly bind to the promoters of OsCesA4, OsCesA7, and OsCesA9 via EMSA and ChIP-qPCR assay, and activate their expressions through transient expression assay (Figure 6). Additionally, the expression levels of OsCesA4, OsCesA7, and OsCesA9 were significantly increased in OsMYB63 overexpressing plants, but markedly decreased in osmyb63 knockout mutants (Figure 7). Although OsMYB63 could activate these three CESA genes, it was probable that OsCesA7 had the relative highest expression accumulation, compared with OsCesA4 and OsCesA9, in OsMYB63 overexpressing plants. Simultaneously, OsMYB63 could bind both AC-II and SMRE3 sites at promoters of three CESA genes, while there was probably higher binding affinity for AC-II than SMRE3 (Supplemental Figure S14). The underlying molecular mechanism should be further elucidated, which will fully uncover the function of OsMYB63. Apart from increasing resistance to bacterial blight, the OsMYB63 overexpressing plants had similar seed-related agronomic traits as wild-type (Supplemental Figure S16), indicating the potential usage of OsMYB63 for genetic improvement.

OsWRKY53 negatively confers resistance to Xoo by strengthening sclerenchyma cell wall

Plant cell walls provide not only mechanical support that is essential for plant growth and development but also physical barrier that offers basal defense against pathogen invasion (Keegstra, 2010; Underwood, 2012; Bacete et al., 2018). Overexpression of OsMYB30 promotes accumulation of ferulic acid and lignin, resulting in increasing the thickness of sclerenchyma cell walls that provide the physical barrier against fungal pathogen infection (Li et al., 2020). Conversely, knocking out of OsCesA4, OsCesA7, and OsCesA9 reduces the thickness of cell walls, increasing rice plant susceptibility to bacterial blight (Hu et al., 2017). The OsWRKY53-oe and osmyb63 plants had decreased cellulose content and thinner sclerenchyma cell walls in vascular bundles where Xoo multiply and proliferate, resulting susceptibility to Xoo. In turn, the oswrky53 and OsMYB63-oe plants had increased cellulose content and thicker sclerenchyma cell walls, conferring resistance to Xoo. Along with the enhanced thickness of sclerenchyma cell walls, the oswrky53 and OsMYB63-oe plants had increased leaf mechanical strength (Supplemental Figure S17), suggesting that the thickened sclerenchyma cell wall in leaf xylem vessels may correspond with the strengthened plant leaf architecture.

Because OsWRKY53 could regulate a large number of genes, we could not eliminate the possibility of other targeting genes that also play roles in OsWRKY53-mediated resistance to Xoo through altering other defense response signal transduction pathways. Taken together, we proposed a working model for OsWRKY53 that OsWRKY53 regulates a number of targeting genes by altering BR signaling pathway to modulate plant growth and development, while upon bacterial pathogen infection, OsWRKY53 targets and suppresses the expression of OsMYB63, and in turn, OsMYB63 binds and activates the expression of three secondary cell wall-related cellulose synthase genes to strengthen the sclerenchyma cell walls, strengthening physical barrier to defend bacterial pathogen invasion (Figure 9). Knock out of OsWRKY53 and overexpression of OsMYB63 can enhance rice resistance to bacterial pathogen without causing obvious morphology defects. Thus, modifying these candidate genes provides an applicable strategy to improve rice resistance to bacterial pathogens, accompanied by strengthened plant leaf architecture.

Figure 9.

Figure 9

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Proposed working model of OsWRKY53. OsWRKY53 binds and suppresses the expression of OsMYB63; in turn OsMYB63 binds and promotes the expression of three secondary cell wall-related cellulose synthase genes to strength sclerenchyma cell walls, resulting in enhanced resistance to bacterial blight. On the other hand, OsWRKY53 regulates unidentified genes to alter BR signaling pathways to modulate plant development. Solid arrows and T line indicate promotion and inhibition of gene expression, respectively. Dashed arrows indicate potentially regulated pathways.

Materials and methods

Generation of constructs and rice transformation

To generate the overexpressing vectors of OsWRKY53 and OsMYB63-FLAG, the full-length cDNAs of these genes were amplified from ZH11 using the specific primers (Supplemental Table S1), and then inserted into pU1301 (Cao et al., 2007) and pU1301-FLAG (Yuan et al., 2010) vectors, respectively. To generate the overexpressing OsWRKY53-GFP construct, the full-length cDNA of OsWRKY53 was fused with GFP and inserted into pU1301 vector. To generate oswrky53 and osmyb63 mutants, two sgRNAs targeting the specific regions at their exons or introns were ligated into the pCXUN-Cas9 vector (He et al., 2017). To generate the functional complementation of OsWRKY53-GFP and OsMYB63-FLAG plants, the full length cDNA of OsWRKY53 fused with GFP and OsMYB63 fused with FLAG were driven under their native promoter, respectively, and were inserted into pCAMBIA1301 vector. These constructs were transferred into Agrobacterium tumefaciens strain EHA105, which were further transformed into rice calli from mature embryos of ZH11 according to the protocol (Lin and Zhang, 2005).

