Spermidine Synthase Emerges as a Master Switch Behind a Devastating Cotton Wilt Fungus

Deleting a single gene called VdSPDS from the soil-borne fungus Verticillium dahliae leaves the pathogen unable to grow, unable to sporulate, exquisitely sensitive to a battery of stresses, and dramatically less able to cause wilt disease in cotton, according to a new study published on 23 March 2026 in Discover Genetics and Evolution. The gene encodes spermidine synthase, a key enzyme in the polyamine biosynthesis pathway, and the findings establish it as a central pillar of the fungus’s biology and virulence machinery.

The research team, led by Rui Hai and Yong Song with colleagues at Chinese institutions, worked with the highly virulent V. dahliae strain Vd991, which they obtained from the Institute of Cotton Research at the Chinese Academy of Agricultural Sciences. Using Agrobacterium tumefaciens–mediated transformation based on homologous recombination, they generated a gene deletion mutant, designated ΔVdSPDS, and a complemented strain in which the native gene was restored. Two independent knockout mutants with identical phenotypes were obtained, and one representative strain was selected for detailed characterization.

The most striking result was the mutant’s strict dependence on exogenous spermidine. On standard potato dextrose agar without supplementation, the ΔVdSPDS mutant failed to grow at all, a phenotype the authors describe as complete loss of vegetative growth and conidiation. Adding 0.5 millimolar or 1 millimolar spermidine to the medium fully rescued the growth defect, while the wild-type strain showed no growth inhibition at these concentrations. Based on preliminary experiments, the team used 0.5 millimolar spermidine in all subsequent experiments to ensure comparable growth conditions among the wild-type, mutant, and complemented strains.

Even with spermidine supplied, the mutant was not fully restored to normal behavior. At three days post-inoculation on supplemented medium, its colony diameter was significantly smaller than that of the wild-type and complemented strains, indicating an impaired initial growth rate, although this deficit was gradually alleviated with prolonged incubation, and by seven and eleven days no significant differences in colony diameter remained. The mutant also produced significantly fewer conidia than the wild type at three, five, and seven days, while the complemented strain produced conidial counts similar to the wild type, confirming that the defect was due to the deletion itself. Interestingly, conidia from the mutant showed a significantly higher germination rate than those of the control strains, and microscopic examination revealed sparse aerial hyphae in the mutant, in contrast to the dense aerial mycelia of the wild type and complemented strains.

Stress testing revealed a broad vulnerability. When colonies were assessed at seven days post-inoculation, a time point chosen because baseline growth of the mutant on control medium was comparable to the wild type, the mutant showed pronounced growth inhibition under osmotic stress imposed by 1 molar sodium chloride, sorbitol, or potassium chloride, with sodium chloride causing nearly complete growth arrest. It was also significantly more sensitive to cell wall stress triggered by Congo Red and to membrane stress induced by sodium dodecyl sulfate. In separate assays, the mutant exhibited a significantly larger inhibition zone around filter paper disks saturated with 30 percent hydrogen peroxide, indicating enhanced sensitivity to oxidative stress, and showed markedly reduced colony diameter under nitrosative stress induced by the nitric oxide donor sodium nitroprusside, at concentrations the paper lists as 10 and 30 millimolar in the methods section.

The team next probed the mutant’s invasive capacity using a cellophane penetration assay. Three days after removal of the cellophane membrane, the colony diameter of the mutant was significantly smaller than that of the wild-type and complemented strains, indicating impaired penetration-associated growth. To explore the molecular basis of this defect, the researchers measured the relative expression of genes associated with infection peg development, appressorium formation, host penetration, and root colonization. The mutant showed significant downregulation of several pathogenicity-related genes, including VdNox1, VdPls1, VdCrz1, VdSep5, VdCSIN1, Som1, and Vta3, compared with both the wild type and the complemented strain, suggesting that VdSPDS contributes to pathogenic development by modulating the transcription of genes required for infection structure formation and host invasion.

