Tem1 is a small GTPase, a class of molecular switches that alternate between an active GTP-bound state and an inactive GDP-bound state. In budding yeast, Tem1 sits at the top of the mitotic exit network, or MEN, a signaling cascade that couples the completion of nuclear division to cytokinesis. The Bfa1-Bub2 complex acts as a GTPase-activating protein, or GAP, that hydrolyzes Tem1-bound GTP, keeping the pathway off until the mitotic spindle is correctly positioned. When the spindle pole body, the fungal equivalent of the centrosome, reaches the right location, the polo-like kinase Cdc5 disables the GAP, allowing GTP-loaded Tem1 to trigger a kinase cascade that ultimately releases the phosphatase Cdc14 and drives the cell out of mitosis. Homologs of this pathway exist across fungi, but until now M. oryzae lacked a confirmed Tem1 ortholog, leaving a critical gap in understanding how the pathogen coordinates its unusual cell biology with infection.

To find the missing piece, the team searched the M. oryzae genome using yeast and Fusarium graminearum Tem1 proteins as queries. The search pinpointed MGG_04862, annotated as a septum-promoting GTP-binding protein, which shares 60.21 percent sequence identity with yeast Tem1 and a striking 72.28 percent identity with the Fusarium homolog. Phylogenetic analysis of Tem1 proteins from 24 fungal species placed MoTem1 in a clade with homologs from other devastating plant pathogens, including Botrytis cinerea and Sclerotinia sclerotiorum. The researchers then built a panel of isogenic strains in the Guy11 wild-type background: a knockout lacking the gene entirely, an overexpression strain, and two point mutants locked into opposite states. The Q182L substitution produces a constitutively active GTP-bound protein, while T137N creates a dominant-negative, inactive version. Yeast two-hybrid and pull-down assays confirmed that MoTem1 interacts with the fungal GAP components MoBfa1 and MoBub2 in a state-dependent manner, with the active form recruiting the full complex and the inactive form binding only MoBfa1.

The phenotypes of these mutants revealed that MoTem1’s activity cycling, rather than its mere presence or abundance, is what matters. The constitutively active strain grew about 41.56 percent more slowly than wild type, while the knockout and both locked-state mutants produced dramatically fewer conidia, the three-celled spores that launch infections, with reductions of 35 to 67 percent. Overexpression alone left conidiation untouched, demonstrating that dynamic GDP/GTP cycling is essential for asexual reproduction. Germination assays told a similar story: strains with enhanced or reduced GTPase activity germinated faster than wild type, reaching up to nearly 90 percent germination within two hours, while the constitutively active strain germinated poorly, formed multiple germ tubes, developed thickened cell walls and produced abnormally elongated germ tubes. These defects in pre-infection morphogenesis foreshadowed even more dramatic consequences on the plant itself.

When the researchers spray-inoculated rice seedlings, the virulence patterns were striking. The knockout and constitutively active strains were both attenuated, with the active mutant restricted to small lesion types and never producing the largest class of lesions. The dominant-negative strain, however, was hypervirulent, generating large Type IV lesions that were absent in wild-type infections at the same time point. The authors propose that inactivating MoTem1 promotes invasive hyphal growth, the phase of infection during which the fungus spreads from cell to cell through plasmodesmata, and lesion expansion directly depends on how extensively those hyphae extend. Constitutive activation, by contrast, disrupts the timing of morphogenic programs and likely the deployment of effectors, undermining the finely choreographed sequence of appressorium formation, cuticle penetration and biotrophic establishment that M. oryzae requires.

Stress experiments added another layer of complexity. Under oxidative stress from hydrogen peroxide, the dominant-negative strain was significantly more tolerant than wild type, with a higher half-maximal inhibitory concentration, while the constitutively active strain was hypersensitive. Cell wall stress from Congo Red produced the same split: overexpression conferred tolerance, constitutive activation caused severe sensitivity. Under osmotic stress with sorbitol, the knockout, overexpression and dominant-negative strains all showed enhanced tolerance, but responses to ionic stress with sodium and potassium chloride varied by allele. The picture that emerges is of a cell cycle regulator whose nucleotide-binding state feeds into entirely separate stress signaling pathways, with the GTP-bound form promoting oxidative resilience and the cycling state maintaining cell wall and membrane integrity.