Pathogen inoculation

Rice flag leaves were inoculated with X. oryzae pv. oryzae (Xoo) strains at the booting (panicle development) stage by the leaf-clipping method as previously described (Hui et al., 2019). Xoo strains used in this study included Philippine strains PXO61, PXO71, PXO99, PXO341, and PXO347, and Chinese strains ZHE173 and GD1358. Disease was scored by measuring the lesion length at 14 d after inoculation. The bacterial growth rate in rice leaves was determined by counting colony-forming units as described previously (Yuan et al., 2016).

Gene expression analysis

For gene expression analysis, the total RNA was isolated using Trizol reagent (Invitrogen). To examine the influence of bacterial pathogen infection on gene expression, 3-cm leaf fragments next to bacterial infection sites were used for RNA isolation. The cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Quantitative RT-qPCR was performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) as described previously (Yuan et al., 2016), using gene-specific primers (Supplemental Table S1). The expression level of rice actin gene was used to standardize the RNA sample as inner control. The expression levels of examined target genes were quantified by a relative quantitation method (2−ΔΔCT). Each RT-qPCR assay was repeated at least twice with a similar result, with each repetition having three replicates.

In situ hybridization

The midrib of rice flag leaf was sampled and cut into 0.5-cm length fragment, then fixed in 70% (v/v) FAA solutions (absolute ethanol:37% [v/v] formaldehyde:acetic acid:DEPC H2O = 70:10:5:15). The samples were fixed, dehydrated, embedded, sliced, and attached to slides as previously reported (Zhao et al., 2009). For preparation of the digoxigenin-labeled RNA probes, the specific coding regions of OsWRKY53 and OsMYB63 were amplified via PCR using gene-specific primers (Supplemental Table S1). The PCR products fused with T7 or SP6 promoters were used as templates for amplifying digoxigenin-labeled sense and antisense RNA probes. Tissue sections were cleared, dehydrated, dried, hybridized, and washed as previously reported (Zhao et al., 2009). The labeled probes were detected and images were photographed with a microscope (Axio Scope A1, Carl Zeiss, Germany).

Western blot assay

For Western blot assay, the total proteins were extracted from rice leaves in extraction buffer (25 mM Tris–HCl [pH 7.5], 1 mM EDTA [pH 8.0], 150 mM NaCl, 1% NP40, 5% glycerol, and complete EDTA-free protease inhibitor cocktail). The protein samples were separated on an SDS-PAGE gel and transferred onto a nitrocellulose membrane, then analyzed by blotting with different antibodies. Antibodies used for immunoblotting analyses included anti-GFP (AE012, ABclonal) and anti-FLAG (AE005, ABclonal).

ChIP-qPCR

The ChIP experiment was performed as described previously (Ke et al., 2020). Chromatin was extracted and fragmented via ultrasound to 200–400 bp. Antibodies anti-GFP and anti-FLAG were incubated with 40 µL protein A Dynabeads (Invitrogen, Norway) at 4°C for 4 h after which beads were washed, then 100 µL fragmented chromatin suspension was added, followed by incubation at 4°C overnight. After extensive washing and de-crosslink, the precipitated and input DNA samples were analyzed by qPCR using specific primers (Supplemental Table S1).

Transient expression assay in protoplasts

To determine the transcriptional regulation activity, the effector and reporter constructs were co-transfected into rice protoplasts as described previously (Ke et al., 2020). The transfected protoplasts were cultured for 12–16 h at 25°C in the dark. The LUC activities were measured using the Dual LUC Reporter Assay System (Promega, USA) according to the manufacturer’s instructions. The relative reporter gene expression levels were expressed as the ratio of firefly LUC to the renilla LUC (REN).

Protein expression and EMSA

The coding sequences of OsWRKY53 and OsMYB63 were amplified and cloned into the pCold and pMAL vectors, respectively, using the gene-specific primers (Supplemental Table S1). The recombinant plasmid was introduced to Escherichia coli BL21(DE3) cells, and then cells were induced with 0.2 mM isopropylthio-b-galactoside overnight at 16°C and collected by centrifugation. The His-OsWRKY53 and MBP-OsMYB63 proteins were purified using Ni Sepharose 6 fast Flow (GE-Healthcare) and Amylose Resin (BioLabs, #E8021V), respectively. A single-stranded DNA oligonucleotide was labeled with 5-carboxyfluorescein (FAM) at the 5′-end. The dsDNA was generated by mixing equal molar quantities of forward and reverse strands. The mixture was subsequently boiled and cooled to room temperature. For the EMSAs, increasing amounts of the unlabeled oligonucleotides (10-, 50-, and 100-fold of labeled probes) were incubated with FAM-labeled dsDNA on ice in binding buffer (10 mM Tris–HCl [pH 7.5], 50 mM KCl, 10 mM MgCl2, 5% [v/v] glycerol, 1 mM DTT) for 20 min. The reactions were then resolved on 6% native acrylamide gels in 0.25 Tris borate buffer under an 4°C electrical field of 110 V for 2 h. Gels were visualized using a FUJIFILM (FLA-5100).

