Fungal pH Sensor Revealed as Master Switch Behind Wheat Scab Toxin

A devastating wheat pathogen listens to the acidity of its surroundings with far more sophistication than scientists previously appreciated, and the consequences reach all the way into the grain on our tables. In a study published in Crop Health, researchers report that the conserved pH signaling pathway Pal/PacC in Fusarium graminearum, the fungus behind Fusarium head blight, orchestrates fungal growth, tolerance of environmental stresses, membrane penetration, and the production of deoxynivalenol, one of the world’s most problematic mycotoxins. By systematically deleting each component of the pathway and tracing the molecular chain of command, the team has assembled the most complete picture yet of how a plant pathogen converts an environmental cue into a virulence decision. The findings suggest that the pH regulatory system is not a peripheral curiosity but a central node in the pathogen’s arsenal, opening a new front in the effort to protect cereal crops.

Fusarium head blight is a scourge of wheat, barley, maize, and other cereals worldwide. Beyond the direct yield losses, infected grains become contaminated with mycotoxins, most notably deoxynivalenol, commonly abbreviated DON, and zearalenone. DON is not merely a food safety hazard; it is also a genuine virulence factor that helps the fungus spread through host tissue. Understanding how the fungus regulates its production has therefore been a long-standing goal of plant pathology. The new work, led by Yujie Wang, Tong Cao, and Dekun Liu with senior authors Jun Tian and Qin Gu, builds on the team’s earlier discovery that F. graminearum actively alkalinizes wheat tissue during infection, raising the pH of its surroundings by nearly three units as it invades.

The Pal/PacC pathway is a classic piece of fungal machinery, first dissected in the bread mold Aspergillus nidulans and the yeast Saccharomyces cerevisiae. In its canonical form, the pathway consists of six Pal proteins, PalA, PalB, PalC, PalF, PalH, and PalI, together with a zinc-finger transcription factor called PacC. Under acidic conditions, the full-length PacC protein folds into a closed conformation that shields it from processing enzymes and keeps it trapped in the cytoplasm. When the environment turns alkaline, the transmembrane sensor PalH detects the shift and triggers phosphorylation and ubiquitination of PalF, which recruits the endosomal sorting complexes required for transport to the plasma membrane. A proteolytic cascade then clips the inhibitory tail from PacC, and the truncated form migrates into the nucleus, where it switches on alkaline-responsive genes and suppresses those favored by acidity.

Whether this elegant system operated the same way in F. graminearum was largely unknown. To find out, the researchers used homologous recombination to construct targeted deletions of each pathway component, generating mutants lacking FgPalA, FgPalB, FgPalC, FgPalF, FgPalH, FgPalI, or the transcription factor FgPacC itself. When the mutants were grown on potato dextrose agar, complete medium, and minimal medium, a consistent pattern emerged. Every deletion except FgPalI impaired hyphal growth, and even where colony diameters looked normal, the aerial hyphae of the mutants were strikingly stunted when cultured in tubes. Complemented strains carrying the intact genes regained wild-type growth, confirming that the defects were genuinely attributable to the deleted genes. Notably, spore germination and sexual reproduction, measured by perithecium formation on carrot agar, were unaffected, indicating that the pathway is crucial for vegetative growth but dispensable for reproduction.

The stress experiments delivered some of the most striking results. Mutants lacking FgPalA, FgPalB, FgPalC, FgPalF, FgPalH, or FgPacC were significantly more sensitive to alkaline pH 8.0 than the wild-type strain, while the FgPalI mutant was indifferent, marking FgPalI as a pathway member uninvolved in alkaline response. The same mutants also showed heightened vulnerability to a battery of osmotic and membrane stresses, including 1.0 M sodium chloride, 1.0 M potassium chloride, 0.15 M lithium chloride, 1.0 M sorbitol, and 0.01 percent sodium dodecyl sulfate, as well as oxidative stress from 0.05 percent hydrogen peroxide. In other words, the pH sensing apparatus doubles as a general stress survival system, a finding that helps explain how the fungus copes with the chemically hostile environments it encounters inside plants and in alkaline or saline soils.

Perhaps the most unexpected discovery came from the western blot analyses. The researchers had previously shown that GFP-tagged FgPacC accumulates in the nucleus under alkaline conditions. Now they found that high salt does the same job: under 1.0 M NaCl, FgPacC was cleaved into a 30-kilodalton active isoform, dubbed FgPacC30, independently of alkaline pH. This means the pathway integrates multiple environmental signals, not just acidity, and that osmotic stress alone is sufficient to activate the transcription factor. Sequence analysis supported the idea, revealing that FgPacC shares conserved protease recognition sites and cleavage regions with homologs in Aspergillus nidulans and Neurospora crassa, sharing roughly 45 and 51 percent similarity respectively.

