Journal: bioRxiv

Article Title: ABA-regulated JAZ1 Proteins Bind NAC42 Transcription Factors to Suppress the Activation of Phytoalexin Biosynthesis in Plants

doi: 10.1101/2024.09.26.615281

Figure Lengend Snippet: ABA and ABA-upregulated GmJAZ1 genes suppress the elicitation of glyceollin biosynthesis. (A) Isoflavonoid metabolite amounts in Harosoy 63 seedlings treated for 9 d with pH 3.0 medium with and without ABA. Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. Error bars = standard error (SE), n ≥ 3 per condition. For a list of statistical values for this experiment, see Supplementary Table S2. (B) Glyceollin amounts in W82 soybean hairy roots after 24 h treatment with Phytophrhora sojae wall glucan elicitor (WGE) with and without ABA. Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. Error bars = standard error (SE), n ≥ 3 per condition. A repeat of this experiment is shown in Supplementary Figures S2. For a list of statistical values from both experiments, see Supplementary Table S3. (C) Glyceollin metabolite amounts in Harosoy 63 soybean seedlings after 6 d dehydration with or without norflurazone treatment measured by UPLC-PDA. Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. Error bars = standard error (SE), n ≥ 3 per condition. A repeat of this experiment is shown in Supplementary Figures S3. For a list of statistical values for this experiment, see Supplementary Table S4. (D) Expression levels of GmJAZ1 genes in W82 seedlings upon exposure to 6 d dehydration, dehydration+norflurazon, or ABA treatment measured by qRT-PCR. Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. Error bars represent SE (n = 5 per condition). For statistical values see Supplementary Table S5. (E) Schematic diagram of the GmJAZ1-9 gene and the region used go clone the 302 bp RNAi trigger site. (F) Expression levels of GmJAZ1 genes and glyceollin biosynthesis genes in transgenic W82 soybean seedlings after 6 days of dehydration treatment, measured by qRT-PCR. Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. Error bars represent SE (n = 5 per condition). For a list of statistical values from both experiments, see Supplementary Table S7. (G) Glyceollin levels in transgenic W82 soybean hairy roots transformed with an empty vector or an RNA interference construct targeting the JAZ1 gene, measured after 6 days of treatment using UPLC-PDA. Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. Error bars = standard error (SE), n ≥ 3 per condition. For a list of statistical values for this experiment, see Supplementary Table S8.

Article Snippet: For overexpressing GmJAZ1-9 into hairy roots, entry clone (GmJAZ1-9-pDONR221) were LR (Invitrogen, ON, Canada) recombined downstream of the CaMV 35S promoter in the destination vector pGWB2.

Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay, Transformation Assay, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: ABA-regulated JAZ1 Proteins Bind NAC42 Transcription Factors to Suppress the Activation of Phytoalexin Biosynthesis in Plants

doi: 10.1101/2024.09.26.615281

Figure Lengend Snippet: JAZ1 proteins from soybean, Arabidopsis and grapevine interact with NAC42 protein activators of phytoalexin biosynthesis. (A) Fluorescence microscopy of GmJAZ1-9 translationally fused to an N-terminal green fluorescent protein (GFP) tag in transgenic W82 hairy roots. A nuclear localization signal translationally fused to GFP (NLS-GFP) was used as positive control. An empty vector was used as negative control. DAPI (6 µg/ml) demonstrates nuclear staining. (B) Yeast two-hybrid analysis testing for interactions among GmJAZ1-9 and GmNAC42-1 or GmMYB29A2 proteins. Yeast strain PJ69-4a was co-transformed with genes that were translationally fused to the GAL4 activation domain (AD) or binding domain (BD). Yeast containing plasmid pairs were plated on SD-Leu-Trp (SD-L-T) and SD-Leu-Trp-His (SD-L-T-H) ±3-aminotriazole (3AT). Growth on SD-L-T or SD-L-T-H+ 0 mM 3AT (top rows) are plating controls, on SD-L-T-H or SD-L-T+H + 10 mM 3AT (bottom rows) indicates positive protein-protein interactions. pDEST-GADT7 (AD) was used as an empty ‘vector’ negative control. 3AT is used for attenuating auto-activation. (C) BIFC assays in N. benthamiana to confirm the interactions of GmJAZ1-9 and GmNAC42-1. (D) Yeast two-hybrid analysis testing for interactions between Arabidopsis AtJAZ1 and ANAC042 or AtMYB14. Yeast strain PJ69-4a was co-transformed and plated on SD-L-T-H and SD-L-T-H ±3AT. Yeast two-hybrid analysis testing for interactions between VvJAZ9 and VvNAC42 or VvMYB14. Yeast strain PJ69-4a was transformed and plated on SD-L-T-H and SD-L-T-H ±3AT. Growth on SD-L-T-H+ 0 mM 3AT (top rows) are plating controls, on SD-L-T+H + 10 mM 3AT (bottom rows) indicates positive protein-protein interactions. F , BIFC assays to confirm the interactions of AtJAZ1 and ANAC042, and VvJAZ9 and VvNAC42. All images were collected using Zeiss confocal microscope. Scale bars are 20 µm. Additional images and negative controls for C and F are shown in Supplementary Figure S6.

Article Snippet: For overexpressing GmJAZ1-9 into hairy roots, entry clone (GmJAZ1-9-pDONR221) were LR (Invitrogen, ON, Canada) recombined downstream of the CaMV 35S promoter in the destination vector pGWB2.

