Natural products largely produced by Pseudomonads-like soil-dwelling microorganisms are a consistent source of antimicrobial metabolites and pesticides. Herein we report the isolation of Pseudomonas mosselii strain 923 from rice rhizosphere soils of paddy fields, which specifically inhibit the growth of plant bacterial pathogens Xanthomonas species and the fungal pathogen Magnaporthe oryzae. The antimicrobial compound is purified and identified as pseudoiodinine using high-resolution mass spectra, nuclear magnetic resonance and single-crystal X-ray diffraction. Genome-wide random mutagenesis, transcriptome analysis and biochemical assays define the pseudoiodinine biosynthetic cluster as psdABCDEFG. Pseudoiodinine biosynthesis is proposed to initiate from guanosine triphosphate and 1,6-didesmethyltoxoflavin is a biosynthetic intermediate. Transposon mutagenesis indicate that GacA is the global regulator. Furthermore, two noncoding small RNAs, rsmY and rsmZ, positively regulate pseudoiodinine transcription, and the carbon storage regulators CsrA2 and CsrA3, which negatively regulate the expression of psdA. A 22.4-fold increase in pseudoiodinine production is achieved by optimizing the media used for fermentation, overexpressing the biosynthetic operon, and removing the CsrA binding sites. Both of the strain 923 and purified pseudoiodinine in planta inhibit the pathogens without affecting the rice host, suggesting that pseudoiodinine can be used to control plant diseases.

Multiple-trait genome-wide association study based on principal component analysis for residual covariance matrix

Pseudoiodinine is an antimicrobial compound produced by Pseudomonas mosselii that exhibits strong inhibitory activity against bacterial and fungal pathogens of rice, highlighting its potential as a green biopesticide. However, the regulatory mechanisms underlying pseudoiodinine production remain poorly understood. In this study, genome-wide random mutagenesis identified two insertion mutants of 277-3 and 277-23 that completely abolished antagonistic activity against Xanthomonas oryzae pv. oryzicola (Xoc) RS105, with transposon insertions mapped to tolB and tolR, respectively. Further analysis showed that mutations in tolB and tolR significantly impaired bacterial growth during the exponential phase, suggesting that the Tol-Pal system is required for the optimal growth of P. mosselii. Consistently, overexpression of the tol-pal operon resulted in a 3.2-fold increase in pseudoiodinine production compared with the original strain. These findings indicate that the Tol-Pal system contributes to pseudoiodinine production primarily by maintaining efficient bacterial growth. Collectively, this study provides the first insight into the Tol-Pal system in promoting pseudoiodinine production and suggests its potential application in engineering P. mosselii strains.