Pathogen-Responsive MPK3 and MPK6 Reprogram the Biosynthesis of Indole Glucosinolates and Their Derivatives in Arabidopsis Immunity

Abstract

Antimicrobial compounds have critical roles in plant immunity; for example, Arabidopsis thaliana and other crucifers deploy phytoalexins and glucosinolate derivatives in defense against pathogens. The pathogen-responsive MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 have essential functions in the induction of camalexin, the major phytoalexin in Arabidopsis. In search of cyanide, a coproduct of ethylene and camalexin biosynthesis, we found that MPK3 and MPK6 also affect the accumulation of extracellular thiocyanate ion derived from the indole glucosinolate (IGS) pathway. Botrytis cinerea infection activates MPK3/MPK6, which promote indole-3-yl-methylglucosinolate (I3G) biosynthesis and its conversion to 4-methoxyindole-3-yl-methylglucosinolate (4MI3G). Gain- and loss-of-function analyses demonstrated that MPK3/MPK6 regulate the expression of MYB51 and MYB122, two key regulators of IGS biosynthesis, as well as CYP81F2 and IGMT1/IGMT2, which encode enzymes in the conversion of I3G to 4MI3G, through ETHYLENE RESPONSE FACTOR6 (ERF6), a substrate of MPK3/MPK6. Under the action of PENETRATION2 (PEN2), an atypical myrosinase, and PEN3, an ATP binding cassette transporter, 4MI3G is converted to extracellular unstable antimicrobial compounds, possibly isothiocyanates that can react with nucleophiles and release the stable thiocyanate ion. Recent studies demonstrated the importance of PEN2/PEN3-dependent IGS derivatives in plant immunity. Here, we report that MPK3/MPK6 and their substrate ERF6 promote the biosynthesis of IGSs and the conversion of I3G to 4MI3G, a target of PEN2/PEN3-dependent chemical defenses in plant immunity.

Figure 1.

Induction of Extracellular Thiocyanate after MPK3/MPK6 Activation or B. cinerea Infection Is Dependent on IGS Biosynthetic Pathway. (A) Activation of MPK3/MPK6 in the gain-of-function GVG-Nt-MEK2^DD transgenic plants (DD) seedlings after DEX treatment induces the accumulation of thiocyanate in culture medium. Twelve-day-old DD, DD mpk3, and DD mpk6 seedlings were treated with DEX (1 μM). FW, fresh weight. (B) B. cinerea-induced accumulation of thiocyanate is dependent on CYP79B2/CYP79B3 and other components in the IGS biosynthesis pathway. Twelve-day-old seedlings were inoculated with B. cinerea (Bc) spores (4.0 × 10⁵ spores per vial). Medium samples were collected at indicated times. The levels of thiocyanate ion in culture medium were quantified using the isonicotinic-barbituric acid method. Error bars indicate sd (n = 3). Two-way ANOVA was performed to compare the levels of thiocyanate ions in different genotypes after treatment. Lowercase letters above the columns indicate statistically different groups (P < 0.05).

Figure 2.

Key Enzymes, Regulators, and Major Intermediates in the IGS Biosynthetic Pathway. Indole-3-yl-methyl-glucosinolate (I3G), 1MI3G, and 4MI3G are three major IGSs found in Arabidopsis. Their de novo biosynthesis is dependent on MYB34, MYB51, and MYB122, three key transcription factors that control the expression of IGS biosynthetic genes. Solid arrows indicate single enzymatic steps, whereas dashed lines stand for several enzymatic steps. IAOx is also the key intermediate leading to camalexin and auxin (adapted from Figure 8 in Frerigmann and Gigolashvili, 2014).

Figure 3.

Accumulation of 4MI3G in Arabidopsis Seedlings Infected with B. cinerea and DD Seedlings after DEX Treatment. (A) Accumulation of selected GSs in Col-0 seedlings after inoculation with B. cinerea (Bc) spores (left panel). Twelve-day-old Col-0 seedlings were inoculated with B. cinerea spores (4 × 10⁵ spores/vial). Samples were collected at indicated times. Glucosinolates were measured using the HPLC assay. Ratios of steady state levels of 4MI3G and I3G were calculated and presented (right panel). (B) Accumulation of selected glucosinolates in 12-d-old DD, DD mpk3, and DD mpk6 seedlings after DEX (1 μM) treatment. Glucosinolates were measured at indicated times. Ratios of steady state levels of 4MI3G and I3G were calculated and presented (right panels). Error bars indicate sd (n = 3). One-way ANOVA was performed to compare the levels of GSs at different time point after treatment. Lowercase letters above the columns indicate statistically different time points (P < 0.05). FW, fresh weight. (C) Activation of MPK3/MPK6 in response to B. cinerea and in DD, DD mpk3, and DD mpk6 seedlings after DEX treatment. Activation of MPK6 and MPK3 was determined by immunoblot analysis using anti-pTEpY antibody.

