Brassinosteroids Antagonize Jasmonate-Activated Plant Defense Responses through BRI1-EMS-SUPPRESSOR1 (BES1)

Abstract

Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense tradeoff is unclear. Here, we identified the Arabidopsis (Arabidopsis thaliana) dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1- D, which was highly susceptible to S. exigua and B. cinerea BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1- D to B. cinerea but did not improve resistance of bes1- D to S. exigua In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122 and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense tradeoff by suppressing expression of defensin and glucosinolate biosynthesis genes.

Figure 1.

Isolation and phenotypic characterization of dwe1. A, Phenotypes of 4-week-old wild-type (WT) and dwe1 plants. B, Morphology of wild-type and dwe1 leaves. C, Senescent phenotypes of wild-type and dwe1 mutant. D and E, Stomata on the leaves of 3-week-old wild-type and dwe1 plants revealed by SEM (D) and calculation of stomatal densities (E). Experiments were repeated three times independently. For each experiment, five leaves from independent plants were used for each genotype. Error bars represent SD (n = 5). Asterisk indicates Student’s t test significance between wild type and dwe1 mutant (*P < 0.05). Bars = 50 μm. F, Reverse transcription quantitative PCR (RT-qPCR) analysis of the JA-inducible genes PDF1.2a, PDF1.2b, VSP1, and VSP2 in 6-d-old wild-type and dwe1 seedlings treated without (control) or with MeJA treatment (100 µm MeJA) for 6 h. The expression levels of PDF1.2s and VSPs of plants harvested at 0 h are shown at the top left. The experiments were biologically repeated three times with similar results. For one experiment, three technical replicates (30 seedlings were pooled for each replicate) were used for each genotype. Error bars represent sd (n = 3 technical replicates). Asterisks indicate Student’s t-test significance between indicated samples (**P < 0.01 and *P < 0.05). NS, no significant difference.

Figure 2.

The dwe1 mutant partially reduces the insensitivity of coi1-2 to MeJA and suppresses the hypersensitivity of coi1-2 to insect herbivory and pathogen infection. A and B, Root lengths (A) of 10-d-old wild-type (WT), dwe1, dwe1 coi1, and coi1-2 seedlings grown on MS or MS medium containing 50 μm MeJA. Relative root elongation is expressed as a percentage of root elongation on MS medium. The experiments were biologically repeated three times with similar results. Error bars represent sd (n > 20 roots). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test). C, Phenotypes of leaves from 4-week-old wild-type, dwe1, dwe1 coi1, and coi1-2 plants after 7 d of herbivory by S. exigua. Representative insects taken from the corresponding plants are shown below the rosette leaves. D, S. exigua larval weights for each genotype after 7 d feeding as shown in C is expressed as a percentage of wild-type. The experiments were biologically repeated three times with similar results. Error bars represent sd (n > 30 larvae). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test). E, Phenotypes of leaves from 4-week-old wild type, dwe1, dwe1 coi1, and coi1-2 at 0 (D0) and 7 (D7) days after inoculation with B. cinerea (B.c.). The corresponding leaves inoculated with buffer without B. cinerea were treated as controls (CK). F, Statistics of lesion diameters for each genotype as shown in E is expressed as a percentage of wild type. The experiments were biologically repeated three times with similar results. Error bars represent sd (n > 15 leaves). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test).

Figure 3.

Map-based cloning and complementation test of DWE1. A, The DWE1 locus is located on chromosome IV between markers A9791 (20 recombinants) and B18036 (five recombinants). The gene was further mapped to two bacterial artificial chromosomes (F23E13 and AP22) between the markers A17120 (two recombinants) and B17230 (one recombinant). cM, centimorgans; kb, kilobases. T19K4 is a BAC clone overlapped with F23E13. B, Diagram of the genomic region flanking the T-DNA insertion site in dwe1. The gray and black boxes indicate untranslated regions and exons, respectively. P1, P2, and P3 are primers used for PCR analysis. C, PCR analysis of the wild-type (WT) and dwe1 showing the T-DNA insertion site at −182 bp on 5′-untranslated regions in dwe1. D, RT-PCR analysis showing presence of ROT3 mRNA in wild-type and dwe1 plants. E and F, Complementation of the dwe1 mutation by ROT3. Four-week-old wild-type, dwe1, and two independent complemented transformants (dwe1 35S::ROT3 #1, and #9) were exposed to S. exigua and B. cinerea. Larval weights (E) and lesion diameters (F) at 7 d after treatment is expressed as a percentage of wild-type. The experiments were biologically repeated three times with similar results. Error bars represent sd (n > 30 larvae for S. exigua feeding, and n > 15 leaves for B. cinerea infection). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test).

