A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis - PubMed (original) (raw)

A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis

Dongtao Ren et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Plant recognition of pathogens leads to rapid activation of MPK3 and MPK6, two Arabidopsis mitogen-activated protein kinases (MAPKs), and their orthologs in other species. Here, we report that synthesis of camalexin, the major phytoalexin in Arabidopsis, is regulated by the MPK3/MPK6 cascade. Activation of MPK3/MPK6 by expression of active upstream MAPK kinase (MAPKK) or MAPKK kinase (MAPKKK) was sufficient to induce camalexin synthesis in the absence of pathogen attack. Induction of camalexin by Botrytis cinerea was preceded by MPK3/MPK6 activation, and compromised in mpk3 and mpk6 mutants. Genetic analysis placed the MPK3/MPK6 cascade upstream of PHYTOALEXIN DEFICIENT 2 (PAD2) and PAD3, but independent or downstream of PAD1 and PAD4. Camalexin induction after MPK3/MPK6 activation was preceded by rapid and coordinated up-regulation of multiple genes encoding enzymes in the tryptophan (Trp) biosynthetic pathway, in the conversion of Trp to indole-3-acetaldoxime (IAOx, a branch point between primary and secondary metabolism), and in the camalexin biosynthetic pathway downstream of IAOx. These results indicate that the MPK3/MPK6 cascade regulates camalexin synthesis through transcriptional regulation of the biosynthetic genes after pathogen infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Activation of MPK3 and MPK6 induces camalexin in conditional gain-of-function GVG-NtMEK2 DD transgenic plants. (A) Endogenous MPK3 and MPK6 are required for the full induction of camalexin in GVG-NtMEK2 DD plants. Two-week-old GVG-NtMEK2 DD, GVG-NtMEK2DD/mpk3, and GVG-NtMEK2DD/mpk6 seedlings were treated with 1 μM DEX. Camalexin was quantitated by fluorospectrometry at the indicated times. Error bars indicate standard deviations (n = 3). (B) MPK3 and/or MPK6 activation (Upper), as determined by the in-gel kinase assay, and NtMEK2DD induction (Lower), as determined by immunoblot analysis using anti-Flag antibody. The asterisk indicates a nonspecific band that is recognized by the secondary antibody.

Fig. 2.

Fig. 2.

Camalexin induction after MPK3/MPK6 activation in conditional gain-of-function MAPKKK transgenic plants. (A) Activation of endogenous MAPKs by constitutively active ΔMEKK1 and ΔMAPKKKα. Five-day-old hygromycin-resistant T2 seedlings were transferred to GC vials. When the seedlings were 2 weeks old, three vials were treated with 1 μM DEX, and three were treated with an equal volume of ethanol as controls (−DEX). Seedlings were collected 24 h later, and MAPK activation was determined by the in-gel kinase assay. (B) Activation of MPK3/MPK6 by ΔMEKK1 and ΔMAPKKKα leads to camalexin accumulation. After the seedlings were collected, camalexin accumulation in the medium was quantitated by fluorospectrometry. Bars represent means and standard deviations (n = 3).

Fig. 3.

Fig. 3.

Function of MPK3 and MPK6 in pathogen-induced camalexin biosynthesis and resistance against B. cinerea. (A) MAPK activation in Arabidopsis after B. cinerea inoculation. Two-week-old WT (Col-0), mpk3, mpk6, and rescued mpk3/mpk6 double-mutant seedlings were inoculated with Botrytis spores. At the indicated times, seedlings were collected for in-gel kinase assay. (B) Camalexin in the medium was quantitated by fluorospectrometry. Error bars indicate standard deviations (n = 3). (C) Five-week-old soil-grown Arabidopsis plants were inoculated with 10 μl of spore suspension (1 × 105 spores per milliliter). Disease symptoms were scored 3 days later. More than 20 plants per genotype were assayed in each of three independent experiments. Representative images are shown.

Fig. 4.

Fig. 4.

PAD2 and PAD3, but not PAD1 and PAD4, are required for camalexin induction in GVG-NtMEK2 DD plants. (A) Two-week-old seedlings homozygous for the GVG-NtMEK2 DD transgene and pad1, pad2, pad3, or pad4 were treated with 1 μM DEX, samples were taken at the indicated times, and camalexin levels in the culture medium were determined by fluorospectrometry. Bars represent means and standard deviations (n = 3). (B) Induction of PAD3 expression in pad mutants after MPK3/MPK6 activation, as determined by RNA blot analysis (Upper). Equal loading was confirmed by ethidium bromide (EtBr)-staining of the gel (Lower).

Fig. 5.

Fig. 5.

Coordinated induction of Trp biosynthetic genes and cytochrome P450 genes in the camalexin pathway after MPK3/MPK6 activation. Total RNA was extracted from GVG-NtMEK2 DD seedlings before (0 h) and at 3 and 6 h after DEX treatment and from GVG-NtMEK2DD/mpk3 and GVG-NtMEK2DD/mpk6 seedlings 6 h after DEX treatment. After reverse transcription, the levels of each gene were determined by real-time qRT-PCR analysis. The comparative Ct method was used to calculate the levels of transcripts relative to that in GVG-NtMEK2 DD plants without DEX treatment (0 h), which was set at 1. Levels of _EF1_α transcript were used to normalize different samples. Bars represent means and standard deviations (n = 3).

Fig. 6.

Fig. 6.

A model of the role of the MAPKKKα/MEKK1–MKK4/MKK5–MPK3/MPK6 cascade in regulating camalexin biosynthesis in plants challenged by pathogens. A simplified camalexin biosynthetic pathway and its regulatory pathway are placed in separate rectangular boxes with dashed outlines. Genes identified by genetic screens (PAD1 to PAD4) are boxed. Enzymes whose encoding genes are induced by pathogen infection and MPK3/MPK6 activation are marked by bold font. One arrow may represent multiple steps because of unknown components.

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