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Pathogens secrete numerous effectors to overcome plant‐derived reactive oxygen species (ROS), but how pathogens modulate effector secretion during infection remains unclear. In this study, we showed that the nucleotide diphosphate kinase Ndk1 in Fusarium graminearum plays important roles in vegetative growth, conidiation, sexual reproduction and pathogenicity. The species‐specific N‐terminus and three active sites of FgNdk1 functioned in the development and virulence of F. graminearum and contributed to its enzymatic activity. Protein structure results showed that the N‐terminus is rich in proline, and subcellular localisation and enzymatic activity assays confirmed that it was responsible for endoplasmic reticulum (ER)‐anchoring; additionally, the proline‐rich feature contributed to the role of the N‐terminus in enzymatic activity. We further revealed that the N‐terminus of FgNdk1 exhibited a loosened structural conformation, likely facilitating the activity of FgNdk1 anchored in the ER. Moreover, FgNdk1 significantly suppressed ROS in planta. Comparative transcription analysis showed that 16 effector genes were differentially expressed in the Fgndk1 mutant, particularly during the infection stage. FgNdk1 greatly promoted the secretion of effectors FgSp10, FgSp16 and FgSp24 FgSP, which were important for the virulence of F. graminearum and ROS detoxification. Overall, FgNdk1 contributes to virulence by promoting effector secretion to scavenge ROS in planta, and its proline‐rich species‐specific N‐terminus enhances enzymatic activity, further influencing the development and virulence of F. graminearum. These findings elucidate the mechanism by which effector secretion is modulated by an ER‐anchored protein during plant fungal pathogen invasion. The ER‐anchored nucleoside diphosphate kinase FgNdk1 promotes the secretion of effectors FgSp10, FgSp16 and FgSp24 to scavenge plant‐derived ROS during Fusarium graminearum infection.

This article is a Commentary on Wang et al., 216: 205–215.

Background The type IV secretion system is widely present in various bacteria, such as Salmonella , Escherichia coli , and Helicobacter pylori . These bacteria use the type IV secretion system to secrete type IV secretion effectors, infect host cells, and disrupt or modulate the communication pathways. In this study, type III and type VI secretion effectors were used as negative samples to train a robust model. Results The area under the curve of T4Seeker on the validation and independent test sets were 0.947 and 0.970, respectively, demonstrating the strong predictive capacity and robustness of T4Seeker. After comparing with the classic and state-of-the-art T4SE identification models, we found that T4Seeker, which is based on traditional features and large language model features, had a higher predictive ability. Conclusion The T4Seeker proposed in this study demonstrates superior performance in the field of T4SEs prediction. By integrating features at multiple levels, it achieves higher predictive accuracy and strong generalization capability, providing an effective tool for future T4SE research.

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell’s metabolism for the benefit of the bacterium to bypassing the host’s defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging, potentially fatal tick-borne infectious disease. The bacterium enters human cells via the binding of its unique outer-membrane invasin EtpE to the cognate receptor DNase X on the host-cell plasma membrane; this triggers actin polymerization and filopodia formation at the site of E. chaffeensis binding, and blocks activation of phagocyte NADPH oxidase that catalyzes the generation of microbicidal reactive oxygen species. Subsequently, the bacterium replicates by hijacking/dysregulating host-cell functions using Type IV secretion effectors. For example, the Ehrlichia translocated factor (Etf)-1 enters mitochondria and inhibits mitochondria-mediated apoptosis of host cells. Etf-1 also induces autophagy mediated by the small GTPase RAB5, the result being the liberation of catabolites for proliferation inside host cells. Moreover, Etf-2 competes with the RAB5 GTPase-activating protein, for binding to RAB5-GTP on the surface of E. chaffeensis inclusions, which blocks GTP hydrolysis and consequently prevents the fusion of inclusions with host-cell lysosomes. Etf-3 binds ferritin light chain to induce ferritinophagy to obtain intracellular iron. To enable E. chaffeensis to rapidly adapt to the host environment and proliferate, the bacterium must acquire host membrane cholesterol and glycerophospholipids for the purpose of producing large amounts of its own membrane. Future studies on the arsenal of unique Ehrlichia molecules and their interplay with host-cell components will undoubtedly advance our understanding of the molecular mechanisms of obligatory intracellular infection and may identify hitherto unrecognized signaling pathways of human hosts. Such data could be exploited for development of treatment and control measures for ehrlichiosis as well as other ailments that potentially could involve the same host-cell signaling pathways that are appropriated by E. chaffeensis .

Bordetella bronchiseptica and Bordetella pertussis are two closely related respiratory pathogens that employ their T3SS injectisome to deliver the BteA effector into host cells. In this study, we visualized the needle tip filament of their T3SS injectisome, a structure formed by the Bsp22 protein. We demonstrate that during Bordetella cultivation in Stainer-Scholte medium, Bsp22 filaments are abundant and can dynamically extend up to several micrometers in length through the incorporation of new subunits at their distal ends. In contrast, these filaments become shorter and/or less abundant during infection of host cells. This reduction correlates with decreased bsp22 mRNA expression and lower Bsp22 protein levels, while the levels of bscD mRNA, which encodes the inner membrane ring protein of the injectisome, remain stable. These results highlight the adaptability of the Bordetella T3SS injectisome and show how its tip filament structure changes in response to different environments.

The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in . To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in , namely . The Δ mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the Δ mutant. Further studies showed that deletion of the gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with a role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus.

The maize pathogenic fungus Ustilago maydis experiences endoplasmic reticulum (ER) stress during plant colonization and relies on the unfolded protein response (UPR) to cope with this stress. We identified the U. maydis co‐chaperone, designated Dnj1, as part of this conserved cellular response to ER stress. ∆dnj1 cells are sensitive to the ER stressor tunicamycin and display a severe virulence defect in maize infection assays. A dnj1 mutant allele unable to stimulate the ATPase activity of chaperones phenocopies the null allele. A Dnj1‐mCherry fusion protein localizes in the ER and interacts with the luminal chaperone Bip1. The Fusarium oxysporum Dnj1 ortholog contributes to the virulence of this fungal pathogen in tomato plants. Unlike the human ortholog, F. oxysporum Dnj1 partially rescues the virulence defect of the Ustilago dnj1 mutant. By enabling the fungus to restore ER homeostasis and maintain a high secretory activity, Dnj1 contributes to the establishment of a compatible interaction with the host. Dnj1 orthologs are present in many filamentous fungi, but are absent in budding and fission yeasts. We postulate a conserved and essential role during virulence for this class of co‐chaperones.

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

Macrophages are one of the key target cells of pathogenic Yersinia , where central immune response pathways, such as phagocytosis, gene expression, and inflammasome assembly, are suppressed by secreted bacterial effectors (Yops) in a highly coordinated fashion. Most studies analyzing cooperation between Yop proteins have utilized cell lines and mouse-derived macrophages, which strongly differ from human macrophages. This study employed primary human macrophages and analyzed cooperation between different Yersinia enterocolitica effector proteins on gene expression, histone phosphorylation, calcium signaling, and inflammasome assembly. We reveal synergistic, antagonistic, and individual roles of different Yersinia effector proteins. This work highlights how highly coordinated activities of a limited set of effectors can efficiently disarm macrophage immune responses and lead to a successful infection.