Figure 4.

BES1 is essential for BR-mediated suppression of plant resistance to necrotrophic pathogen infection. A, RT-qPCR analysis of the JA-inducible genes PDF1.2a, PDF1.2b, and VSP1 in 7-d-old wild-type (WT), bes1-d, and bzr1-1D plants treated with water (control), 100 µm MeJA (MeJA), or 100 µm MeJA with 200 nm BL (MeJA + BL) for 6 h. Transcripts were normalized to levels of ACTIN2. The experiments were biologically repeated three times with similar results. Error bars represent sd (n = 3 technical replicates). Different letters indicate significant differences within the same treatment at P < 0.05 (one-way ANOVA with a Tukey HSD test). B, Phenotypes of leaves from 4-week-old plants of wild-type, bes1-d, bes1-2, and coi1-2 plants at 5 d after inoculation with B. cinerea (B.c.) or buffer without B. cinerea (CK). C, Statistics of mean lesion diameter for the genotypes shown in B is expressed as a percentage of wild-type. The experiments were biologically repeated three times with similar results. Error bars represent sd (n > 15 leaves). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test).

Figure 5.

BES1 interacts with the downstream terminator regions of PDF1.2a and PDF1.2b and suppresses their transcriptional activities. A, Schematic diagrams indicating the locations of putative BES1 binding sites (BRRE, E-box, and G-box elements) in the promoter, coding, and downstream terminator regions of PDF1.2a (P1 to P7) and PDF1.2b (P1 to P3) genomic sequences. Numbers indicate the nucleotide positions relative to their corresponding translational start site (ATG), which is shown as +1 with arrows. B, ChIP-qPCR assays showing the interactions between BES1 and BRRE/G-box elements at the terminator regions of PDF1.2a and PDF1.2b. The assays were performed using 12-d-old BES1-l-GFP transgenic plants treated with 1 μm BL for 2 h. Primers used for ChIP-qPCR were specific to the regions containing the BRRE, E-box, or G-box motif sites shown in A. ACT2 was used as a negative control. The qPCR results were normalized against the input samples. The experiments were biologically repeated three times with similar results. Error bars represent sd (n = 3 technical replicates). C and D, EMSA assays detecting the binding of BES1 to the downstream terminators of PDF1.2a (C) and PDF1.2b (D). E, Schematic diagrams of effector and reporter constructs used in the transient LUC assays. The 35SPPDK promoter driving BES1 (35S::BES1), ORA59 (35S::ORA59), and ERF1 (35S::ERF1) were used as effectors. The empty vector was used as a control. The gene structures of PDF1.2a and PDF1.2b are shown in the bottom. Exons are shown as black boxes; promoter, introns, and downstream regions of terminator are indicated as lines; and 5′ and 3′ untranslated regions are shown as light gray and dark gray boxes, respectively.The dual-luciferase reporter constructs consist of 35S driving the Renilla luciferase (REN) reporter gene (expressed for internal normalization) and the promoter fragment linking downstream regions of PDF1.2a (p1.2a-TAA+) and PDF1.2b (p1.2b-TAA+) terminators of driving the firefly LUC reporter gene. F, Dual-luciferase (LUC) reporter assays showing the suppression of PDF1.2a and PDF1.2b transcriptions by BES1. Effects of BES1 on ORA59- and ERF1-activated transcription of PDF1.2a and PDF1.2b in wild-type (Col-0) protoplasts. The experiments were biologically repeated three times with similar results. Error bars represent sd (n = 3 technical replicates). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test).

Figure 6.

