H2Bub1 Regulates RbohD-Dependent Hydrogen Peroxide Signal Pathway in the Defense Responses to Verticillium dahliae Toxins

Abstract

Histone H2B monoubiquitination (H2Bub1) plays critical roles in regulating growth and development as well as stress responses in Arabidopsis (Arabidopsis thaliana). In this study, we used wild-type and HUB1 and HUB2 loss-of-function Arabidopsis plants to elucidate the mechanisms involved in the regulation of the plant's defense responses to Verticillium dahliae toxins (Vd-toxins). We demonstrated that HUB-mediated H2Bub1 regulates the expression of the NADPH oxidase RbohD by enhancing the enrichment of histone H3 trimethylated on Lys-4 in response to Vd-toxins. RbohD-dependent hydrogen peroxide (H₂O₂) signaling is a critical modulator in the defense response against Vd-toxins. Moreover, H2Bub1 also affects posttranscriptional mitogen-activated protein kinase (or MPK) signaling. H2Bub1 was required for the activation of MPK3 and MPK6. MPK3 and MPK6 are involved in regulating RbohD-mediated H₂O₂ production. MPK3 and MPK6 are associated with protein tyrosine phosphatases (PTPs), such as Tyr-specific phosphatase1 and mitogen-activated protein kinases phosphatase1, which negatively regulated H₂O₂ production. In addition, H2Bub1 is involved in regulating the expression of WRKY33 WRKY33 directly binds to RbohD promoter and functions as a transcription factor to regulate the expression of RbohD Collectively, our results indicate that H2Bub1 regulates the NADPH oxidase RbohD-dependent H₂O₂ production and that the PTP-MPK3/6-WRKY pathway plays an important role in the regulation of RbohD-dependent H₂O₂ signaling in defense responses to Vd-toxins in Arabidopsis.

Figure 1.

HUB1 and HUB2 are involved in regulating defense responses to Vd-toxins in Arabidopsis. A, Root growth of wild-type Columbia-0 (Col-0), hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and HUB1/hub1-4 complementation line seedlings treated with Vd-toxins. Seedlings (4 d old) of the wild type and mutants were transferred from one-half-strength Murashige and Skoog (MS) medium to one-half-strength MS medium without or with Vd-toxins (100–300 μg mL⁻¹). Photographs were taken 4 d after transfer. B, Quantification of root lengths in A. Error bars indicate sd; n = 30. C, Vd-toxins-induced cell death in cotyledons of the Arabidopsis wild type, the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line. Cotyledons of 7-d-old plants were treated with 150 μg mL⁻¹ Vd-toxins and stained with Trypan Blue. Leaves treated with double distilled water were used as controls. D, Quantification of the cell death rates induced by Vd-toxins in C using ImageJ software. Error bars indicate sd; n = 8. E, Relative expression levels of the defense gene PR1 in the wild type, the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line were determined after treatment with 150 μg mL⁻¹ Vd-toxins. Seedlings treated with double distilled water were used as controls. Total RNA was extracted 24 h after the Vd-toxins treatment for RT-qPCR analysis. Error bars indicate sd; n = 15. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. All experiments were repeated at least three times.

Figure 2.

H₂O₂ production induced by Vd-toxins in the leaves of wild-type Arabidopsis Columbia-0 (Col-0) and mutants. A, H₂O₂ was detected by fluorescence resulting from H₂DCF-DA, as described in “Materials and Methods.” Bar = 100 μm. B, Quantification of the H₂DCF-DA fluorescence intensities in A. Error bars indicate sd; n = 8. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. C, Quantitative measurements of H₂O₂ concentrations in the leaves using the chromogenic peroxidase-coupled assay. Error bars indicate sd; n = 3. D, Relative expression levels of GST1 after treatment with 150 μg mL⁻¹ Vd-toxins in the wild type, the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line. Seedlings treated with double distilled water were used as controls. Total RNA was extracted 24 h after the Vd-toxins treatment for RT-qPCR analysis. Error bars indicate sd; n = 3. All experiments were repeated at least three times.

Figure 3.