Techniques: Fluorescence, Microscopy, Transgenic Assay, Positive Control, Plasmid Preparation, Negative Control, Staining, Transformation Assay, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: ABA-regulated JAZ1 Proteins Bind NAC42 Transcription Factors to Suppress the Activation of Phytoalexin Biosynthesis in Plants

doi: 10.1101/2024.09.26.615281

Figure Lengend Snippet: GmJAZ1s are negative regulators of glyceollin phytoalexin biosynthesis in soybean. (A) Gene expressions in RNAi-GmJAZ1 W82 hairy roots elicited for 24 h with WGE. Results represent the average expression of five independent hairy root transformation events. *Significantly different than control by paired Student’s t -test (P < 0.05). Error bars represent SE (n = 5 per genotype). A repeat of this experiment is shown in Supplementary Figure S5. For a list of statistical values from both experiments, see Supplementary Table S9. (B) Gene expressions in 24 h mock (H2O)-treated RNAi-GmJAZ1 W82 hairy roots. Results represent the average expression of five independent hairy root transformation events. *Significantly different than control by paired Student’s t -test (P < 0.05). Error bars represent SE (n = 5 per genotype). For a list of statistical values, see Supplementary Table S10. (C) Amount of total glyceollin metabolites in RNAi-GmJAZ1 W82 hairy roots elicited for 24 h with WGE measured by UPLC-PDA. Results represent the average expression of five independent hairy root transformation events. *Significantly different than control by paired Student’s t -test (P < 0.05). Error bars represent SE (n = 5 per genotype). A repeat of this experiment is shown in Supplementary Figures S5. For a list of statistical values from both experiments, see Supplementary Table S11. (D) Amounts of total glyceollin metabolites in 24 h mock-treated RNAi-GmJAZ1 W82 hairy roots. *Significantly different than control by paired Student’s t -test (P < 0.05). Error bars represent SE (n = 5 per genotype). For a list of statistical values, see Supplementary Table S12. (E) Gene expressions in W82 hairy roots overexpressing GmJAZ1-9 elicited for 24 h with WGE. Results represent the average expression of five independent hairy root transformation events. *Significantly different than control by paired Student’s t -test (P < 0.05). Error bars represent SE (n = 5 per genotype). A repeat of this experiment is shown in Supplementary Figures S5. For a list of statistical values from both experiments, see Supplementary Table S13. (F) Total glyceollin amounts in W82 hairy roots overexpressing GmJAZ1-9 after 24 h WGE treatment. *Significantly different than control by paired Student’s t -test (P < 0.05). Error bars represent SE (n = 5 per genotype). A repeat of this experiment is shown in Supplementary Figures S5. For a list of statistical values from both experiments, see Supplementary Table S14.

Article Snippet: For overexpressing GmJAZ1-9 into hairy roots, entry clone (GmJAZ1-9-pDONR221) were LR (Invitrogen, ON, Canada) recombined downstream of the CaMV 35S promoter in the destination vector pGWB2.

Techniques: Expressing, Transformation Assay, Control

Journal: bioRxiv

Article Title: ABA-regulated JAZ1 Proteins Bind NAC42 Transcription Factors to Suppress the Activation of Phytoalexin Biosynthesis in Plants

doi: 10.1101/2024.09.26.615281

Figure Lengend Snippet: GmJAZ1-9 inhibits GmNAC42-1 transactivation of glyceollin biosynthesis gene promoters by inhibiting its DNA binding. (A) Effects of GmJAZ1-9 on GmNAC42-1’s transactivation of pHIDH and pPTS1 promoters in transfected cucumber protoplasts. Values on the y axis are normalized to co-transfected Renilla luciferase reporter construct. The error bars designate SE (n = 4 per construct). Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. All promoters comprise the 500-bp region upstream of their respective translational start sites. (B) Effects of GmJAZ1-9 on GmMYB29A2 transactivation of pHIDH and pPTS1 promoters in transfected cucumber protoplasts. The error bars designate SE (n = 4 per construct). Different letters show significant differences by single factor ANOVA, Tukey post hoc test, P < 0.05, α = 0.05. (C) Yeast three-hybrid analysis of strain PJ69-4a transformed with GmNAC42-1-Gal4AD and GmJAZ1-9 on SD-Leu-His-Ura (SD-L-H-U) and SD-L-H-U ±3-aminotriazole (3AT). Growth on SD-L-H-U + 10 mM 3AT (top rows) are plating controls, and SD-L-H-U + 10 mM 3AT (bottom rows) indicate positive protein-DNA interactions. pDR-GW was used as the empty ‘Vector’ control.

Article Snippet: For overexpressing GmJAZ1-9 into hairy roots, entry clone (GmJAZ1-9-pDONR221) were LR (Invitrogen, ON, Canada) recombined downstream of the CaMV 35S promoter in the destination vector pGWB2.

Techniques: Binding Assay, Transfection, Luciferase, Construct, Transformation Assay, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: ABA-regulated JAZ1 Proteins Bind NAC42 Transcription Factors to Suppress the Activation of Phytoalexin Biosynthesis in Plants

doi: 10.1101/2024.09.26.615281

Figure Lengend Snippet: Schematic diagram of the negative regulation of glyceollin biosynthesis by the GmJAZ1-9-GmNAC42 protein-protein interaction. Drought induces ABA accumulation which stimulates GmJAZ1 gene transcription via an unknown regulator. The resulting mRNA is translated and the GmJAZ1-9 protein physically interacts with GmNAC42 proteins to prevent them from activating glyceollin gene transcription. Broken arrows, multiple steps; solid arrows, single steps; colored boxes, gene promoters; bent arrows, gene transcription; brown globular structure, GmJAZ1-9 protein; colored bell-shaped structures, transcription factors.

Article Snippet: For overexpressing GmJAZ1-9 into hairy roots, entry clone (GmJAZ1-9-pDONR221) were LR (Invitrogen, ON, Canada) recombined downstream of the CaMV 35S promoter in the destination vector pGWB2.

Techniques: