Fig 1.
Cloning and characterization of the IbNAC1 promoter.
(A) A schematic feature of the IbNAC1 promoter. Prediction of the cis-acting elements in the 1665-bp promoter region of IbNAC1 was performed using the PLACE and PlantCare databases. (B) Activity analysis of the IbNAC1 promoter upon wounding in transgenic sweet potato leaves. The GUS reporter gene driven by the IbNAC1 promoter was transferred into sweet potato. Transgenic sweet potato leaves that had been mechanically wounded for 30 min using tweezers were stained with GUS staining buffer. The leaves were stained after 24 hours to analyze the systemic response of the IbNAC1 promoter. (C) GUS activity in transgenic sweet potato with the GUS reporter gene driven by the IbNAC1 promoter after wounding for 30 min or treatment with 50 μM MeJA for 1 hour, 10 mM ethephon for 1 hour, or 1% H2O2 for 1 hour. Error bars indicate standard deviations (SDs) (n = 10). Different letters represent significance as determined by one-way ANOVA (P<0.05). (D) GUS staining assay in transgenic Arabidopsis with the GUS reporter gene driven by the IbNAC1 promoter. The wounded leaves were stained with GUS staining buffer after 30 min of treatment with a wheel.
Fig 2.
Position -1506 to -1463 bp (NWRE) determines the activity of the IbNAC1 promoter in response to wounding.
(A) A schematic model of the 5’ deletion of the primary promoter of IbNAC1. (B) GUS activity assay of each promoter fragment in transgenic Arabidopsis. Four independent lines of each construct were used to determine the GUS activity after wounding treatment for 30 min. Error bars indicate SDs from three independent replicates. Asterisks represent the significant differences from non-wounded plants by Student t-test’s (*, P<0.05; **, P<0.01). (C) A schematic model of a 5’ promoter deletion from -1665 to -1395 bp. (D) GUS activity assay of each promoter fragment fused to the 35S minimal promoter (35Sm) in transgenic Arabidopsis. Four independent lines of each construct were used to determine the GUS activity after wounding treatment for 30 min. Error bars indicate SDs from four independent replicates. Asterisks represent the significant differences from non-wounded plants by Student’s t-test (**, P<0.01).
Fig 3.
G-box within the NWRE plays an essential role in the wound response.
(A) G-box plays an essential role in NWRE activation. The intact NWRE region or G-box-mutated NWRE region fused to the 35S minimal promoter (35Sm) and GUS reporter were transformed into Arabidopsis. The GUS activity was determined in three independent T4 transgenic lines under wound stress. Error bars indicate standard deviations (SDs) from four independent replicates. Asterisks represent significant differences from non-wounded plants by Student’s t-test (P<0.05). (B) NWRE region fused to 35Sm and the GUS reporter was transformed into sweet potato. Three independent transgenic lines were used to analyze the GUS activity under wounding. Transgenic lines containing 35Sm::GUS were used as negative controls for wound activation. Error bars indicate SDs from four independent replicates. Asterisks represent significant differences from non-wounded plants by Student’s t-test (P<0.05). (C) Activity assay of NWRE in sweet potato leaves upon wounding. Three independent transgenic lines with NWRE-35Sm::GUS (SPNWRE) were wounded by tweezers. After wounding for 30 min, the wounded leaves were stained with GUS staining buffer at 25°C. (D) Microscopic observation of the wounded region in SPNWRE leaves after GUS staining. The wounded regions were monitored under a stereo microscope. 35Sm::GUS transgenic lines (35Sm) were used as negative controls for GUS analysis.
Fig 4.
IbbHLH3 and IbbHLH4 physically bind to the NWRE region.
(A) Expression levels of bHLH transcription factors in sweet potato leaves upon wounding. The bHLH transcription factors that were selected from the transcriptome dataset of wounded leaves were analyzed by a wounding time-course treatment. IbNAC1 was shown as reference. Error bars indicate standard deviations (SDs) from four independent replicates (B) NWRE binding analysis. Yeast one-hybrid (Y1H) screening was used to analyze the binding ability of IbbHLHs with NWRE DNA cis-elements. NWRE fragments were cloned into the pHIS2.1 vector as bait. Then, the bait and pGADT7-IbbHLHs (AD-IbbHLHs) were co-transformed into yeast strain Y187. The growth of the yeast transformants is shown on both +His medium and -His medium with 100 mM 3-AT. The G-box-mutated NWRE was used as a control to verify the binding abilities of IbbHLH3 and IbbHLH4. (C) NWRE binding analysis of IbbHLH4 by Y1H screening. (D) NWRE binding analysis of IbbHLH3 by EMSA. GST-IbbHLH3 binds to 6FAM-NWRE in the presence of changing protein concentrations. The 50x unlabeled NWRE was included as a competitor (CP), and the GST protein was used as a negative control for NWRE binding. (E) NWRE binding analysis of IbbHLH4 by EMSA. GST-IbbHLH4 binds to 6FAM-NWRE depending on the IbbHLH4 protein concentration. The GST protein was used as a negative control for NWRE binding.
Fig 5.
IbbHLH4 antagonizes IbbHLH3 to negatively regulate IbNAC1.
