Table_1_Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.XLSX

Abscisic acid (ABA) is a conserved and important “sesquiterpene signaling molecule” widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.</p

Data_Sheet_1_Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.pdf

Abscisic acid (ABA) is a conserved and important “sesquiterpene signaling molecule” widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.</p

Data_Sheet_2_Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.ZIP

Abscisic acid (ABA) is a conserved and important “sesquiterpene signaling molecule” widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.</p

E3d HPC-induced reduction of extracellular adenosine levels were totally reversed by ENT1 inhibitor NBTI.

<p>After the last hypoxic exposure, animals received either CD73 inhibitor (AOPCP) or ENT1 inhibitor (NBTI) through the microdialysis infusate. AOPCP blocked the elevation of extracellular adenosine levels induced by HPC or E6d HPC (A), while NBTI totally reversed the E3d HPC-induced extracellular adenosine reduction (B). Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

E3d HPC failed to provide protection against cerebral ischemia.

<p>Stroke volume was evaluated 72 hours after MCAO. E3d HPC failed to protect from ischemia 3 days after the last hypoxic exposure (A). Neurological deficits were also determined 72 hours after MCAO. Prominent improvements were observed 3 days after the last hypoxic exposure in the HPC and E6d HPC group (C, D). However, E3d HPC failed to ameliorate the neurological deficits induced by MCAO (C). Data are shown as mean ± SD; <i>n</i> = 6–8 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p

Table_2_Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.XLSX

Abscisic acid (ABA) is a conserved and important “sesquiterpene signaling molecule” widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.</p

E3d HPC reversed the upregulation of CD39 and CD73 and induced ENT-1 mRNA expression.

<p>A single episode of HPC and E6d HPC significantly increased the mRNA expression levels of CD39 (A), CD73 (B) and had no effect on ENT-1 (C) expression. E3d HPC had the opposite effect, having no effect on CD39 (A) and CD73 (B) expression, but significantly increasing ENT-1 (C) expression. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

Data_Sheet_3_Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.ZIP

Abscisic acid (ABA) is a conserved and important “sesquiterpene signaling molecule” widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.</p

E3d HPC reversed the upregulation of CD39 and CD73 and induced ENT-1 protein expression.

<p>Consistent with our mRNA data, a single episode of HPC and E6d HPC significantly increased the protein expression levels of CD39 (A) and CD73 (B) and had no effect on ENT-1 (C) expression. E3d HPC had the opposite effect, having no effect on CD39 (A) and CD73 (B) expression, but significantly increasing ENT-1 (C) protein expression. Representative Western blots are shown in panel D. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

CD39 and CD73 were highly upregulated after HPC and were mainly distributed in neurons.

<p>Immunofluorescence assay was used to show the expression of CD39 (A, B, C) and CD73 (D, E, F) in neurons and astrocytes in different groups. Neuron and astrocyte cytoskeletal proteins, MAP-2 and GFAP, were labeled with green fluorescence, while CD39 or CD73 were labeled with red fluorescence. CD39 and CD73 were highly upregulated after HPC but not after E3d HPC (A, B, D, E). Quantitative analysis of receptor expression showed that both CD39 and CD73 were mainly localized in neurons (C, F). Data are shown as mean ± SD; <i>n</i> = 4 mice in each group.</p