AtRbohD and AtRbohF expression affect H₂O₂ production and the HUB1/HUB2-regulated AtRbohD expression level in response to the Vd-toxins treatment in Arabidopsis. A, H₂O₂ was detected in the leaves of wild-type Columbia-0 (Col-0) and the atrbohD, atrbohF, and atrbohD/F mutants by assessing the fluorescence resulting from H₂DCF-DA, as described in “Materials and Methods.” Bar = 20 μm. B, Quantification of the H₂DCF-DA fluorescence intensities in A. Error bars indicate sd; n = 8. C and D, Relative expression levels of AtRbohD and AtRbohF after treatment with 150 μg mL⁻¹ Vd-toxins in the wild type and the hub1-4, hub2-2, and hub1-4 hub2-2 mutants. Total RNA was extracted 24 h after the Vd-toxins treatment for RT-qPCR analysis. Seedlings treated with double distilled water were used as controls. Error bars indicate sd; n = 3. E, Schematic diagram of the AtRbohD and AtRbohF genes. AtRbohD P1, AtRbohD P2, and AtRbohF P1 are gene body regions; arrows indicate transcription start sites. F and G, Relative enrichments of H2Bub1 and H3K4me3 in the AtRbohD locus after treatment with Vd-toxins in the wild type and the hub1-4, hub2-2, and hub1-4 hub2-2 mutants. Chromatin was extracted 12 h after the Vd-toxins treatment, and immunoprecipitated DNA was analyzed using qPCR. Data were determined as percentages of H2Bub1/H3 and H3K4me3/H3 for each individual gene position. Relative enrichments of H2Bub1 and H3K4me3 in the AtRbohF P1 locus were used as negative controls. Error bars indicate sd; n = 3. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. All experiments were repeated three times.

Figure 4.

HUB1 and HUB2 affect the activation of MPK3 and MPK6 in response to the Vd-toxins treatment in Arabidopsis. A, The kinase activities of MPK3 and MPK6 were detected by immunoblotting using anti-phospho-p44/42 MAPK antibodies (p-MPK6 and p-MPK3). Seedlings (7 d old) of wild-type Columbia-0 (Col-0) and the hub1-4 mutant were treated with 200 μg mL⁻¹ Vd-toxins, and then total proteins were extracted after various times for immunoblot analysis. β-Actin was used as the loading control. Results are presented three independent biological replicates. B, Quantification of the kinase activities of MPK3 and MPK6 in A using ImageJ software. Error bars indicate sd; n = 3. C, The kinase activities of MPK3 and MPK6 were measured in the wild type and the hub1-4, hub2-2, and hub1-4 hub2-2 mutants. Seedlings (7 d old) of the wild type and mutants were treated with 200 μg mL⁻¹ Vd-toxins, and then total proteins were extracted after 30 min for immunoblot analysis. Results are presented three independent biological replicates. D, Quantification of the kinase activities of MPK3 and MPK6 in C using ImageJ software. Error bars indicate sd; n = 3. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests.

Figure 5.

Activation states of MPK3 and MPK6 induced by Vd-toxins in wild-type Arabidopsis Columbia-0 (Col-0) and atrbohD, atrbohF, and atrbohD/F mutants. A, The kinase activities of MPK3 and MPK6 were measured in the wild type and the atrbohD, atrbohF, and atrbohD/F mutants. Seedlings (7 d old) of the wild type and mutants were treated with 200 μg mL⁻¹ Vd-toxins, and total proteins were then extracted after 30 min for immunoblot analysis. β-Actin was used as the loading control. Results are presented three independent biological replicates. B, Quantification of the kinase activities of MPK3 and MPK6 in A using ImageJ software. Error bars indicate sd; n = 3. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests.

Figure 6.

MPK3 and MPK6 regulate H₂O₂ production and RbohD expression in Arabidopsis. A, H₂O₂ was detected in the leaves of wild-type Columbia-0 (Col-0), the mpk3 and mpk6 mutants, the 35S:MPK3 and 35S:MPK6 lines, and the MKK5^DD mutant using fluorescence resulting from H₂DCF-DA, as described in “Materials and Methods.” Leaves were treated with Vd-toxins plus 15 μm DEX. Bar = 20 μm. B, Quantification of the H₂DCF-DA fluorescence intensities in A. Error bars indicate sd; n = 8. C, Cell death in cotyledons of wild-type, the mpk3 and mpk6 mutants, the 35S:MPK3 and 35S:MPK6 lines, and MKK5^DD Arabidopsis seedlings. Cotyledons of 14-d-old seedlings were treated 14 h with 150 μg mL⁻¹ Vd-toxins plus 15 μm DEX and stained with Trypan Blue. Seedlings treated with 15 μm DEX were used as controls. D, Quantification of the cell death rates induced by Vd-toxins in C using ImageJ software. Error bars indicate sd; n = 15. E, Relative expression levels of AtRbohD after treatment in wild-type, the mpk3 and mpk6 mutants, the 35S:MPK3 and 35S:MPK6 lines, and MKK5^DD seedlings. Total RNA was extracted 12 h after the Vd-toxins treatment for RT-qPCR analysis. Error bars indicate sd; n = 9. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. All experiments were repeated three times.

Figure 7.