Transmission electron microscopy

The ultrastructure of rice leaf cell walls in vascular bundles of the xylem vessels was studied by transmission electron microscopy. The leaf tissues were cut into 1 × 3 mm pieces and fixed in 2.5% (w/v) glutaraldehyde in 0.1 M phosphate buffer solution (PBS) (pH 7.2) at 4°C overnight. The fixed tissues were washed in PBS three times for 30 min each at room temperature (20–25°C), postfixed for 2 h in 1% osmium tetroxide, dehydrated in a graded series of acetone, then infiltrated with Spurr resin (SPI, SPI Chem, West Chester, USA), and polymerized at 65°C for 48 h. The samples were cut into ultrathin sections (60–70 nm thick), stained with 2% uranyl acetate, and examined with a Hitachi transmission electron microscope (H-7650; Hitachi, Japan) at 80 kV.

Determination of cellulose content and measurement of mechanical properties

The rice flag leaf was first heated at 110–120°C for about 10 min to inactivate the enzymes, then ground in a mortar and pestle with liquid nitrogen and dried to constant weight at 65°C for about 2 d. The extraction and fractionation of the cell wall polysaccharides were performed with acetic acid:nitric acid:water (8:1:2, v/v/v) and 67% H2SO4, and the extraction was measured using colorimetric assays according to the published method (Peng et al., 2000). The force required to break fresh leaf was measured with a digital testing device (RH-K300, China).

Microarray data analysis

The processed microarray data were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33411). The relative expression of selected WRKY genes used the R package to generate the heatmap. For analysis of OsWRKY53 regulated DEGs, the processed microarray data were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48500) and analyzed by DEseq2 package. Genes with relative expression |log2 fold change|>1 and padj < 0.01 were considered as DEGs.

Statistical analysis

Statistical parameters are reported in the figures and figure legends. The differences between samples were analyzed for statistical significance using the Student’s t test in the Microsoft Office Excel program. The correlation analysis between lesion area and gene expression levels was analyzed using the CORREL analysis in the Microsoft Office Excel program.

Accession numbers

Sequence data from this article can be found in the Rice Genome Annotation Project (RGAP) data libraries under the following accession numbers: OsWRKY53 (Os05g27730), OsMYB63 (Os04g50770), OsMYB61 (Os05g04820), OsMYB103 (Os08g05520), OsCesA4 (Os01g54620), OsCesA7 (Os10g32980), and OsCesA9 (Os09g25490).

Supplemental data

Supplemental Figure S1. Transcriptional profiles of rice WRKY genes in response to Xoo.

Supplemental Figure S2. Response of two representative OsWRKY53-oe T1 families to Xoo.

Supplemental Figure S3. OsWRKY53 negatively regulates rice resistance to Xoo.

Supplemental Figure S4. Characterization of oswrky53 mutant.

Supplemental Figure S5. Data represent agronomic performance of OsWRKY53-oe and oswrky53 plants.

Supplemental Figure S6. The DEGs in OsWRKY53 overexpressing plants.

Supplemental Figure S7. Relative transcriptional levels of defense-related genes and cell wall-related genes in OsWRKY53-oe and oswrky53 plants based on RT-qPCR analysis.

Supplemental Figure S8. OsWRKY53 does not regulate expression of CESA genes.

Supplemental Figure S9. Transcription level of OsMYB61, OsMYB63, and OsMYB103 genes.

Supplemental Figure S10. Data represent phenotype of OsWRKY53-Com lines in response to Xoo.

Supplemental Figure S11. Data represent lesion length and relative transcription level of two OsMYB63-FLAG-oe T1 and T2 families.

Supplemental Figure S12. Characterization of osmyb63 mutant.

Supplemental Figure S13. Phenotype of OsMYB63-Com lines in response to Xoo.

Supplemental Figure S14. OsMYB63 binds and promotes expression of three CESA genes.

Supplemental Figure S15. Phenotype of OsWRKY53-oe/OsMYB63-FLAG-oe plants in response to Xoo.

Supplemental Figure S16. Agronomic traits of the OsMYB63-FLAG-oe plants.

Supplemental Figure S17. Mechanical strength of flag leaf of OsWRKY53 and OsMYB63 transgenic plants.

Supplemental Table S1. Primers used in this study.

Supplementary Material

kiab400_Supplementary_Data
Click here for additional data file. (1.7MB, pdf)

Acknowledgments

We are grateful to Professor Yunde Zhao (University of California, San Diego) for providing the CRISPR vector, Professor Yu Zhao (Huazhong Agricultural University) for analyzing in situ hybridization assay, and Professor Liangcai Peng (Huazhong Agricultural University) for analyzing cell wall composition.

Funding

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0100600), the National Natural Science Foundation of China (31821005, 31822042, 31871946), and the Fundamental Research Funds for the Central Universities (2662019FW006).

Conflict of interest statement. None declared.

 

W.X. and M.Y. designed the research. W.X. performed most of the experiments, analyzed the data, and drafted the manuscript. Y.K. and J.C. assisted the experiments. S.W. and M.Y. supervised the project. M.Y. designed some of the experiments and revised the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Meng Yuan (myuan@mail.hzau.edu.cn).

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