Phytoalexins are plant defense metabolites that are biosynthesized transiently in response to pathogens. Despite that their biosynthesis is highly restricted in plant tissues, the transcription factors that negatively regulate phytoalexin biosynthesis remain largely unknown. Glyceollins are isoflavonoid-derived phytoalexins that have critical roles in protecting soybean crops from the oomycete pathogen Phytophthora sojae. To identify regulators of glyceollin biosynthesis, we used a transcriptomics approach to search for transcription factors that are co-expressed with glyceollin biosynthesis in soybean and stilbene synthase phytoalexin genes in grapevine. We identified and functionally characterized the WRKY family protein GmWRKY72, which is one of four WRKY72-type transcription factors of soybean. Overexpressing and RNA interference silencing of GmWRKY72 in the soybean hairy root system decreased and increased expression of glyceollin biosynthetic genes and metabolites, respectively, in response to wall glucan elicitor from P. sojae. A translational fusion with green fluorescent protein demonstrated that GFP-GmWRKY72 localizes mainly to the nucleus of soybean cells. The GmWRKY72 protein directly interacts with several glyceollin biosynthetic gene promoters and the glyceollin transcription factor proteins GmNAC42-1 and GmMYB29A1 in yeast hybrid systems. The results show that GmWRKY72 is a negative regulator of glyceollin biosynthesis that may repress biosynthetic gene expression by interacting with transcription factor proteins and the DNA of glyceollin biosynthetic genes.

Phytoalexins are specialized metabolites that are synthesized by plants in response to pathogens. A paradigm in transcription factor (TF) biology is that conserved TFs have dedicated roles across plant lineages in regulating specific branches of specialized metabolism. However, the Arabidopsis (Arabidopsis thaliana) NAC family TF ANAC042 (a.k.a. JUNGBRUNNEN1 or JUB1) regulates the synthesis of camalexin, a Trp-derived phytoalexin specifically produced by several Brassicaceae species, whereas its homolog in soybean (Glycine max) regulates the synthesis of glyceollins, which are Phe-derived phytoalexins specific to soybean. The question addressed by this research is whether ANAC042 broadly regulates phytoalexin biosynthetic pathways in Arabidopsis. Using a novel matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS) method, we found that the Arabidopsis loss-of-function mutant anac042–1 elicited with bacterial flagellin (Flg22) is deficient in lineage-specific Trp- and conserved Phe-derived phytoalexins—namely camalexin and 4-hydroxyindole-3-carbonyl nitrile (4OH-ICN), and pathogen-inducible monolignols and scopoletin, respectively. Overexpressing ANAC042 in the anac042-1 mutant restored or exceeded wildtype amounts of the metabolites. The expression of phytoalexin biosynthetic genes in mutant and overexpression lines mirrored the accumulation of metabolites. Yeast-one hybrid and promoter-reporter assays in Nicotiana benthamiana found that the ANAC042 protein directly binds and activates the promoters of CYP71B15, CYP71A12, and PAL1 genes for the synthesis of camalexin, 4OH-ICN, and pathogen-inducible monolignol/scopoletin, respectively. Our results demonstrate that ANAC042 regulates conserved and lineage-specific phytoalexin pathways in Arabidopsis. The latter suggests that it is an opportunistic TF that has coopted lineage-specific genes into phytoalexin metabolism, thus providing an exception to the current paradigm.

Phytoalexins are plant defense metabolites that are biosynthesized transiently in response to pathogens. Despite that their biosynthesis is highly restricted in plant tissues, the transcription factors that negatively regulate phytoalexin biosynthesis remain largely unknown. Glyceollins are isoflavonoid-derived phytoalexins that have critical roles in protecting soybean crops from the oomycete pathogen Phytophthora sojae. To identify regulators of glyceollin biosynthesis, we used a transcriptomics approach to search for transcription factors that are co-expressed with glyceollin biosynthesis in soybean and stilbene synthase phytoalexin genes in grapevine. We identified and functionally characterized the WRKY family protein GmWRKY72, which is one of four soybean WRKY72-type transcription factors of soybean. Overexpressing and RNA interference silencing of GmWRKY72 in the soybean hairy root system decreased and increased expression of glyceollin biosynthetic genes and metabolites, respectively, in response to wall glucan elicitor from P. sojae. A translational fusion with green fluorescent protein demonstrated that GFP-GmWRKY72 localizes mainly to the nucleus of soybean cells. The GmWRKY72 protein directly interacts with several glyceollin biosynthetic gene promoters and the glyceollin transcription factor proteins GmNAC42-1 and GmMYB29A1 in yeast hybrid systems. The results show that GmWRKY72 is a negative regulator of glyceollin biosynthesis that may repress biosynthetic gene expression by interacting with transcription factor proteins and the DNA of glyceollin biosynthetic genes.

Phytoalexins are specialized metabolites that are synthesized by plants in response to pathogens. A paradigm in transcription factor (TF) biology is that conserved TFs have dedicated roles across plant lineages in regulating specific branches of specialized metabolism. However, the Arabidopsis (Arabidopsis thaliana) NAC family TF ANAC042 (a.k.a. JUNGBRUNNEN1 or JUB1) regulates the synthesis of camalexin, a Trp-derived phytoalexin specifically produced by several Brassicaceae species, whereas its homolog in soybean (Glycine max) regulates the synthesis of glyceollins, which are Phe-derived phytoalexins specific to soybean. The question addressed by this research is whether ANAC042 broadly regulates phytoalexin biosynthetic pathways in Arabidopsis. Using a novel matrix-assisted laser desorption ionization high resolution mass spectrometry (MALDI-HRMS) method we found that the Arabidopsis loss-of-function mutant anac042-1 elicited with bacterial flagellin (Flg22) is deficient in lineage-specific Trp- and conserved Phe-derived phytoalexins - namely camalexin and 4-hydroxyindole-3-carbonyl nitrile (4OH-ICN), and pathogen-inducible monolignols and scopoletin, respectively. Overexpressing ANAC042 in the anac042-1 mutant restored or exceeded wildtype amounts of the metabolites. The expression of phytoalexin biosynthetic genes in mutant and overexpression lines mirrored the accumulation of metabolites. Yeast-one hybrid and promoter-reporter assays in Nicotiana benthamiana found that the ANAC042 protein directly binds and activates the promoters of CYP71B15, CYP71A12, and PAL1 genes for the synthesis of camalexin, 4OH-ICN, and pathogen-inducible monolignol/scopoletin, respectively. Our results demonstrate that ANAC042 regulates conserved and lineage-specific phytoalexin pathways in Arabidopsis. The latter suggests that it is an opportunistic TF that has coopted lineage-specific genes into phytoalexin metabolism, thus providing an exception to the current paradigm.