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ABSTRACT The fungusAspergillus melleusis an important biosynthesis host for varied commercial applications. Gene annotation of a previously published genome produced 12,841 protein-coding genes and identified 102 biosynthetic gene clusters. 

Abstract Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we introduce ALLEGRO, a combinatorial optimization algorithm designed to compose minimal, yet highly effective sgRNA libraries targeting thousands of species at the same time. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting multiple genes of interest for over 2000 species across the fungal kingdom. We experimentally validated sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, Yarrowia lipolytica, and Saccharomyces cerevisiae, confirming successful genome edits. Additionally, we employed a generalized Cas9–ribonucleoprotein delivery system to apply ALLEGRO’s sgRNA libraries to untested fungal genomes, such as Rhodotorula araucariae. Our experimental findings, together with cross-validation, demonstrate that ALLEGRO facilitates efficient CRISPR genome editing, enabling the development of universal sgRNA libraries applicable to entire taxonomic groups. 

Abstract The host microbiome is integral to metabolism, immune function, and pathogen resistance. Yet, reliance on relative abundance in microbiome studies introduces compositional biases that obscure ecological interpretation, while the absence of robust tools for absolute abundance quantification has limited biological discovery. Here, we apply absolute abundance profiling to uncover host-specific microbial patterns across herpetofauna orders that are masked in relative abundance data. Relative- and absolute abundance-derived bacterial and fungal microbiomes exhibit divergent profiles shaped by compositional bias and multifactorial effects. Absolute abundance identified key genera, Lactococcus, Parabacteroides, and Cetobacterium in salamanders, and Basidiobolus and Mortierella in lizards, turtles, snakes, and tortoises, that consistently emerged as core taxa, revealing host-associated patterns previously obscured by compositional constraints. In closely related Desmognathus species, where environmental and phylogenetic variation was minimized, absolute abundance enabled finer resolution of microbiome dynamics and significantly reduced false discovery rates. Absolute abundance-based network analyses further revealed distinct keystone taxa between the relative and absolute abundance datasets. Despite low redundancy, Basidiobolus exhibited high network betweenness, efficiency, and degree, suggesting its role as a key connector between microbial modules and a contributor to overall network robustness. This predicted structural role aligns with Burt’s structural hole theory, which suggests that nodes linking otherwise disconnected modules occupy influential network positions. These findings underscore the value of absolute abundance in resolving microbial dynamics and supporting meaningful interpretation of host-microbiome associations. This advance is made possible by DspikeIn, a flexible wet-lab and computational framework that enhances ecological resolution and cross-study comparability. 

ABSTRACT The fungal genusNeonectriacontains many phytopathogenic species currently impacting forests and fruit trees worldwide. Despite their importance, a majority ofNeonectriaspp. lack sufficient genomic resources to resolve suspected cryptic species. Here, we report draft genomes and assemblies forNeonectria magnoliaeNRRL 64651 andNeonectria puniceaNRRL 64653. 

Over the last billion years, the fungal kingdom has diversified to over 10 million species with over 95% still undescribed. Beyond the well-known macroscopic mushrooms and microscopic yeast, fungi are heterotrophs that feed on almost any organic carbon, recycling nutrients through decay of dead plants and animals and sequestering carbon into the Earth’s ecosystems. Human directed applications of fungi extend from yeast responsible for leavened bread, alcoholic beverages, and biofuels to pharmaceuticals, including antibiotics and psychoactive compounds. Conversely fungal infections pose risks to ecosystems ranging from crops to wildlife to humans, driven in part by human and animal movement and potentially accelerating with climate change. Genomic surveys are expanding our knowledge of the true biodiversity of the fungal kingdom while genome editing tools make it possible to imagine harnessing these organisms to fuel the bioeconomy. Here, we explore the fungal threats facing civilization and opportunities to harness fungi to combat these threats. 

EndofungalMycetohabitans(formerlyBurkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus,Rhizopus microsporus. The one known secreted effector family fromMycetohabitansconsists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenicXanthomonasandRalstoniaspp. to activate host genes that promote disease. These ‘BurkholderiaTAL-like (Btl)’ proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of twoMycetohabitansstrains revealed that the single Btl in oneMycetohabitans endofungorumstrain enhances fungal membrane stress tolerance, while others in aMycetohabitans rhizoxinicastrain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nineMycetohabitansspp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal–bacterial metagenomes. We show thatbtlgenes are present across diverseMycetohabitansstrains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested thatMycetohabitansmight contain more species than previously recognized. Within our data set, Btl proteins were more conserved acrossM. rhizoxinicastrains than acrossM. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial–fungal symbioses in myriad ways. 

Every fungal cell is encapsulated in a cell wall, essential for cell viability, morphogenesis, and pathogenesis. Most knowledge of the cell wall composition in fungi has focused on ascomycetes, especially human pathogens, but considerably less is known about early divergent fungal groups, such as species in the Zoopagomycota and Mucoromycota phyla. To shed light on evolutionary changes in the fungal cell wall, we studied the monosaccharide composition of the cell wall of 18 species including early diverging fungi and species in the Basidiomycota and Ascomycota phyla with a focus on those with pathogenic lifestyles and interactions with plants. Our data revealed that chitin is the most characteristic component of the fungal cell wall, and was found to be in a higher proportion in the early divergent groups. The Mucoromycota species possess few glucans, but instead have other monosaccharides such as fucose and glucuronic acid that are almost exclusively found in their cell walls. Additionally, we observed that hexoses (glucose, mannose and galactose) accumulate in much higher proportions in species belonging to Dikarya. Our data demonstrate a clear relationship between phylogenetic position and fungal cell wall carbohydrate composition and lay the foundation for a better understanding of their evolution and their role in plant interactions. 

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractableEntomophthora muscaeisolate that infects fruit flies. OurE. muscaeassembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find thatE. muscaeand its closest allies possess genes that are likely homologs to the blue-light sensorwhite-collar 1, aNeurospora crassagene that has a well-established role in maintaining circadian rhythms. We uncover evidence thatE. muscaediverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent withE. muscae’s species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within theE. muscaespecies complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi. 

This work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.