Fluorescence microscopy explained part of the mechanism. EGFP-tagged MoTem1 localized as puncta to spindle pole bodies, confirmed by co-localization with the SPB marker MoAlp6-mCherry. The constitutively active variant accumulated intensely at these structures in both hyphae and conidia, while the inactive variant showed completely diffuse cytoplasmic fluorescence, demonstrating that only the GTP-bound form is recruited to spindle pole bodies. Biochemical assays confirmed that purified MoTem1 has genuine GTP-hydrolyzing activity, roughly four times that of a tag-only control, and that the T137N substitution abolishes it. Critically, both the knockout and the constitutively active strain produced multinucleate hyphal compartments, indicating that nuclear division and septum formation had fallen out of sync. In the knockout, cytokinesis fails outright; in the active mutant, premature MEN activation bypasses the spindle position checkpoint, or SPOC, the surveillance system that normally delays mitotic exit until the spindle is correctly aligned. The dominant-negative strain maintained normal nuclear segregation, which the authors attribute to residual wild-type MoTem1 activity in that background being sufficient for cytokinesis.

Transcriptomic profiling across all five strains revealed the full scope of MoTem1’s influence. Principal component analysis separated the knockout and constitutively active strains sharply from wild type, and differential expression analysis identified 72 genes consistently altered across all mutants, including core cell cycle regulators and stress-responsive transcription factors. Weighted gene co-expression network analysis resolved eight modules, three of which stood out: a brown module correlated with oxidative stress resistance and negatively with growth rate, suggesting a trade-off between stress adaptation and proliferation; a yellow module tied to hyphal growth and enriched for ribosome and glycolysis genes; and a tan module most strongly correlated with pathogenicity, containing 38 virulence-associated genes encoding secreted effectors and cutinases. Among the tan module’s hub genes was MoCHS1, a class V chitin synthase, one of three of M. oryzae’s seven chitin synthases known to be required for pathogenicity.

The link to MoCHS1 proved to be the study’s most consequential finding. Expression of this chitin synthase was downregulated 3.07-fold in the constitutively active strain and upregulated 1.26-fold in the dominant-negative strain, mirroring their virulence phenotypes. Pharmacological validation followed: the chitin synthase inhibitor Polyoxin B suppressed growth of the constitutively active strain by over 40 percent while barely touching wild type, and the dominant-negative strain was strongly resistant. Reactive oxygen species assays on rice leaf disks added a plant immunity dimension. Culture filtrates from the dominant-negative strain elicited the strongest ROS burst in rice tissue, followed by wild type, with the constitutively active strain eliciting the weakest response, and chitinase pretreatment abolished all responses, confirming that chitin oligomers acting as pathogen-associated molecular patterns were responsible. The authors suggest that altered MoCHS1 expression changes how much chitin is exposed or released from the fungal cell wall, modulating the plant’s early immune perception and potentially accelerating the transition to the necrotrophic phase that produces large necrotic lesions.

Taken together, the study repositions MoTem1 from a narrow cell cycle component to a signaling hub with genome-wide reach. Its GTP-dependent recruitment to spindle pole bodies safeguards mitotic fidelity, while its activity states ripple outward through transcriptional networks governing redox balance, cell wall architecture, osmotic adaptation and virulence. The work also highlights evolutionary divergence within the MEN pathway: unlike in Fusarium graminearum, where inactive Tem1 localizes to septa, inactive MoTem1 disperses into the cytoplasm, and M. oryzae’s MoSep1 activates MoMob1 directly, bypassing MoDbf2. For a pathogen responsible for one of agriculture’s most serious diseases, these findings open a conceptually new angle: the machinery that ends mitosis is also a master regulator of how the fungus grows, withstands stress and attacks its host, and perturbing its nucleotide cycle can either cripple the pathogen or, paradoxically, make it more dangerous.

