IbBBX24 Promotes the Jasmonic Acid Pathway and Enhances Fusarium Wilt Resistance in Sweet Potato

Abstract

Cultivated sweet potato (Ipomoea batatas) is an important source of food for both humans and domesticated animals. Here, we show that the B-box (BBX) family transcription factor IbBBX24 regulates the jasmonic acid (JA) pathway in sweet potato. When IbBBX24 was overexpressed in sweet potato, JA accumulation increased, whereas silencing this gene decreased JA levels. RNA sequencing analysis revealed that IbBBX24 modulates the expression of genes involved in the JA pathway. IbBBX24 regulates JA responses by antagonizing the JA signaling repressor IbJAZ10, which relieves IbJAZ10's repression of IbMYC2, a JA signaling activator. IbBBX24 binds to the IbJAZ10 promoter and activates its transcription, whereas it represses the transcription of IbMYC2 The interaction between IbBBX24 and IbJAZ10 interferes with IbJAZ10's repression of IbMYC2, thereby promoting the transcriptional activity of IbMYC2. Overexpressing IbBBX24 significantly increased Fusarium wilt disease resistance, suggesting that JA responses play a crucial role in regulating Fusarium wilt resistance in sweet potato. Finally, overexpressing IbBBX24 led to increased yields in sweet potato. Together, our findings indicate that IbBBX24 plays a pivotal role in regulating JA biosynthesis and signaling and increasing Fusarium wilt resistance and yield in sweet potato, thus providing a candidate gene for developing elite crop varieties with enhanced pathogen resistance but without yield penalty.

Figure 1.

The Responses of IbBBX24 to Fob and MeJA Treatment. (A) Expression of IbBBX24 in Fob-susceptible Lizixiang and Fob-resistant ND98 sweet potato after infection with Fob. The leaves of pot-grown Lizixiang and ND98 plants were sampled 0, 0.5, 1, 2, and 3 d after inoculation with Fob spores at a density of 1.5 × 10⁷ mL⁻¹. The values were determined by RT-qPCR from three biological replicates consisting of pools of five plants. The error bars indicate ±sd (n = 3). Different lowercase letters indicate a significant difference at P < 0.05 based on Student’s t test. (B) Expression of IbBBX24 in Fob-susceptible Lizixiang and Fob-resistant ND98 under 100 μM of MeJA treatment. Four-week–old in vitro-grown plants were submerged in half strength MS medium containing 100 μM of MeJA and sampled at 0, 0.5, 1, 3, 6, and 12 h after treatment. The error bars indicate ±sd (n = 3). The values were determined by RT-qPCR from three biological replicates consisting of pools of five plants. (C) Phylogenetic analysis of BBX proteins from sweet potato (IbBBX24) and Arabidopsis using the neighbor-joining method in MEGA6.0 with 1,000 bootstrap iterations. The numbers at the nodes of the tree indicate bootstrap values from 1,000 replicates. IbBBX24 is marked with a black box. (D) Comparison of the genomic structures of IbBBX24 and AtBBX24. Boxes indicate exons, and lines indicate introns. (E) Immunoblots showing that Fob and MeJA induce the accumulation of IbBBX24 protein in Fob-resistant ND98. Anti-HSP was used as a sample loading control.

Figure 2.

Overexpression of IbBBX24 Enhances Fob Resistance in Sweet Potato. (A) Development of disease symptoms in Fob-resistant line ND98, wild-type (WT), and IbBBX24 transgenic pot-grown plants after Fob inoculation by the mycelia infection method. Sterile PDA tablets (mock, shown in Supplemental Figure 6A) or Fob mycelia tablets at the same growth state were placed on a 1-cm–long wound on the stems of plants. Surface and transverse sections of diseased stems are shown. The images were taken at 15 DAI. (B) Development of disease symptoms in ND98, wild-type (WT), and IbBBX24 transgenic plants after Fob inoculation by the spore infection method. The pot-grown plants were inoculated with water (mock, shown in Supplemental Figure 6B) or an Fob spore solution at a density of 1.5 × 10⁷ mL⁻¹ for 9 d of treatment. (C) Histological examination of transverse sections of stems of ND98, wild-type (WT), and IbBBX24 transgenic plants infected with Fob by the spore infection method at 0, 5, and 9 DAI. The red arrows indicate loose cells. Scale bars = 100 μm. (D) Scanning electron microscopy images of fungus growing in the leaf stomata of ND98, wild-type (WT), and IbBBX24 transgenic plants infected with Fob at 3 DAI by the spore infection method. The red circles indicate fungus growing from the stomata, and the blue arrows indicate Fob spores. Scale bars = 10 μm. (E) JA contents in ND98, wild-type (WT), and IbBBX24 transgenic plants at 0 DAI and 1 DAI by the spore infection method. The pot-grown plants were inoculated with water (mock, shown in Supplemental Figure 6B) or an Fob spore solution at a density of 1.5 × 10⁷ mL⁻¹ (Fob, shown in Figure 2B) for 9 d of treatment. Leaves located in the same position (the fourth leaf) from three plants were pooled as one replicate. Three biological replicates were performed. Different letters indicate statistically significant differences. Multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post hoc tests (P < 0.05). FW, fresh weight.