(A) Transactivation assay indicates that the activation of the NWRE region in the IbNAC1 promoter by IbbHLH3 was suppressed by IbbHLH4. RLUC driven by the 35S promoter was used as an internal control. Error bars represent standard deviations (SDs) (n = 10). Different letters represent significance as determined by one-way ANOVA (P<0.05). (B) Transactivation assay indicates that the activation of the IbNAC1 promoter by IbbHLH3 was suppressed by IbbHLH4. Error bars represent SDs (n = 10). Different letters represent significance as determined by one-way ANOVA (P<0.05). (C) In vivo transactivation assay. 35S::IbbHLH3 or 35S::IbbHLH4 was expressed in NWRE-A Arabidopsis. Total protein extracts from 21-day old plants were used for GUS activity analysis. Error bars represent SDs from four biological replicates. Different letters mean the significant difference determined by one-way ANOVA (P>0.05). (D) In vivo transactivation assay 35S::IbbHLH3 or 35S::IbbHLH4 was expressed in mNWRE-A Arabidopsis to examine the regulatory function of IbbHLH3 and IbbHLH4 on G-box in NWRE. Error bars represent SDs from three biological replicates. Different letters represent significance as determined by one-way ANOVA (P<0.05). (E) In vivo competing assay. IbbHLH4 was transiently expressed in IbbHLH3/NWRE-A plants by agro-infiltration. Error bars represent SDs from four biological replicates. Asterisk represents the significant difference from IbbHLH3/NWRE-A plants (Student’s t-test, P<0.05). (F) Overexpression of IbbHLH3 and IbbHLH4 in sweet potato. 35S::YFP (EV), 35S::YFP-IbbHLH3, or 35S::YFP-IbbHLH4 was expressed in sweet potato leaves by particle bombardment. The expression level of IbNAC1 was quantified by qRT-PCR. Error bars indicate SDs from three biological replicates. Different letters represent significance as determined by one-way ANOVA (P<0.05).
Fig 6.
IbbHLH4 interacts with IbbHLH3 to repress the transcriptional activation function.
(A) Bimolecular fluorescence complementation (BiFC) assay to detect the interactions of IbbHLH3 and IbbHLH4. The NLS-mCherry construct was co-expressed into protoplasts as a nuclei marker. The empty vectors of nYFP and cYFP interacted with IbbHLH3 and IbbHLH4, respectively, as negative controls. GUS protein was used as negative interaction protein of BiFC. (B) BiFC assay to detect the protein interaction between IbbHLH3 and IbbHLH4. (C) Co-IP assay to verify the interaction of IbbHLH3 with IbbHLH4 in plant. The 35S::GST-IbbHLH4 (GST-IbbHLH4) was co-expressed with 35S::GFP-IbbHLH3 (GFP-IbbHLH3) or 35S::GFP (GFP, control) in tobacco leaves. The total protein from the tobacco leaves were immunoprecipitated with the GST resin, and were further analyzed by western blot using anti-GST antibody and anti-GFP antibody. 35S::GST (GST) was used as negative control for IbbHLH3 interaction. (D) Transactivation assay using the GAL4DB-based expression system. The activation of Gal4BS by BD-IbbHLH3 binding was suppressed by IbbHLH4, while IbbHLH4 could not affect the activity of Gal4BS. RLUC driven by the 35S promoter was used as internal control to normalize the transfection efficiency. Error bars mean standard deviations (SDs) (n = 10). Different letters mean statistically significance determined by one-way ANOVA (P<0.05).
Fig 7.
The protein interaction of IbbHLH3 with, JAZs, IbEIL1 and MAPKs.
(A) BiFC assay to detect the heterodimeric interaction of IbbHLH3 with JAZs (IbJAZ1, IbJAZ2a, and IbJAZ2b), IbEIL1, and IbMAPK (IbWIPK1 and IbWIPK2). The NLS-mCherry construct was co-expressed into protoplasts as a nuclear marker. (B) Yeast two-hybrid assays. The interactions were indicated by the ability of yeast transformants to grow on SD medium lacking Leu/Trp/His (SD -L-T/-H) with 5 mM 3-AT. The coding region of IbbHLH3 was cloned into pGADT7 (AD), and IbJAZs, IbEIL1, and IbWIPKs were cloned into pGBKT7 (BD). Empty vector was used as negative control for IbbHLH3 interaction. (C) Competing assay of IbbHLH3. The activation of NWRE by the expression of IbbHLH3 was inhibited by the co-expression with JAZ1, JAZ2a, JAZ2b and IbEIL1. Error bars mean standard deviations (SDs) (n = 10). Different letters represent the significance determined by one-way ANOVA (P<0.05).
Fig 8.
A proposed model in sweet potato leaves of wound signaling regulating IbNAC1 expression against herbivory.
The early wounding signals trigger the expression of IbbHLH3 and repress the expression of JAZs and EIL1, which are repressors of IbbHLH3 that inhibit its transactivation function before wounding stress. The released IbbHLH3 activates the expression of IbNAC1 by binding to the G-box motif in the promoter region. Subsequently, IbNAC1 up-regulates the expression of the sporamin gene and participates in the JA response against insect feeding. IbbHLH4 and JAZ1, induced in the late wound response, repress IbNAC1 by competing with IbbHLH3 to avoid IbNAC1-mediated injury.