Microbiology and Plant Pathology

Reassortment between different influenza strains occurs when they co-infect the same host cell. The emergence of a reassortant virus depends on both its intrinsic fitness and extrinsic factors, including preexisting humoral immunity. The generation of pandemic strains, such as H2N2 and H3N2, and zoonotic influenza A viruses, such as H5N6, H5N8, and H7N9, in birds is suggested to be the result of extensive selection by preexisting antibodies. To further explore the role of humoral immunity in reassortment, we generated two divergent fluorescent protein-expressing viruses and used strain-specific and cross-reactive monoclonal antibodies (mAbs) to assess the impact of cross-immunity on reassortment. Our results indicate that all mAbs altered the genotypic diversity and significantly reduced the release of progeny virions in co-infected cells both in vitro and in vivo. Moreover, antibody transfer studies in mice revealed protection from challenge with divergent pathogenicity profiles. Notably, selection driven by a strain-specific mAb depended on its neutralizing specificity, whereas the selection driven by broadly reactive mAbs was independent of neutralization specificity. Our findings demonstrate that preexisting neutralizing antibodies shape reassortment and that strain-specific neutralizing antibodies promote antigenic shift during co-infection, which is not the case for broadly cross-reactive antibodies that recognize influenza viruses from different subtypes.

Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.

Phyling is a fast, scalable, and user-friendly tool supporting phylogenomic reconstruction of species phylogenies directly from protein-encoded genomic data. It identifies orthologous genes by searching protein sequences against a curated set of hidden Markov model profiles, consisting of single-copy orthologs derived from the BUSCO database. To optimize the speed of the final inference, Phyling includes a module to filter aligned orthologs based on their phylogenetic informativeness. Finally, Phyling provides a companion wrapper for automated species tree construction using either consensus or concatenation strategies. Phyling efficiently resolves large phylogenies by optimizing memory usage and data processing. Its checkpoint system enables users to incrementally add or remove samples without repeating the entire search process. For analyses involving closely related taxa, Phyling supports the use of nucleotide coding sequences, which may capture phylogenetic signals missed by protein sequences. The benchmark results show that Phyling substantially runs faster than OrthoFinder, a reciprocal best hit based method, while achieving equal or better accuracy.

BackgroundEastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have undergone substantial population declines throughout their range, leading them to become the focus of increased conservation efforts, including care in zoo and university settings. However, effective implementation of such conservation strategies often relies on a comprehensive understanding of host health, which can be directly influenced by the gut microbiome, yet characterization of gut microbiota often remains overlooked in ex situ conservation facilities. Additionally, effects on the gut microbiome associated with releasing zoo-reared animals into the wild are poorly understood. Therefore, these circumstances make hellbenders an ideal species to examine the relationship between zoo management strategies and gut microbial dynamics.Methods16S rRNA sequencing was used to investigate dissimilarities between the gut microbiome of hellbenders in zoo and wild settings and to evaluate the impact of implementing a wild diet in zoo-reared hellbenders. Additionally, the bacterial composition of zoo-released individuals and wild resident hellbenders was compared to examine the response of the gut microbiome upon release into natural habitat. Selected samples were also chosen for ITS1 rDNA sequencing as a preliminary investigation of the hellbender gut mycobiome.ResultsHuman rearing strongly affected the gut microbiome, leading to reduced bacterial richness as well as differing community structure than wild hellbenders. However, implementation of a wild diet in a zoo setting modulated the microbiome and appeared to be mainly driven by bacterial turnover. Additionally, both bacterial and fungal gut assemblages demonstrated the capacity for restructuring upon release into native habitat to become more reflective of a wild-type microbiome.ConclusionsWe completed the first study elucidating the gut microbial composition patterns of hellbenders, across both zoo and wild settings. These results provide an understanding of the potential impacts of conservation populations in zoos on gut microbial communities and also inform headstart programs of the transition of the gut microbiome post-reintroduction to the wild.

Pseudomonas species are Gram-negative bacterial pathogens that affect a wide range of economically important crops, including almond. Increasing resistance of Pseudomonas syringae to conventional management strategies highlights the need for alternative disease control options. In this study, we isolated and characterized three lytic bacteriophages, including vB_PsyP_Mobley, vB_PsyP_Plaza, and vB_PsyP_Mission, targeting almond-infecting Pseudomonas strains. Host-range analysis across 36 isolates revealed partially overlapping infectivity profiles, with strongest activity against phylogroup 2 (PG2) almond-associated P. syringae pv. syringae and reduced infectivity against phylogenetically distinct isolates, including PG7 P. viridiflava and other crop-associated pathovars. Efficiency-of-plating assays quantitatively supported these host-range patterns. Plaque morphology and transmission electron microscopy demonstrated icosahedral capsids with short, non-contractile tails consistent with tailed dsDNA bacteriophages of the class Caudoviricetes, with vB_PsyP_Mission producing halo-associated plaques consistent with predicted extracellular polysaccharide-modifying proteins. Genomic analyses revealed compact (~ 40 kb) genomes lacking integrases, tRNAs, lysogeny-associated genes, and known virulence or antimicrobial resistance determinants. Comparative genomics showed that vB_PsyP_Plaza and vB_PsyP_Mission likely represent novel isolates within previously described species-level groups, whereas vB_PsyP_Mobley is more genomically divergent. Collectively, these results define the phenotypic and genomic characteristics of three related but distinguishable Pseudomonas phages and provide a basis for future in planta evaluation of their potential utility against almond bacterial blast.

