Lsm8 is the defining subunit of the nuclear Lsm2-8 complex, a heptameric ring of Like-Sm proteins that binds the 3-prime end of U6 small nuclear RNA, the catalytic core of the spliceosome. The spliceosome is the macromolecular machine that removes introns from pre-messenger RNA, and without properly assembled Lsm2-8, U6 is left unprotected and splicing cycles falter. Sequence analysis showed that the Fusarium Lsm8 protein shares roughly half its identity with its yeast counterpart and about 65 percent with the human version, underscoring how ancient this module is. Although Lsm8 lacks a predicted nuclear localization signal, a fluorescent Lsm8-GFP fusion co-localized precisely with a histone marker in fungal nuclei, confirming where the protein operates.

To map the complex’s architecture, the team deployed a battery of interaction assays. Yeast two-hybrid tests revealed direct contacts between Lsm8 and Lsm2 and Lsm3, bimolecular fluorescence complementation captured a nuclear interaction between Lsm8 and Lsm4, and co-immunoprecipitation validated all of these associations in living fungal cells. Most decisively, affinity purification followed by mass spectrometry, using Lsm8-GFP as bait, co-purified all six remaining Lsm subunits. The picture that emerged is of Lsm8 as a central architectural keystone. When the researchers deleted the LSM8 gene, GFP-tagged Lsm2, Lsm3 and Lsm4 abandoned the nucleus entirely and diffused throughout the cytoplasm, demonstrating that Lsm8 is indispensable for both assembling the ring and escorting it to its workplace.

The consequences of losing this keystone were dramatic. Deletion mutants grew slowly on every medium tested, from rich potato dextrose agar to minimal medium. Transcriptome sequencing revealed massive reprogramming: 1,710 genes were significantly upregulated and 1,796 downregulated. The upregulated set was enriched in ribosome biogenesis, spliceosome function and RNA degradation, which the authors interpret as a compensatory feedback response to the missing splicing machinery. The downregulated genes clustered in carbon and nitrogen metabolism, including glycolysis and the pentose phosphate pathway, providing a molecular explanation for the growth collapse. Quantitative PCR confirmed the sequencing results for splicing factors such as PRP4 and PRP5, degradation factors such as RRP6 and XRN2, and glycolytic genes such as FBA1 and ENO1.

The most striking defect, however, lay in splicing itself. Across 13,982 detected introns, the median intron retention rate jumped from 0.011 in the wild type to 0.089 in the mutant, a highly significant eight-fold increase. In total, 2,344 introns from 1,821 genes showed significantly elevated retention, while only 45 introns became better spliced. Crucially, most of the affected genes, 1,270 of them, showed no change in overall transcript abundance, meaning the damage was purely post-transcriptional: transcripts were made normally but never spliced correctly. Retained introns disrupt reading frames, producing truncated or non-functional proteins, or triggering nonsense-mediated decay. The affected genes were enriched in chromatin remodeling, RNA polymerase activity, nucleocytoplasmic transport and DNA repair, processes essential to cellular integrity.

The splicing failures mapped directly onto the fungus’s environmental vulnerabilities. When challenged with a panel of eight stress agents, the mutant proved hypersensitive to all of them, with the strongest effects under osmotic stress from salt and the fungicide fludioxonil, which works by hyperactivating the osmotic stress pathway. The team traced this to the high osmolarity glycerol, or HOG, MAPK cascade. HOG1 carries eight introns, and nearly all of them were inefficiently spliced in the mutant, with defects worsening under stress. Total HOG1 mRNA levels were unchanged, but Hog1 protein levels dropped significantly, showing that the defect lies in protein production rather than transcription. Intriguingly, the residual Hog1 that remained was hyperphosphorylated, and intracellular glycerol accumulated to roughly three times wild-type levels, suggesting distorted pathway feedback. The downstream transcription factor ATF1 also showed splicing abnormalities.

