Regulation of Root Colonization by the Beneficial Bacterium Pseudomonas chlororaphis via Soybean Transcription Factor GmWRKY23a

doi:10.13560/j.cnki.biotech.bull.1985.2025-1356

Abstract

Abstract:

Objective This study aims to elucidate the role of the soybean transcription factor GmWRKY23a in regulating root colonization by the beneficial bacterium Pseudomonas chlororaphis IRHB3. Methods Using the soybean cultivar Glycine max cv. Nandou 12, we systematically investigated the structural features of the GmWRKY23 gene and its encoded protein through a combination of bioinformatic analysis, molecular biology approaches, and the soybean hairy root-based transient genetic transformation. The regulatory mechanism of GmWRKY23a in response to IRHB3 root colonization was further characterized. Results Four gene sequences annotated as GmWRKY23 were retrieved from the NCBI and Phytozome databases. All encoded proteins contained the conserved WRKY domain and localized to the nucleus, but exhibited variations in protein structure and promoter cis-acting element composition. Among them, GmWRKY23a showed distinct tissue-specific expression and was significantly induced by IRHB3, the pathogen Fusarium oxysporum B3S1, phytohormones (SA, BR, MeJA), and salt stress (NaCl). Functional assays revealed that overexpression of GmWRKY23a promoted IRHB3 colonization in soybean roots. GmWRKY23a positively regulated the expression of key isoflavone biosynthetic genes, including chalcone synthase (CHS8), chalcone reductase (CHR1), and isoflavone synthase (IFS1), alleviated the transcriptional repression of IFS1 caused by IRHB3 colonization, and synergistically enhanced the expression of CHS8 and CHR1 in coordination with IRHB3. Conclusion GmWRKY23a positively modulates root colonization by the beneficial P. chlororaphis viatranscriptional activation of the isoflavone biosynthesis pathway. These findings provide new insights into the coordinated regulation of transcription and secondary metabolism in plant-beneficial microbe interactions.

Key words: GmWRKY23a, Pseudomonas chlororaphis IRHB3, root colonization, plant immunity, isoflavone biosynthesis

XU Yan, CHEN Ling, HUANG Ling-lin, CHANG Xiao-li. Regulation of Root Colonization by the Beneficial Bacterium Pseudomonas chlororaphis via Soybean Transcription Factor GmWRKY23a[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1356.

Figures/Tables 7

Fig. 1 Phylogenetic tree based on amino acid sequences of soybean GmWRKY23A phylogenetic tree was constructed based on the amino acid sequences of WRKY23 from soybean and other species, including soybean (Glycine max), cowpea (Vigna unguiculata), mung bean (Vigna radiata var. radiata), cashew (Cajanus cajan), adzuki bean (Vigna angularis), wild soybean (Glycine soja), peanut (Arachis hypogaea), Arabidopsis thaliana, tobacco (Nicotiana tabacum), and rice (Oryza sativa)

Fig. 2 Analysis of gene structures, conserved domains and motifs of soybean GmWRKY23 genesA: Structure of GmWRKY23 genes; B: Conserved structures of GmWRKY23; C: Motif of GmWRKY23 genes

Fig. 3 Analysis of cis-acting elements in the promoters of soybean GmWRKY23 genes

Fig. 4 Sequence alignment of four soybean GmWRKY23 proteins* indicate an interval of 10 amino acids

Fig. 5 Subcellular localization ofGmWRKY23a in Nicotinan tabacum leavesBright: bright field; eYFP: enhanced yellow fluorescent protein tag; mCherry: red fluorescent nuclear localization marker; Merge: merged field, where complete overlap of green and red fluorescence appears as orange. p1300-YFP: empty vector control; GmWRKY23a-YFP: fusion protein of GmWRKY23a and YFP

Fig. 6 Expression pattern of GmWRKY23a in soybeanA: Tissue-specific expression. B: Relative expression of GmWRKY23a under biotic stress treatment. C: Relative expression of GmWRKY23a under abiotic stress treatment. Data are presented as mean ± standard deviation (Mean ± SD). Differences between groups were analyzed using one-way ANOVA or two-way ANOVA, and different lowercase letters indicate statistically significant differences (P<0.05)

Fig. 8 Expression of genes related to isoflavone biosynthesis and metabolism pathwayDifferences between groups were tested by one-way ANOVA. Different lowercase letters indicate significant differences at P<0.05

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