Black fungi are among the most stress-resistant organisms known, yet the genetic and ecological foundations of their extraordinary resilience remain poorly understood. This study explores the adaptation strategies of the melanised fungus Elasticomyces elasticus by integrating genomic and ecological data. To uncover the mechanisms of adaptation, we combined whole-genome sequencing, functional annotation, environmental metadata, and large-scale soil metabarcoding analyses. Phylogenomic approaches were employed to delineate evolutionary lineages and assess ploidy levels. The results revealed that the global distribution of Elasticomyces phylotypes is primarily influenced by temperature, UV radiation, and soil organic carbon, suggesting that different phylotypes have evolved heterogeneous strategies for stress resistance. Comparative genomic analyses identified a set of 'sentinel pathways,' notably glutathione metabolism and nucleotide biosynthesis, which were enriched in strains inhabiting the most extreme environments and showed significant correlations with abiotic stressors such as aridity and UV exposure. Furthermore, phylogenomic reconstructions uncovered two independent diploid lineages associated with the harshest environments, pointing to diploidisation as a potential adaptive mechanism to cope with multiple stressors. Overall, the integration of genomic and ecological perspectives provides new insights into how black fungi persist at the edge of habitability. The study highlights specific pathways and genomic traits that underpin resilience to extreme conditions, offering implications that extend beyond terrestrial ecology.

The small interfering RNA (siRNA) pathway directs broad-spectrum antiviral defense through RNA silencing so that virulent infection requires efficient suppression of the defense mechanism. Here, we show that strigolactone (SL) hormone signaling promotes antiviral silencing in rice plants by transcriptional activation of RNA-dependent RNA polymerase 1 (RDR1) and RDR6. We demonstrate that protein P3 of the rice grassy stunt virus (RGSV) blocks SL signaling by directly sequestering the receptor DWARF14 from DWARF3. Structural and functional analyses of the P3-DWARF14 complex reveal that the aspartic acid at position 102 (D102) of DWARF14 is essential for the P3 interaction but not for SL perception. Notably, a single D102N substitution of DWARF14, introduced into two rice cultivars by cytosine base editing (CBE) confers resistance against RGSV by blocking viral suppression of SL signaling-dependent antiviral silencing. Our findings establish a transgene-free strategy for engineering disease resistance by precise genome editing of the SL receptor to escape pathogen suppression of the endogenous defense pathway.

Gene expression patterns are determined to a large extent by transcription factor (TF) binding to noncoding regulatory regions in the genome. However, gene expression cannot yet be systematically predicted from genome sequences, in part because nonfunctional matches to the sequence patterns (motifs) recognized by TFs occur frequently throughout the genome. Large-scale functional genomics data for many TFs has enabled characterization of regulatory networks in experimentally accessible cells such as budding yeast. Beyond yeast, fungi are important industrial organisms and pathogens, but large-scale functional data is only sporadically available. Uncharacterized regulatory networks control key pathways and gene expression programs associated with fungal phenotypes. Here, we explore a sequence-only approach to inferring regulatory networks by leveraging the 100s of genomes now available for many clades of fungi. We use gene orthology as the learning signal to infer interpretable, TF motif-based representations of noncoding regulatory regions. Using these representations to identify conserved signals for motifs, comparative genomics can be scaled to evolutionary comparisons where sequence similarity cannot be detected. We show that similarity of these conserved motif signals predicts gene expression and regulation better than using experimental data, and that we can infer known and novel regulatory connections in diverse fungi. Our new predictions include a pathway for recombination in Candida albicans and pathways for mating and an RNAi immune response in Neurospora. Taken together, our results indicate that specific hypotheses about transcriptional regulation in fungi can be obtained for many genes from genome sequence analysis alone.

Wildfires significantly alter soil carbon (C) and nitrogen (N), reducing microbial richness and biomass, while selecting for "fire-loving" pyrophilous microbes that drive postfire nutrient cycling. However, the genomic strategies and functional trade-offs (balancing gains in one trait with costs in another) underlying the traits that enable pyrophilous microbes to survive and thrive postfire are virtually unknown. We hypothesized that pyrophilous fungi employ specialized genomic adaptations for C and N cycling, with evolutionary trade-offs between traits governing aromatic C degradation, N acquisition pathways, and rapid growth. To test these hypotheses, we performed complementary comparative genomics, transcriptomics after pyrogenic organic matter amendment, and growth rate bioassays for 18 pyrophilous fungi from five Ascomycota (Eurotiales, Pleosporales, Sordariales, Coniochaetales, and Pezizales) and three Basidiomycota (Agaricales, Holtermanniales, and Geminibasidiales) orders isolated from burned soils. We found a dramatic trait trade-off between fast growth and number of genes responsible for aromatic C degradation, implying burned environments select for metabolically costly genes despite their evolutionary cost. We used the comparative genomics framework to evaluate genomic signatures of evolution and found that either gene duplication and somatic mutation, or recombination via sexual reproduction, were the primary drivers of fungal genomic variation in aromatic C degradation and N acquisition genes. Finally, we identified cross-kingdom bacterial to fungal horizontal gene transfer (HGT) as a secondary strategy producing novel aromatic C degradation genes. Overall, we found that trait trade-offs and genome evolutionary strategies are key drivers that may predict the persistence and contribution of pyrophilous fungi to global C and N cycling.

Correction to: Nature Microbiologyhttps://doi.org/10.1038/s41564-025-02116-2, published online 26 September 2025. In the version of this article initially published, in the first paragraph of the “Survey on data reuse” section, a note on participant consent, confidentiality and institutional review was missing and has now been inserted in the HTML and PDF versions of the article.