Reproduction fared no better. The mutant produced fewer conidia, the asexual spores that spread infection, and those spores were shorter and abnormally septated. Sexual development was abolished outright: on carrot agar under black light, the mutant formed no perithecia at all, the fruiting bodies that generate overwintering ascospores. The molecular culprits were again splicing defects, this time in two developmental regulators. GPA1, encoding a heterotrimeric G protein alpha subunit, retained all three of its introns at elevated rates, and STE12, a transcription factor essential for sexual reproduction, retained both of its introns. As with HOG1, total mRNA levels were largely unaffected, reinforcing the theme that Lsm8’s influence operates after transcription.

Virulence and mycotoxin synthesis collapsed in parallel. Infection assays on wheat heads, wheat coleoptiles and corn silks showed the mutant was nearly nonpathogenic, and deoxynivalenol production fell sharply. The TRI gene cluster that manufactures the toxin was hit twice: key genes including TRI1, TRI4, TRI5, TRI6, TRI10 and TRI101 were transcriptionally downregulated, and the splicing efficiency of TRI1, TRI4 and TRI5 transcripts was significantly reduced. Microscopy of a Tri1-GFP reporter delivered perhaps the most visually compelling result: in the wild type, Tri1 aggregates into the spherical toxisomes, specialized compartments where deoxynivalenol is synthesized, but in the mutant almost no toxisomes formed and Tri1-GFP protein levels plummeted. Lsm8 thus guards the fungus’s chemical arsenal at every regulatory level simultaneously.

Finally, the study answered a long-standing question about what happens to all those defective transcripts. In eukaryotes, aberrant RNAs are normally cleared by nonsense-mediated decay, the RNA exosome, which degrades transcripts in the 3-prime to 5-prime direction, or by Xrn enzymes working in the opposite direction. Mass spectrometry showed that Lsm8 physically interacts with core exosome subunits including Rrp45, Rrp4 and Dis3, and the mutant compensated by upregulating exosome components. Genetic dissection proved decisive: intron-retained HOG1 transcripts accumulated massively in an exosome-subunit deletion mutant and even more in an Lsm8-exosome double mutant, while accumulation in an Xrn1 mutant was comparatively modest. The exosome, the authors conclude, is the predominant degrader of intron-retained transcripts arising from Lsm2-8 dysfunction, with Xrn1 playing a supporting role. Because both the Lsm2-8 complex and the exosome are conserved across eukaryotes, from yeast and plants to worms and humans, this coupled splicing-and-surveillance module appears to be a fundamental feature of eukaryotic biology. In Candida albicans, a human fungal pathogen, Lsm loss already impairs filamentation and pathogenicity, suggesting the Fusarium findings generalize. Targeting this conserved module, the researchers argue, could yield antifungal strategies that strike at core RNA-processing machinery shared by diverse pathogens, simultaneously curbing crop disease and mycotoxin contamination while sparing the host, whose own cells process RNA through homologous but distinct regulatory contexts.

Subject of Research: RNA splicing fidelity and the Lsm8-exosome surveillance module in fungal stress adaptation and virulence

Article Title: A conserved Lsm8–exosome module maintains RNA splicing fidelity to control fungal stress adaptation and virulence

Article References: Ren, Y., Cheng, H., Han, X., Guo, M., Xu, C., Yan, J., Ge, Z., Ma, Z., & Chen, Y. (2026). A conserved Lsm8–exosome module maintains RNA splicing fidelity to control fungal stress adaptation and virulence. Stress Biology, 6(1), Article 14. https://doi.org/10.1007/s44154-026-00285-6

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00285-6

Keywords: Fusarium graminearum, Lsm8, Lsm2-8 complex, RNA splicing, intron retention, RNA exosome, deoxynivalenol, mycotoxin biosynthesis, HOG-MAPK pathway, fungal virulence, post-transcriptional regulation, antifungal targets

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