Enzyme assays added a second mechanistic layer. Culture supernatants collected from the mutant, wild type, and complemented strains grown in Czapek medium were analyzed spectrophotometrically, with cutinase activity measured using p-nitrophenyl butyrate as a substrate and cellulase activity quantified by the dinitrosalicylic acid method. Both activities were significantly decreased in the mutant compared with the wild type and complemented strains. Because successful colonization of plant tissues depends on the secretion of cell wall-degrading enzymes that dismantle host physical barriers, the authors argue that the combination of impaired expression of penetration-related genes and diminished enzyme activity provides a dual mechanistic explanation for the mutant’s reduced virulence.

Pathogenicity assays on seedlings of the susceptible cotton cultivar Jimian 11 confirmed the field-level consequence. Because the mutant is auxotrophic for spermidine, exogenous spermidine was supplied during inoculation to maintain fungal viability. At eighteen days post-inoculation, mock-inoculated control plants remained healthy, while plants inoculated with the wild-type and complemented strains developed typical Verticillium wilt symptoms, including chlorosis, necrosis, and vascular discoloration. Plants infected with the mutant, despite the spermidine supplementation, displayed significantly attenuated symptoms, with a markedly lower disease index than plants infected with the wild-type and complemented strains. Fungal re-isolation assays revealed decreased colonization of mutant-inoculated stems, and quantitative PCR analysis demonstrated that fungal biomass in cotton stems infected with the mutant was significantly lower than in those infected with the control strains. The authors conclude that although exogenous spermidine can sustain vegetative growth, the endogenous function of VdSPDS is indispensable for full virulence.

The study also examined the gene’s structure. Identified by searching the V. dahliae genome database using the Saccharomyces cerevisiae spermidine synthase sequence as a query, the 274-amino-acid VdSPDS protein contains two conserved domains, the AdoMet-MTase superfamily domain and the spermine synthase N-terminal domain, and showed the highest sequence similarity to the ortholog from Colletotrichum graminicola. Notably, whereas most examined fungal SPDS proteins contain nine conserved motifs, VdSPDS lacks motifs 3, 6, and 8, suggesting that although the core catalytic framework is conserved, the protein may possess unique structural features relative to other fungal orthologs.

The findings place V. dahliae within a growing body of fungal work showing that polyamine biosynthesis is critical for pathogenicity. Previous studies cited by the authors report that deletion of spermidine synthase in Aspergillus flavus abolishes vegetative growth and conidiation in vitro, that knockdown of the ortholog in Alternaria alternata significantly reduces pathogenicity toward pear fruit, and that deletion in Fusarium graminearum impairs both sexual and asexual reproduction and decreases stress tolerance. In the new study, the authors note that V. dahliae causes Verticillium wilt in cotton and a wide range of other plant species, and that in cotton the disease can reduce yield by approximately 10 to 35 percent, with management remaining challenging because microsclerotia persist in soil and highly effective fungicides are limited. They caution that the work was conducted in a single laboratory strain background under controlled greenhouse and in vitro conditions, and that the mutant’s defects were assessed with spermidine supplementation to compensate for its auxotrophy. Within those constraints, the study demonstrates that VdSPDS is essential for fungal growth, development, stress tolerance, and virulence in V. dahliae, and offers a foundation for understanding how polyamine metabolism orchestrates the pathogenic machinery of fungal-plant interactions.

Subject of Research: Functional characterization of the spermidine synthase gene VdSPDS in the fungal cotton pathogen Verticillium dahliae

Article Title: Spermidine synthase is essential for the trophic growth, stress response, and pathogenicity of Verticillium dahliae

Article References: Hai, R., Song, Y., Lv, J., Feng, Z., Han, J., Li, Y., Feng, H., & Sun, J. (2026). Spermidine synthase is essential for the trophic growth, stress response, and pathogenicity of Verticillium dahliae. Discover Genetics and Evolution, 72(1), Article 1. https://doi.org/10.1007/s00294-026-01335-2

Image Credits: AI Generated

DOI: 10.1007/s00294-026-01335-2

Keywords: Verticillium dahliae, spermidine synthase, polyamine biosynthesis, gene knockout, fungal pathogenicity, cotton wilt, stress tolerance, cell wall-degrading enzymes, cellophane penetration, disease index, Vd991, virulence