Figure 3.

Effects of MeJA Treatment on Fob Resistance in Sweet Potato. (A) Development of disease symptoms in Fob-resistant line ND98, wild-type (WT), and IbBBX24 transgenic plants after Fob inoculation by the spore infection method with or without MeJA treatment. Pot-grown plants were inoculated with Fob spore solution at a density of 1.5 × 10⁷ mL⁻¹ for 7 d of treatment. Plants were irrigated with 100 mL of 0 mM or 0.5 mM of MeJA solution per pot once a day. The disease phenotypes were photographed at 0, 3, 5, and 7 DAI. The experiment was independently repeated four times with three plants per replicate. (B) and (C) Statistical analysis of the number of diseased leaves (B) and the length of the necrotic regions of stems (C) of ND98, wild-type (WT), and IbBBX24 transgenic plants at 7 DAI by the spore infection method with or without MeJA treatment. The values represent ±sd (n = 12) from four independent biological replicates with three plants per replicate. The dots represent outlier points. Different letters indicate statistically significant differences. Multiple comparisons were calculated by two-way ANOVA followed by Bonferroni post hoc tests (P < 0.05).

Figure 4.

Genome-wide Analysis of the Role of the IbBBX24 Regulon in the Fob Response. (A) Venn diagram of the number of expressed genes in OE-16, Ri-3, and wild type (WT) at 1 DAI with Fob by RNA-seq. (B) The number of DEGs among OE-16, Ri-3, and wild type (WT) at 1 DAI with Fob by RNA-seq. (C) Heat map of DEGs involved in the JA pathway based on RNA-seq analysis of OE-16 and Ri-3 at 1 DAI of Fob. Higher transcript levels are shown in red (0 to 2), and lower transcript levels are shown in blue (−2 to 0). WT, wild type. (D) Relative expression levels of IbMYC2, IbJAZ10, and IbCHI in wild-type (WT) and IbBBX24 transgenic plants at 0 DAI and 1 DAI with Fob. The values were determined by RT-qPCR from three biological replicates consisting of pools of five plants. The error bars indicate ±sd (n = 3). Different lowercase letters indicate a significant difference at P < 0.05 based on Student’s t test. (E) Distribution of IbBBX24 binding regions in the sweetpotato genome. ChIP was performed in line OE-16 at 1 DAI with Fob with anti-IbBBX24 antibody. Promoter 5-k region, −5 kb to TSS; terminator 2-kb region, 2 kb downstream of the terminator. (F) Peak distance from the TSS of IbBBX24. The peaks were highly enriched from −1 kb to 0 from the TSS. (G) Venn diagram showing the number and overlap of DEGs detected by RNA-Seq and ChIP-Seq at 1 DAI with Fob. a, 2,470 DEGs were upregulated in OE-16 and downregulated in Ri-3 compared with the wild type, as detected by RNA-seq; b, 1,407 DEGs were downregulated in OE-16 and upregulated in Ri-3 line compared with the wild type, as detected by RNA-seq; c, 3,778 putative targets containing peaks associated with a gene model detected by ChIP-seq.

Figure 5.

IbBBX24 Regulates IbJAZ10 and IbMYC2 Expression by Binding Directly to Their Promoters. (A) and (B) Expression analysis of IbJAZ10 (A) and IbMYC2 (B) in IbBBX24 transgenic and wild-type (WT) plants at different DAI with Fob by the spore infection method. The values were determined by RT-qPCR from three biological replicates consisting of pools of three plants. The error bars indicate ±sd (n = 3). Different lowercase letters indicate a significant difference at P < 0.05 based on Student’s t test. (C) and (D) Y1H assays of IbBBX24 binding to the IbJAZ10 and IbMYC2 promoters. (E) Using EMSAs, it was found that the recombinant protein 6His-IbBBX24 retarded the shift of the probe, indicating that IbBBX24 binds to the IbJAZ10 promoter. 100× indicates the usage of excess nonlabeled probe as a competitor. “+” and “−” indicate presence and absence, respectively. (F) EMSAs using 6His-IbBBX24 and wild-type (wt) or various mutated versions of the IbMYC2 promoter subfragments as probes. The terms “mut1,” “mut2,” and “mut3” stand for mutated probes in which the various T/G-box “ACGT” motifs were replaced with “GGGG.” (G) and (H) IbBBX24 inhibits IbJAZ10pro-LUC (G) but activates IbMYC2pro-LUC (H) activity, as determined by dual-LUC assays in sweetpotato protoplasts. The expression level of REN was used as an internal control. The LUC/REN ratio represents the relative activity of the IbJAZ10 promoter. Data are values from four independent experiments. The error bars indicate ±sd (n = 4). ^**P < 0.01; Student’s t test.

Figure 6.

