Stronger together: Ethylene, jasmonic acid, and MAPK signaling pathways synergistically induce camalexin synthesis for plant disease resistance

Plants have innate immunity that allows them to recognize pathogen attacks and trigger defense responses. Upon the detection of microbial invaders, plants initiate early signaling events including the activation of kinases and the induction of defense hormones. These activated early signals lead to the induction of later and more sustained defense responses. Among these sustained defenses are phytoalexins, which are antimicrobial metabolites synthesized by plants upon pathogen infection. Plants have many classes of phytoalexins that play a role in abiotic and biotic stress signaling (Jeandet et al., 2014).

One of the important phytoalexins in Arabidopsis thaliana is camalexin. Camalexin is a sulfur-containing tryptophan-derived secondary metabolite involved in disease resistance, as Arabidopsis mutants defective in camalexin synthesis are more susceptible to pathogens (Lemarie et al., 2015). Camelexin synthesis is induced by the plant defense hormones ethylene (ET) and jasmonic acid (JA). While the camalexin biosynthetic pathway is well studied, defense hormone signaling pathways that regulate camalexin biosynthesis are not completely understood. In this issue, Jinggeng Zhou and colleagues show that ET and JA signaling pathways act synergistically to positively regulate pathogen-induced camalexin synthesis in Arabidopsis (Zhou et al., 2022).

Prior work has shown that ET/JA-responsive genes are regulated by the APETALA2/ET RESPONSE FACTOR (ERF) family of transcription factors (Huang et al., 2016). To identify the ERF transcription factors that regulate pathogen-induced camalexin synthesis, the authors generated and screened erf transcription factor mutants and infected them with the necrotrophic fungal pathogen Botrytis cinerea. The erf1 mutant exhibited compromised B. cinerea-induced camalexin production and was more susceptible to B. cinerea infection. Furthermore, overexpressing ERF1 in Arabidopsis increased B. cinerea-induced camalexin production and increased resistance to B. cinerea, demonstrating that ERF1 is a positive regulator in B. cinerea-induced camalexin production and resistance. Importantly, the ET/JA signaling pathway appears to work synergistically to induce camalexin synthesis via ERF1, as cotreatment of Arabidopsis seedlings with 1-aminocyclopropane-1-carboxylic acid (an ET precursor) and methyl jasmonate upregulated ERF1 expression and induced camalexin biosynthesis.

Another transcription factor in Arabidopsis that can activate camalexin biosynthetic genes is WRKY33. To determine if there was a genetic relationship between WRKY33 and ERF1, the authors transformed an ERF1 overexpression construct into the wrky33 mutant. These resulting ERF1-OE wrky33 plants were unable to produce as much camalexin in the presence of B. cinerea compared with the ERF1-OE plants. This result was similar when transforming a WRKY33 overexpression construct into the erf1 mutant. Biochemical assays and a bimolecular fluorescence complementation assay in Arabidopsis protoplasts revealed that ERF1 and WRKY33 directly interact in the nucleus. Interestingly, overexpressing ERF1 in WRKY33 overexpression plants enhanced ERF1-induced camalexin synthesis. These results suggest that these two transcription factors act as a transcriptional complex to interdependently and cooperatively induce camalexin production.

Surprisingly, the authors discovered that ERF1 had two protein bands when expressed in Arabidopsis protoplasts, suggesting that the protein was post-translationally modified. Further examination of ERF1 protein sequence showed that there are mitogen-activated protein kinases (MAPK) phosphorylation sites. In vitro and in vivo phosphorylation assays revealed that the MAPKs MPK3 and MPK6 phosphorylate ERF1. Additionally, this phosphorylation enhances ERF1 transactivation activity as demonstrated in the transactivation assays in Arabidopsis protoplasts. Finally, the authors used gain- and loss-of-function analyses to demonstrate that ET/JA and MPK3/MPK6 signaling pathways work synergistically to induce camalexin biosynthesis via ERF1 and WRKY33 transcription factors (see Figure).

Figure.

Synergistic induction of camalexin biosynthesis by ET/JA and MPK3/MPK6 signaling pathways via ERF1 and WRKY33 transcription factors in Arabidopsis. ET and JA pathways act through the ERF1 transcription factor to synergistically induce pathogen-responsive camalexin biosynthesis, while MPK3/MPK6 phosphoactivate ERF1 and therefore act synergistically with ET/JA pathways via ERF1 to induce camalexin biosynthesis. MPK3/MPK6 also phosphoactivate the WRKY33 transcription factor to induce camalexin biosynthesis. Moreover, ERF1 and WRKY33 form transcriptional complexes to cooperatively activate camalexin biosynthetic genes, thereby further mediating the synergy of ET/JA and MPK3/MPK6 signaling pathways to induce camalexin biosynthesis for Arabidopsis disease resistance. Modified from Zhou et al. (2022), TOC icon.

Plant defense signaling is more complex than we can imagine and we still have much more to learn about it. Perhaps the reason ET/JA and MPK3/MPK6 signaling pathways cooperate to produce camalexin is to ensure there is an efficient response to the pathogen attack. Given that there is “strength in numbers,” it is likely there are other uncharacterized plant defense-related signaling pathways that work synergistically to produce a strong defense response.