The WRKY Family Transcription Factor GmWRKY72 Represses Glyceollin Phytoalexin Biosynthesis in Soybean

Abstract

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.

Keywords: Glycine max; glyceollin; phytoalexin; transcription factor; transcriptional repressor.

Figure 1

The glyceollin biosynthetic pathway in soybean. CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone synthase; PTS, pterocarpan synthase; P6αH, dihydroxypterocarpan-6α-hydroxylase; G4DT, trihydroxypterocarpan dimethylallyltransferase; G2DT, dimethylallylpyrophosphate:trihydroxypterocarpan dimethylallyl transferase; GLS, glyceollin synthase.

Figure 2

Identification and protein similarity analysis of GmWRKY72. (A) Number of transcription factors in soybean that were up-regulated by pH 3.0 medium or WGE and down-regulated by dehydration that were co-expressed with grapevine VviSTS genes (p < 0.05) by RNA-seq. (B) Cluster analysis of deduced amino sequences of GmWRKY72 and other closely related WRKY transcription factors. (C) Multiple alignments of GmWRKY72 and WRKY72 proteins from soybean, Arabidopsis, and tomato. Glycine max: GmWRKY72 (Glyma.17G097900), Glyma.09G240000, Glyma.18G256500, Glyma.19G020600. Arabidopsis thaliana: WRKY72 (At5G15130), Solanum lycopersicum: SlWRKY72a (GU017421), SlWRKY72b (GU017422), are from GeneBank. Identical and similar amino acids were in red and blue, respectively. Conserved motifs are underlined in red.

Figure 3

GmWRKY72 is a negative regulator of glyceollin biosynthesis in soybean. (A) Expression level of GmWRKY72 gene in W82 hairy roots elicited for 24 h with H2O or WGE by qRT-PCR. The significance test was performed by paired Student’s t-test (p < 0.05), which is indicated by asterisks. Error bars indicate SE (n = 3). (B) Gene expression in hairy roots silencing GmWRKY72 elicited for 24 h with WGE or mock (H₂O) treatment. The significance test was performed by paired Student’s t-test (p < 0.05), which is indicated by asterisks. Error bars indicate SE (n = 3). (C) Amounts of glyceollins and isoflavonoid metabolites in soybean hairy roots undergoing RNAi silencing of GmWRKY72 24 h after treatment with WGE or H₂O. The significance test was performed by single-factor ANOVA, Tukey post hoc test (p < 0.05, α = 0.05), which is indicated by different letters. Error bars represent SE (n = 5). (D) Transcript levels in hairy roots overexpressing GmWRKY72 elicited for 24 h with WGE or mock (H₂O) treatment. The significance test was performed by paired Student’s t-test (p < 0.05), which is indicated by asterisks. Error bars indicate SE (n = 3). (E) Amounts of glyceollins and isoflavonoids metabolites in hairy roots overexpressing GmWRKY72 elicited for 24 h with WGE or mock (H₂O) treatment. The significance test was performed by single-factor ANOVA, Tukey post hoc test (p < 0.05, α = 0.05), which is indicated by different letters. Error bars represent SE (n = 5).

Figure 4

Characterization of the subcellular localization, protein–protein interactions (PPIs), and protein–DNA interactions (PDIs) of the GmWRKY72 protein. (A) Fluorescence microscopy of GmWRKY72 translationally fused to green fluorescent protein (GFP) in transgenic W82 hairy roots. Green fluorescent protein harboring an N-terminal nuclear localization sequence (NLS-GFP) was used as positive control, and DAPI (6 µg/mL) images indicate nuclear staining. Red arrows indicate some localization of GFP-GmWRKY72 to the cell periphery. Bars are 2 µm and 5 µm, respectively. (B) Schematic diagram demonstrating promoter fragments of IFS2, PTS1, P6αH, and CHR used for yeast one-hybrid assays. W-box elements with sequence 5′-TTGAC-3′ (blue boxes). (C) Yeast one-hybrid analysis of strain YM4271 transformed with GmWRKY72-Gal4AD and pIFS2::HIS3, pPTS1::HIS3, pP6αH::HIS3, pCHR::HIS3 and on SD-Leu-His (SD-L-H). Growth on SD-L-H + 0 mM 3-aminotriazole (3AT, top rows) are plating controls, and SD-L-H + 20 mM 3AT (bottom rows) indicate positive PDIs. (D) Yeast two-hybrid analysis testing for PPIs between GmWRKY72 and GmNAC42-1, GmMYB29A1, GmMYB29A2, and GmHSF6-1. Yeast strain PJ69-4a was transformed and plated on SD-L-T and SD-L-T-H ± 3AT, respectively. Growth on SD-L-T-H+ 0 mM 3AT (top rows) are plating control; growth on SD-L-T+H + 40 mM 3AT (bottom rows) indicates positive PPIs. pDEST-GADT7 was used as the empty ‘Vector’ control.