BR antagonizes JA-dependent indolic GS synthesis in response to S. exigua herbivory. A, Phenotypes of 4-week-old plants of wild-type (WT), bes1-D, and bes1-2 after 7 d of herbivory by S. exigua. The coi1-2 mutant was used as a positive control. B, S. exigua larval weights after 7 d of feeding for the genotypes shown in A are expressed as a percentage of wild type. The experiments were biologically repeated three times with similar results. Error bars represent sd (n > 30 larvae). Different letters indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test). C, Ultra-HPLC/quadrupole time-of-flight measurements showing the levels of indolic GS and aliphatic GS in wild-type, dwe1, coi1-2, bes1-2, and bes1-d untreated plants (control) and in plants treated with S. exigua for 3 d. The experiments were biologically repeated twice with similar results. Error bars represent sd (n = 4 technical replicates). Fw, fresh weight. Different letters within each treatment indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test); capital letters compare with each other, and lowercase letters compare with each other. D, RT-qPCR showing the expression levels of the indolic GS biosynthesis genes (CYP79B2, CYP79B3, and CYP83B1) in wild-type, dwe1, coi1-2, bes1-2, and bes1-D plants in response to water (control) or MeJA (100 μm MeJA) treatment for 12 h. The JA-inducible maker gene VSP2 was used as a positive control. For each genotype, transcript levels relative to wild-type control were normalized to that of ACTIN2. The experiments were biologically repeated three times with similar results. Error bars represent sd (n = 3 technical replicates). Different letters within each treatment indicate significant differences at P < 0.05 (one-way ANOVA with a Tukey HSD test); capital letters compare with each other, and lowercase letters compare with each other.

Figure 7.

BES1 interacts with MYB transcription factors to alleviate the MYB-activated transcription of genes involved in GS biosynthesis. A, In-vivo CoIP assay showing the physical interactions between BES1 and MYB34, MYB51, MYB122, MYB28, and MYB29. HA-tagged BES1 (BES1-HA) was coexpressed with FLAG-tagged MYBs (MYB34-, MYB51-, MYB122-, MYB28-, MYB29-, and MYB76-FLAG) in wild-type (WT; Col-0) Arabidopsis protoplasts and immunoprecipitated by FLAG affinity magnetic beads. EV, empty vector. B, BiFC assay showing the interaction between BES1 and MYBs proteins. The split nYFP and cYFP fusions BES1-cYFP and MYBs-nYFP (MYB34-, MYB51-, MYB122-, MYB28-, MYB29-, and MYB76-nYFP) were coexpressed in wild-type protoplasts for 16 h, followed by confocal microscopy. The ARF4-RFP vector was coexpressed as a nuclear localization marker. BF, Bright field. Bar = 10 μm. C, ChIP-qPCR analysis of the in vivo binding of BES1-l-GFP to the promoters of CYP79B3 (P1 to P6), SUR1 (P1 and P2), UGT74B1 (P1 to P5), MYB34 (P1 to P5), MYB51 (P1 to P3), and MYB122 (P1 and P2). Primers used for ChIP-qPCR were specific to the regions containing the BRRE, E-box, or G-box elements. ACT2 was used as a negative control. The qPCR results were normalized against the input samples. The dashed lines separate the results from the different genes. The experiments were biologically repeated three times with similar results. Error bars represent sd (n = 3 technical replicates). D, Dual-luciferase (LUC) reporter assays showing the suppression of CYP79B3 and UGT74B1 transcription by BES1. Left, Schematic diagrams of effector and reporter constructs used in the transient LUC assays. 35SPPDK promoter driving BES1, MYC2, MYB34, MYB51, and MYB122 were used as an effector. The empty vector was used as a control. The dual-luciferase reporter constructs consist of 35S driving Renilla luciferase (REN) reporter gene for internal normalization, and the promoters of CYP79B3 and UGT74B1 driving firefly LUC reporter gene. Right, Effects of BES1 on the MYC2-, MYB34-, MYB51-, MYB122-activated transcription of CYP79B3 and UGT74B1 in wild-type protoplasts. The experiments were biologically repeated three times with similar results. Error bars represent sd (n = 3 technical replicates). Asterisks denote Student’s t test significance between indicated samples (**P < 0.01).

Figure 8.

A working model showing the role of BES1 in fine-tuning plant growth-defense tradeoffs by suppressing JA-mediated defensin and glucosinolate biosynthesis. Plant growth signals trigger the expression of genes, including DWE1, to induce brassinosteroid (BR) synthesis. BR binds to the cell-surface receptor kinase BRI1 to activate BR signal transduction by sequential transphosphorylation events. The dephosphorylated transcription factor BES1 translocates into the nucleus, leading to the expression of BR-responsive genes and promotion of plant growth and development. By contrast, insect attack and pathogen infection induce the accumulation of JAs. The active form of JA, JA-Ile, is perceived by the COI1 receptor to recruit JAZ repressors for degradation. The downstream transcription factors MYC2/3/4 and ERF1/ORA59 are released, activating biosynthesis of defense compounds, defensins and GS, respectively. In response to pathogen infection and herbivore feeding, BES1 functions in the plant growth-defense trade off by negatively regulating JA-induced transcription of PDF1.2s and indole-GS biosynthetic genes, which helps restore the proper cellular levels of defensin and GS.