Subject of Research: Role of the mitotic exit GTPase MoTem1 in development, stress adaptation and pathogenicity of the rice blast fungus Magnaporthe oryzae

Article Title: Mutational analysis of the mitotic exit GTPase MoTem1 reveals its role in development, stress adaptation, pathogenicity and global gene regulation in Magnaporthe oryzae

Article References: Pei, M., Xie, X., Cao, Y., Chen, J., Yang, F., Wang, Z., Olsson, S., Lu, G.-D., & Li, Y. (2026). Mutational analysis of the mitotic exit GTPase MoTem1 reveals its role in development, stress adaptation, pathogenicity and global gene regulation in Magnaporthe oryzae. Stress Biology, 6(1), Article 38. https://doi.org/10.1007/s44154-026-00310-8

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00310-8

Keywords: Magnaporthe oryzae, MoTem1, mitotic exit network, GTPase, spindle pole body, SPOC, chitin synthase, MoCHS1, pathogenicity, stress adaptation, transcriptomics, WGCNA

Chitin is to fungi what a load-bearing skeleton is to a building. Long chains of N-acetylglucosamine sugars are synthesized at the cell membrane and threaded outward, where they crystallize into microfibrils that reinforce the wall. Because human cells make no chitin at all, the enzymes responsible are theoretically ideal drug targets. Yet despite decades of interest, inhibitor development against chitin synthases has stalled, largely because researchers lacked a detailed picture of how these enzymes work, particularly the class II chitin synthases such as Chs1, which are essential for fungal viability. The new study changes that, presenting structures of CaChs1 at resolutions between 2.93 and 3.38 angstroms, sharp enough to trace individual amino acid side chains, bound substrates and even lipid molecules lodged inside the protein.

The technical achievement behind the structures is considerable. The team purified the enzyme from insect cells, verified its lipid cargo by liquid chromatography and mass spectrometry, and then froze thousands of single-particle specimens for imaging on cryo-electron microscopes. Computational classification of more than a hundred thousand micrographs allowed the researchers to sort the particles into distinct conformational states, effectively producing a molecular movie of the enzyme at work. They captured the enzyme in its resting, ligand-free form, in complexes with the sugar donor UDP-GlcNAc, and in a series of states showing the nascent chito-oligomer chain as it grows inside the catalytic site and begins its journey through the membrane-spanning translocation channel.

What emerges from these snapshots is a picture of a tightly choreographed machine. Chitin elongation and translocation are coupled to coordinated motion of the glycosyltransferase domain, the catalytic engine that adds sugar units one at a time, and the dimer interface, the region where two copies of the enzyme associate. As the growing polymer lengthens, the enzyme shifts between states that position the acceptor sugar for the next bond-forming step while simultaneously opening the channel through which the new chain is pushed toward the cell exterior. Molecular dynamics simulations carried out by collaborators at Lehigh University helped the team validate how the chitobiose product sits within the active site and how the polymer threads through the pore, complementing the static electron density maps with a dynamic view of the process.

The most striking discovery, however, concerns a small molecule called diynyl arylamine, abbreviated DA, a non-competitive inhibitor specific to CaChs1 that was previously known to block the enzyme without a clear mechanism. The structures show that DA does not bind at the catalytic site at all. Instead, it lodges within the chitin translocation channel itself, precisely where a regulatory phospholipid normally resides. By occupying this lipid-binding position, DA physically occludes the route through which the growing polymer must be extruded, jamming the machine in much the same way that a plug stops a pipe. This mode of inhibition, blocking product export rather than the chemical reaction itself, had been suspected for processive glycosyltransferases but had never been visualized in such atomic detail for a fungal chitin synthase.