To map the wiring of the pathway, the team turned to yeast two-hybrid assays, and for the first time in F. graminearum demonstrated direct physical interactions among the essential components. FgPalF, which sits downstream of the plasma membrane complex, interacts with FgPalA and the cysteine protease FgPalB, while FgPalA binds both FgPalB and FgPalC. Crucially, both FgPalA and FgPalB also bind the transcription factor FgPacC. Confocal microscopy tied this network to function: GFP-FgPacC rapidly entered the nucleus under alkaline conditions in the wild type, but in mutants lacking FgPalA, FgPalB, FgPalC, FgPalF, or FgPalH the fluorescent signal stayed diffused in the cytoplasm. The FgPalI mutant, once again, behaved normally. The conclusion is that ambient pH-dependent proteolytic activation governs where FgPacC ends up in the cell, and thus whether it can act on DNA at all.

The pathway’s role in infection was tested with a cellophane penetration assay. When strains were grown on cellophane overlying acidic, neutral, or alkaline medium, the mutants grew more poorly under alkaline conditions than under neutral or acidic ones. More tellingly, after the membrane was removed, the FgPalA, FgPalB, FgPalC, FgPalF, FgPalH, and FgPacC mutants failed to penetrate the cellophane under alkaline pH, while neither the wild type nor the mutants could penetrate under acidic conditions. The authors interpret this as evidence that an acidic host environment does not harm fungal growth but directly blocks penetration, whereas the alkalinized environment that F. graminearum itself creates during wheat infection demands a functional Pal/PacC pathway for the fungus to push through tissue barriers.

The deepest mechanistic insight concerns DON. When wheat kernels were inoculated with the mutants, strains lacking FgPacC, FgPalA, FgPalB, or FgPalH produced significantly more deoxynivalenol than the wild type, and FgPacC was shown to negatively regulate the DON precursors 3-ADON and 15-ADON in trichothecene biosynthesis induction medium. Electrophoretic mobility shift assays revealed that purified FgPacC30 binds directly to the promoter of FgTRI1, a gene encoding a DON biosynthetic enzyme, and reverse transcription quantitative PCR confirmed that FgTRI1 expression rises sharply in the FgPacC deletion mutant under alkaline conditions. The epigenetic story completes the circuit. Chromatin immunoprecipitation followed by quantitative PCR showed that, under alkaline conditions, the FgTRI1 promoter in the FgPacC mutant carried markedly higher levels of the acetylated histone marks H3K18ac and H2BK11ac than in the wild type. Since the team’s earlier work established that FgPacC30 inhibits the histone acetyltransferase FgGcn5, the model is that activated FgPacC30 binds FgGcn5, dampens acetylation at the FgTRI1 promoter, keeps the chromatin closed, and thereby suppresses toxin production during invasive growth.

This is, according to the authors, the first report of a transcription factor within the SAGA complex repressing trichothecene gene expression at the epigenetic level in a Fusarium pathogen, and it resolves a long-standing puzzle: although acidic pH promotes TRI gene transcription in laboratory culture, the fungus alkalinizes wheat tissue during infection yet still times its toxin output precisely. The answer is that host alkalinization itself triggers FgPacC30 activation, which then actively holds DON biosynthesis in check while the mycelium establishes itself. The regulatory duality of FgPacC, simultaneously governing stress adaptation and toxigenesis, positions it as an attractive target for future control strategies. If researchers can find ways to disrupt the Pal/PacC signaling network, or to lock FgPacC in its inactive state, they might simultaneously weaken the fungus’s stress tolerance, its tissue penetration, and its toxin output, a triple blow against one of agriculture’s most destructive pathogens.

Subject of Research: The Pal/PacC pH signaling pathway and its regulation of growth, stress responses, and mycotoxin biosynthesis in Fusarium graminearum

Article Title: The pH signaling pathway Pal/PacC regulates fungal growth, stress responses, and mycotoxin biosynthesis in Fusarium graminearum

Article References: Wang, Y., Cao, T., Liu, D., Zhao, H., Chen, Y., Li, S., Wen, K., Ali, Q., Huang, H., Zhou, S., Wu, H., Gao, X., Tian, J., & Gu, Q. (2025). The pH signaling pathway Pal/PacC regulates fungal growth, stress responses, and mycotoxin biosynthesis in Fusarium graminearum. Crop Health, 3(1), Article 17. https://doi.org/10.1007/s44297-025-00054-3

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00054-3

Keywords: Fusarium graminearum, Pal/PacC pathway, FgPacC, pH signaling, deoxynivalenol, mycotoxin biosynthesis, Fusarium head blight, FgTRI1, histone acetylation, FgGcn5, plant pathology, stress response