Interaction of IbBBX24 with IbJAZ10 In Vitro and In Vivo. (A) The B-box domain of IbBBX24 is necessary and sufficient for interaction with IbJAZ10 in a Y2H system. BD-IbBBX24^N98 contains IbBBX24 amino acid residues 1 to 98, whereas BD-IbBBX24^C133 contains amino acid residues 99 to 231. Yeast cells were plated onto SD/−Ade/−His/−Leu/−Trp + 3 mM of 3AT medium to screen for possible interactions. (B) Confirmation of the interaction of IbBBX24 and IbJAZ10 by BiFC in N. benthamiana leaf epidermal cells, as shown by a yellow fluorescent signal. The N terminus of YFP was respectively fused to IbBBX24 and IbBBX29, while the C terminus of YFP was fused to IbJAZ10. IbBBX29 from the BBX protein family was used as a related noninteracting protein for a negative control. The images were observed under a confocal microscope 2 d later. EV, empty vector. Scale bars = 10 μm. (C) LCI assay showing that IbBBX24 interacts with IbJAZ10. The N terminus of LUC was fused to IbBBX24, and the C terminus of LUC was fused to IbJAZ10. The images were observed using chemiluminescence imaging 2 d later.(D) In vivo interaction between IbBBX24 and IbJAZ10, as revealed by the co-IP assay. Total proteins from N. benthamiana leaf cells expressing HA-IbBBX24 and IbJAZ10-Myc. Total proteins were extracted and incubated with anti-HA agarose beads. Proteins before (input) and after IP were detected with anti-HA and anti-Myc antibodies.

Figure 7.

IbBBX24 Enhances the DNA Binding Activity of IbMYC2 by Releasing It from Suppression by IbJAZ10. (A) Y2H analysis showing that IbJAZ10 interacts with IbBBX24, IbMYC2, and IbCOI1. Yeast cells were plated onto SD/-Ade/-His/-Leu/-Trp and 3 mM 3AT medium for stringent screening of possible interactions. (B) Interaction of IbMYC2, IbJAZ10, and IbBBX24 with the IbNAC72 promoter, as determined by dual-LUC assays in sweetpotato protoplasts. The expression level of REN was used as an internal control. The LUC/REN ratio represents the relative activity of the IbNAC72 promoter. Data are values from three independent experiments. The error bars indicate ±sd (n = 3). Different lowercase letters indicate a significant difference at P < 0.05 based on Student’s t test. (C) EMSA showing that the DNA binding of IbMYC2 is suppressed by IbJAZ10 but stimulated by IbBBX24 in vitro. Biotin-labeled probes were incubated with various combinations of the same amount of purified 6His-IbBBX24, 6His-IbMYC2, and 6His-IbJAZ10 proteins, and the free and bound DNAs were separated on an acrylamide gel. mut, mutated probe in which the G-box motif CACATG was replaced with GGGGGG; wt, wild type.

Figure 8.

Overexpression of IbJAZ10 in Tobacco Increases Plant Susceptibility to Fob. (A) Phenotypes of one-month–old L4 and W38 tobacco plants grown in half strength MS medium. Scale bar = 1.5 cm. (B) Leaf phenotypes of one-month–old L4 and W38 plants grown in half strength MS medium. Scale bar = 1.5 cm. (C) Transcript levels of IbJAZ10 in W38 and transgenic tobacco lines. The results are expressed as relative values with respect to W38, which was set to 1.0. The values were determined by RT-qPCR from three biological replicates consisting of pools of three plants. The error bars indicate ±sd (n = 3). ^**P < 0.01; Student’s t test. (D) to (F) Root length (D), stem length (E), and stem diameter (F) of one-month–old L4 and W38 plants grown in half strength MS medium. The error bars indicate ±sd (n = 3). ^*P < 0.05; ^**P < 0.01; Student’s t test. (G) Development of plant disease symptoms in W38 and IbJAZ10 transgenic plants after Fob inoculation. W38 and IbJAZ10 transgenic plants were inoculated with Fob spores at a density of 1.5 × 10⁷ mL⁻¹ for 14 d. (H) Development of disease symptoms in leaves of W38 and IbJAZ10 transgenic plants after Fob inoculation. W38 and IbJAZ10 transgenic plants were inoculated with Fob spores at a density of 1.5 × 10⁷ mL⁻¹ for 14 d. (I) and (J) Number of diseased leaves (I) and the length of the necrotic regions of stems (J) in W38 and IbJAZ10 transgenic plants at 14 DAI. The error bars indicate ±sd (n = 9). ^*P < 0.05; ^**P < 0.01; Student’s t test.

Figure 9.

Proposed Working Model of the Role of IbBBX24 in JA-mediated Fob Resistance. IbBBX24 binds to the promoters of IbJAZ10 and IbMYC2, repressing IbJAZ10 transcription but activating IbMYC2 transcription. In IbBBX24-OE plants, elevated levels of IbBBX24 compete with IbMYC2 to interact with IbJAZ10 and enhance the ability of IbMYC2 to regulate its target genes. This process results in the activation of JA signaling, leading to Fob resistance. Yellow balls represent IbBBX24, blue balls represent IbJAZ10, and red balls represent IbMYC2. WT, wild type.