Figure 4.

B. cinerea-Induced 4MI3G Biosynthesis Is Compromised in mpk3 mpk6 Double Mutants. (A) and (B) Steady state levels of I3G (A) and 4MI3G (B) in 12-d-old Col-0 and chemical-genetically rescued mpk3 mpk6 double mutant seedlings after B. cinerea (Bc) inoculation. Col-0, MPK6SR, and MPK3SR seedlings were pretreated with DMSO (solvent of NA-PP1 stock) or NA-PP1 (final concentration of 2.5 μM) for 30 min before Bc inoculation. Samples were collected at indicated times for GS assay. (C) Ratios of steady state levels of 4MI3G and I3G in Col-0, MPK6SR, and MPK3SR seedlings after Bc inoculation. Error bars indicate sd (n = 3). Differences in IGS contents between DMSO and NA-PP1 pretreatment groups were analyzed by two-way ANOVA. Different lowercase letters above the brackets indicate statistically different groups (P < 0.01). MPK6SR genotype: mpk3 mpk6 P_MPK6:MPK6^YG line #58; MPK3SR genotype: mpk3 mpk6 P_MPK3:MPK3^TA line #17.

Figure 5.

Activation of 4MI3G Biosynthetic Genes in Response to B. cinerea and Gain-of-Function Activation of MPK3/MPK6 Cascade. (A) Expression of CYP81F2, IGMT1, and IGMT2, genes encoding the two key enzymes in the conversion of I3G to 4MI3G, was highly induced in response to B. cinerea (Bc) inoculation. One-way ANOVA was used to compare gene expression at different time points (P < 0.05). (B) Activation of 4MI3G biosynthetic genes in DEX-treated DD seedlings was compromised in either mpk3 or mpk6 mutant background. Gene expression was quantified by RT-qPCR and calculated as percentages of the EF1α transcript. Error bars indicate sd (n = 3). Gene expression in seedlings of different genotypes was analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01).

Figure 6.

Loss of Function of MPK3 and MPK6 Compromises B. cinerea-Induced Activation of 4MI3G Biosynthetic Genes. Col-0, MPK6SR, and MPK3SR seedlings were pretreated with DMSO (solvent of NA-PP1 inhibitor) or NA-PP1 (2.5 μM) for 30 min before B. cinerea (Bc) inoculation. Samples were collected at indicated times for total RNA preparation. CYP81F2, IGMT1, and IGMT2 gene expression was determined by RT-qPCR and calculated as percentages of the EF1α transcript. Error bars indicate sd (n = 3). Differences in gene expression between DMSO and NA-PP1 pretreatment groups were analyzed by two-way ANOVA. Different lowercase letters above the brackets indicate statistically different groups (P < 0.01). MPK6SR: mpk3 mpk6 P_MPK6:MPK6^YG line #58; MPK3SR: mpk3 mpk6 P_MPK3:MPK3^TA line #17.

Figure 7.

I3G Biosynthesis Is Highly Induced in Response to B. cinerea Infection or MPK3/MPK6 Activation. (A) Steady state levels of I3G and 4MI3G in 12-d-old Col-0, cyp79b2/b3, and cyp81f2 seedlings after inoculation with B. cinerea (Bc) spores. (B) Steady state levels of I3G and 4MI3G in 12-d-old DD, DD cyp81f2 seedlings after DEX treatment. Samples were collected at indicated times after treatment, and IGS levels were determined by HPLC assay. Error bars indicate sd (n = 3). Differences in IGS contents between different genotypes were analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01).

Figure 8.

B. cinerea-Induced MYB51/MYB122 Gene Activation Is Dependent on MPK3/MPK6. (A) Induction of MYB51 and MYB122 gene expression by B. cinerea (Bc) infection is dependent on MPK3/MPK6. Col-0, MPK6SR, and MPK3SR seedlings were pretreated with DMSO or NA-PP1 (2.5 μM) for 30 min before Bc inoculation. Differences in gene expression between DMSO and NA-PP1 pretreatment groups were analyzed by two-way ANOVA. Different lowercase letters above the brackets indicate statistically different groups (P < 0.01). (B) Activation of MYB51 and MYB122 expression in DEX-treated DD seedlings was compromised in mpk3 or mpk6 mutant background. Samples were collected at indicated times. Gene expression was quantified by RT-qPCR and calculated as percentages of the EF1α transcript. Gene expression in seedlings of different genotypes after treatment was analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01). Numbers on the right indicate the induction folds relative to 0 h. Error bars indicate sd (n = 3).

Figure 9.