MPK3 and MPK6 interact with PTP1 and MKP1, which negatively regulate H₂O₂ production in Arabidopsis. A, Interactions of PTP1 and MKP1 with MPK3 and MPK6 in the yeast two-hybrid system. The experiments were performed three times with similar results. B, Co-IP of MPK3 and MPK6 interactions with PTP1 and MKP1. Total proteins were extracted from 15-d-old wild-type Columbia-0 (Col-0) and transgenic 35S:PTP1 and 35S:MKP1 lines. Input and immunoprecipitated proteins were analyzed by independently immunoblotting with anti-MYC, anti-MPK3, and anti-MPK6 antibodies. C, The kinase activities of MPK3 and MPK6 were detected by immunoblotting using anti-phospho-p44/42 MAPK antibodies (p-MPK6 and p-MPK3). Seedlings (7 d old) of the wild type, the ptp1 and mkp1 mutants, and the 35S:PTP1 and 35S:MKP1 lines were treated with 200 μg mL⁻¹ Vd-toxins, and the total proteins were then extracted at various times for immunoblot analysis. β-Actin was used as the loading control. D, Quantification of the kinase activity levels of MPK3 and MPK6 in C using ImageJ software. Results are presented three independent biological replicates. Error bars indicate sd; n = 3. E, H₂O₂ was detected in the leaves of the wild type, the ptp1 and mkp1 mutants, and the 35S:PTP1 and 35S:MKP1 lines using a fluorescence assay with H₂DCF-DA, as described in “Materials and Methods.” Bar = 20 μm. F, Quantification of the H₂DCF-DA fluorescence intensities in E. Error bars indicate sd; n = 8. G, Cell death induced by Vd-toxins in cotyledons of the wild type, the ptp1 and mkp1 mutants, and the 35S:PTP1 and 35S:MKP1 lines. Cotyledons of 14-d-old seedlings were treated 14 h with 150 μg mL⁻¹ Vd-toxins and stained with Trypan Blue. H, Quantification of the cell death rates induced by Vd-toxins in G using ImageJ software. Error bars indicate sd; n = 9. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. All experiments were repeated three times.

Figure 8.

HUB1 and HUB2 regulated the expression of WRKY33 in response to Vd-toxins, and WRKY33 regulated the expression of AtRbohD in Arabidopsis. A, The relative expression levels of WRKY33 induced by 150 μg mL⁻¹ Vd-toxins in wild-type Columbia-0 (Col-0), the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line. Error bars indicate sd. B, Schematic diagram of the WRKY33 gene. P1 and P2 are gene body regions; the arrow indicates the transcription start site. C and D, Relative enrichments of H2Bub1 and H3K4me3 in the WRKY33 locus after treatment with 150 μg mL⁻¹ Vd-toxins in the wild type, the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line. Chromatin was extracted 12 h after the Vd-toxins treatment, and immunoprecipitated DNA was analyzed by qPCR. Data were determined as the percentages of H2Bub1/H3 and H3K4me3/H3 for each individual gene position. Relative enrichments of H2Bub1 and H3K4me3 in the AtRbohF P1 locus were used as negative controls. Error bars indicate sd. Different letters in A, C, and D represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. E, WRKY33 regulates the expression of AtRbohD. The protoplasts of Arabidopsis harboring 35S:WRKY33 were used. The expression of AtRbohD was examined after the protoplasts were exposed to Vd-toxins for 4 h. pSuper1300 was used as the negative control. Error bars indicate sd. F, GUS activity measurement in N. benthamiana leaves after the transient expression of ProRbohD:GUS and 35S:WRKY33. pCAMBIA1391 and pSuper1300 were used as negative controls. LUC was used as an internal control. G, Schematic diagram showing the promoter structure of the AtRbohD gene. The upstream regions and part of the coding region are indicated by black wide lines and a white box, respectively. The solid arrowheads indicate the sites containing W-boxes in the AtRbohD promoter. The two fragments (P1 and P2) used for the yeast one-hybrid assay and EMSA are indicated. H, Yeast one-hybrid assay showing that WRKY33 binds to the AtRbohD promoter. I, EMSA showing WRKY33 binding to the AtRbohD promoter. Biotin-labeled fragments of the AtRbohD promoter that contained W-boxes were used as probes. Each experiment was repeated three times with similar results.

Figure 9.

Model of the H2Bub1 mechanism used to regulate defense responses to Vd-toxins in Arabidopsis. In this model, HUB-mediated H2Bub1 regulates the expression of the NADPH oxidase RbohD and WRKY33 by enhancing the enrichment of H3K4me3 in response to Vd-toxins. H2Bub1 also affects posttranscriptional MAPK signaling. H2Bub1 is required for the activation of MPK3 and MPK6. Phosphorylation of WRKY33 by MPK3 and MPK6 regulates the expression of RbohD. Moreover, MPK3 and MPK6 associate with PTPs, which negatively regulate H₂O₂ production. The PTP-MPK3/6-WRKY pathway regulates H₂O₂ signals in the responses against Vd-toxins in Arabidopsis.