The finding that a regulatory lipid sits inside the translocation channel adds an intriguing layer of biology. Lipids co-purifying with CaChs1 were identified as phosphatidylethanolamine species, and electron density attributable to a phospholipid was observed in the channel of the unliganded enzyme. The researchers propose that this lipid may help regulate the conformation of transmembrane helix 5 and an interfacial helix, elements that shift position when DA binds. In other words, the channel is not merely a passive tube but a gated, lipid-sensitive passage whose opening and closing is coordinated with the catalytic cycle. That a small drug can hijack this regulatory site suggests that channel-lipid interactions may be a general design principle among processive polymer-synthesizing enzymes, which include cellulose synthases in plants and hyaluronan synthases in vertebrates.

Perhaps the most clinically resonant result came from combination experiments. The team tested DA alongside nikkomycin Z, a nucleoside-derived natural product that inhibits class I chitin synthases by mimicking the sugar donor and occupying the catalytic site. Because the two compounds attack different parts of the chitin synthesis machinery, the researchers asked whether they would act synergistically. They did, and potently so. The combination showed strong synergy not only against C. albicans but also against Candida auris, the multidrug-resistant yeast that the World Health Organization has placed on its list of fungal priority pathogens. This matters because single-agent chitin synthase inhibitors have historically struggled: fungi can compensate for partial inhibition of one chitin synthase class by ramping up another. Hitting both the class I catalytic site and the class II translocation channel simultaneously appears to overwhelm that redundancy.

The work also fits into a broader renaissance in antifungal structural biology. In recent years, cryo-EM has illuminated the structures of chitin synthases from several organisms, including earlier studies of C. albicans class I enzymes and structures of fungal beta-1,3-glucan synthase bound to the frontline drug caspofungin, reported by an overlapping Duke team earlier in 2026. Together, these studies are mapping the two great polymer systems of the fungal wall, chitin and glucan, at atomic resolution. For the first time, medicinal chemists can see exactly where existing drugs bind, why some enzymes are resistant, and which pockets remain unexploited. The translocation channel of CaChs1 now joins that short list of validated, structure-guided targets.

Caution is warranted before declaring a new drug on the horizon. DA itself is a research tool rather than a medicine, and translating channel-blocking chemistry into a safe, potent antifungal will require optimization of selectivity, pharmacokinetics and formulation. The authors note that the DA-binding pocket is conserved among class II chitin synthases from Candida species and Saccharomyces cerevisiae, which is encouraging for spectrum but raises the question of how broadly across fungal pathogens such inhibitors could reach. Still, the conceptual advance is clear: the study establishes the chitin translocation channel as a druggable site and provides the structural foundation for rational design. At a time when antifungal drug resistance is rising and the clinical pipeline remains thin, every new angle of attack counts, and this one comes with an atomic-resolution map.

The structures and maps underlying the study have been deposited in public archives, including the Electron Microscopy Data Bank and the Protein Data Bank, so that laboratories worldwide can interrogate the channel, model their own inhibitor designs and test the lipid-regulation hypothesis. For a field that has waited more than four decades since chitin synthetase was first localized on the yeast plasma membrane for a molecular-level view of its target, the arrival of these structures marks a genuine turning point. The fungal wall’s most essential builder has finally been caught in the act, and its most vulnerable passage is now on the map.

Subject of Research: Cryo-EM structural analysis of the Candida albicans chitin synthase Chs1 and its druggable chitin translocation channel

Article Title: Cryo-EM structures of Candida albicans chitin synthase Chs1 reveal a druggable translocation channel

Article References: Cryo-EM structures of Candida albicans chitin synthase Chs1 reveal a druggable translocation channel. (n.d.). https://doi.org/10.1038/s41564-026-02485-2

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02485-2

Keywords: Candida albicans, chitin synthase, cryo-EM, antifungal resistance, translocation channel, diynyl arylamine, nikkomycin Z, Candida auris, fungal cell wall, drug discovery, structural biology, glycosyltransferase