MYB51 and MYB122 Are Essential for I3G and 4MI3G Biosynthesis in Response to B. cinerea and Activation of MPK3/MPK6 Cascades. (A) Steady state levels of I3G (upper panel) and 4MI3G (lower panel) in 12-d-old Col-0, myb51, myb122, and myb51 myb122 single/double mutant seedlings after B. cinerea (Bc) inoculation. (B) Steady state levels of I3G (upper panel) and 4MI3G (lower panel) in 12-d-old DD, DD myb51, DD myb122, and DD myb51 myb122 seedlings after DEX treatment. Samples were collected at indicated times, and IGS levels were determined by HPLC assay. Error bars indicate sd (n = 3). Differences in IGS contents between different genotypes were analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01).

Figure 10.

ERF6, a Substrate of MPK3/MPK6, Plays an Important Role in the B. cinerea-Induced Biosynthesis of 4MI3G from I3G. (A) Steady state levels of I3G and 4MI3G in 12-d-old Col-0, 35S:ERF6^WT, 35S:ERF6^4D, and 35S:ERF6-EAR seedlings. One-way ANOVA was used to compare the levels of IGSs in different genotypes (P < 0.05). (B) Intracellular levels of I3G and 4MI3G in 12-d-old Col-0 and 35S:ERF6-EAR seedlings after B. cinerea (Bc) inoculation. Samples were collected without treatment (A) or at indicated times after treatment (B), and IGS levels were determined by HPLC assay. IGS contents in different genotypes were analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01). Error bars indicate sd (n = 3). ns, not significant.

Figure 11.

ERF6 Regulates the Expression of CYP81F2, IGMT1, and IGMT2 by Directly Interacting with Their Promoters. (A) Gene expression of CYP81F2, IGMT1, and IGMT2 in 12-d-old Col-0, 35S:ERF6^WT, 35S:ERF6^4D, and 35S:ERF6-EAR seedlings was quantified by RT-qPCR and calculated as percentages of the EF1α transcript. One-way ANOVA was used to compare gene expression in different genotypes (P < 0.05). (B) Gene expression of CYP81F2, IGMT1, and IGMT2 in 12-d-old Col-0 and 35S:ERF6-EAR seedlings after B. cinerea (Bc) inoculation. Samples were collected without treatment (A) or at indicated times after treatment (B) for total RNA preparation. The transcript levels were determined by RT-qPCR and calculated as percentages of the EF1α transcript. Error bars indicate sd (n = 3). Gene expression in different genotypes was analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01). (C) Diagrams showing the GCC boxes in the genomic sequences of CYP81F2, IGMT1, and IGMT2 genes. (D) ERF6^4D binds to the GCC-boxes in CYP81F2, IGMT1, and IGMT2 genes in vivo. ChIP-qPCR was performed using 12-d-old 35S:ERF6^4D seedlings. Tagged 4myc-ERF6^4D protein-chromatin complex was immunoprecipitated with an anti-myc antibody. A control reaction was processed side-by-side using mouse IgG. ChIP- and input-DNA samples were quantified by real-time qPCR using primers specific to the GCC-box-containing regions (C). The ChIP results are presented as a percentage of input DNA. Differences in DNA abundance of anti-myc and IgG antibody samples were analyzed by Student’s t test (*P < 0.05 and **P < 0.01). Error bars indicate sd (n = 3).

Figure 12.

PEN2 and PEN3 Are Involved in the Accumulation of Extracellular Thiocyanate Ion in Response to B. cinerea Infection. Intracellular levels of I3G and 4MI3G and extracellular levels of thiocyanate in B. cinerea-inoculated Col-0, pen2-1, and pen3-1 mutant seedlings (A) and DEX-treated DD, DD pen2, and DD pen3 seedlings (B). Twelve-day-old seedlings were treated with B. cinerea spores (4.0 × 10⁵ spores per vial) or DEX (1 µM). Samples were collected at indicated times, and IGSs were measured using HPLC assay. Thiocyanate levels were quantified using the isonicotinic-barbituric acid method. Error bars indicate sd (n = 3). IGS or thiocyanate ion levels in seedlings of different genotypes were analyzed by two-way ANOVA. Different lowercase letters above the columns indicate statistically different groups (P < 0.01).

Figure 13.

A Model Depicting the Function of the MPK3/MPK6 Cascade in the Induction of IGS Biosynthesis and Their Derivatives in Arabidopsis Challenged by B. cinerea. In response to fungal pathogen infection, activation of the MPK3/MPK6 cascade promotes the biosynthesis of I3G and the conversion of I3G to 4MI3G. ERF6, one of the substrates of MPK3 and MPK6, is able to activate gene expression of its direct target genes including CYP81F2, IGMT1, and IGMT2 to drive the biosynthesis of 4MI3G from I3G. ERF6 is also involved in the activation of MYB51/MYB122 expression through unidentified factors, which promotes the biosynthesis of I3G, the precursor for 4MI3G synthesis. After hydrolyzing by PEN2 and/or other myrosinases, IGSs are converted to unidentified unstable compounds, possibly isothiocyanates, which release SCN⁻ in their breakdown process.