Microbiology and Plant Pathology

Viroids occur in plants as swarms of sequence variants clustered around a dominant variant, leading to adoption of the term quasispecies to describe the viroid population in an individual host. The composition of the quasispecies can potentially change according to the age of the infection, the position of the leaf or branch in the canopy, and the host species. The primary aim of this study was to investigate the quasispecies concept for citrus viroid VII (CVd-VII), a recently discovered member of the family Pospiviroidae. Three experiments were conducted to determine factors affecting viroid variability (i) within different tissues of a lemon plant, (ii) among different plants of the same species (citron), and (iii) among different species and hybrids of citrus. Using two primer sets to produce amplicons for high-throughput sequencing, viroid population profiles were generated for each sample. The number of variants that were identified with both primer sets ranged from 2 to 13 per sample, and each sample comprised 1 to 4 major (> 10% sample) variants. The composition of variants differed in samples from different plants and among tissue types of a single plant. Single-nucleotide polymorphisms (SNPs), mostly in the form of substitutions, were the primary source of variation; in this study, SNPs were observed in approximately 10% of the viroid genome. The results of the three experiments indicate that CVd-VII follows the quasispecies model as reported for other viroids and that variability occurs in viroid populations in different tissue types and host species.

UNLABELLED: Heterotrimeric G protein signaling pathways control growth and development in eukaryotes. In the multicellular fungus Neurospora crassa, the guanine nucleotide exchange factor RIC8 regulates heterotrimeric Gα subunits. In this study, we used RNAseq and liquid chromatography-mass spectrometry (LC-MS) to profile the transcriptomes and metabolomes of N. crassa wild type, the Gα subunit mutants Δgna-1 and Δgna-3, and Δric8 strains. These strains exhibit defects in growth and asexual development (conidiation), with wild-type and Δgna-1 mutants producing hyphae in submerged cultures, while Δgna-3 and Δric8 mutants develop conidiophores, particularly in the Δric8 mutant. RNAseq analysis showed that the Δgna-1 mutant possesses 159 mis-regulated genes, while Δgna-3 and Δric8 strains have more than 1,000 each. Many of the mis-regulated genes are involved in energy homeostasis, conidiation, or metabolism. LC-MS revealed changes in levels of primary metabolites in the mutants, with several arginine metabolic intermediates impacted in Δric8 strains. The differences in metabolite levels could not be fully explained by the expression or activity of pathway enzymes. However, transcript levels for two predicted vacuolar arginine transporters were affected in Δric8 mutants. Analysis of arginine and ornithine levels in transporter mutants yielded support for altered compartmentation of arginine and ornithine between the cytosol and vacuole in Δric8 strains. Furthermore, we validated previous reports that arginine and ornithine levels are low in wild-type conidia. Our results suggest that RIC8 regulates asexual sporulation in N. crassa at least in part through altered expression of vacuolar transporter genes and the resultant mis-compartmentation of arginine and ornithine. IMPORTANCE: Resistance to inhibitors of cholinesterase-8 (RIC8) is an important regulator of heterotrimeric Gα proteins in eukaryotes. In the filamentous fungus Neurospora crassa, mutants lacking ric8 undergo inappropriate asexual development (macroconidiation) during submerged growth. Our work identifies a role for RIC8 in regulating expression of transporter genes that retain arginine and ornithine in the vacuole (equivalent of the animal lysosome) and relates this function to the developmental defect. Arginine is a critical cellular metabolite, both as an amino acid for protein synthesis and as a precursor for an array of compounds, including proline, ornithine, citrulline, polyamines, creatine phosphate, and nitric oxide. These results have broad relevance to human physiology and disease, as arginine modulates immune, vascular, hormonal, and other functions in humans.

ABSTRACTHistoplasmosis is an endemic mycosis in North America frequently reported along the Ohio and Mississippi River Valleys, although autochthonous cases occur in non-endemic areas. In the United States, the disease is provoked by two genetically distinct clades of Histoplasma capsulatum sensu lato, Histoplasma mississippiense (Nam1) and H. ohiense (Nam2). To bridge the molecular epidemiological gap, we genotyped 93 Histoplasma isolates (62 novel genomes) including clinical, environmental, and veterinarian samples from a broader geographical range by whole-genome sequencing, followed by evolutionary and species niche modelling analyses. We show that histoplasmosis is caused by two major lineages, H. ohiense and H. mississippiense; with sporadic cases caused by H. suramericanum in California and Texas. While H. ohiense is prevalent in eastern states, H. mississipiense was found to be prevalent in the central and western portions of the United States, but also geographically overlapping in some areas suggesting that these species might co-occur. Species Niche Modelling revealed that H. ohiense thrives in places with warmer and drier conditions, while H. mississippiense is endemic to areas with cooler temperatures and more precipitation. In addition, we predicted multiple areas of secondary contact zones where the two species co-occur, potentially facilitating gene exchange and hybridization. This study provides the most comprehensive understanding of the genomic epidemiology of histoplasmosis in the USA and lays a blueprint for the study of invasive fungal diseases.

UNLABELLED: Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron. Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron. Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide. IMPORTANCE: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.

Alzheimer's disease (AD) and Alzheimer's disease-related dementias (ADRDs) are broad-impact multifactorial neurodegenerative diseases. Their complexity presents unique challenges for developing effective therapies. This review highlights research presented at the 2024 Society for Neuroscience meeting which emphasized the gut microbiome's role in AD pathogenesis by influencing brain function and neurodegeneration through the microbiota–gut–brain axis. This emerging evidence underscores the potential for targeting the gut microbiota to treat AD/ADRD.

Insect herbivores frequently cospeciate with symbionts that enable them to survive on nutritionally unbalanced diets. While ancient symbiont gain and loss events have been pivotal for insect diversification and feeding niche specialization, evidence of recent events is scarce. We examine the recent loss of nutritional symbionts (in as little as 1 MY) in sap-feeding Pariaconus, an endemic Hawaiian insect genus that has undergone adaptive radiation, evolving various galling and free-living ecologies on a single host-plant species, Metrosideros polymorpha within the last ∼5 MY. Using 16S rRNA sequencing, we investigated the bacterial microbiomes of 19 Pariaconus species and identified distinct symbiont profiles associated with specific host-plant ecologies. Phylogenetic analyses and metagenomic reconstructions revealed significant differences in microbial diversity and functions among psyllids with different host-plant ecologies. Within a few millions of years, Pariaconus species convergently evolved the closed-gall habit twice. This shift to enclosed galls coincided with the loss of the Morganella-like symbiont that provides the essential amino acid arginine to free-living and open-gall sister species. After the Pariaconus lineage left Kauai and colonized younger islands, both open- and closed-gall species lost the Dickeya-like symbiont. This symbiont is crucial for synthesizing essential amino acids (phenylalanine, tyrosine, and lysine) as well as B vitamins in free-living species. The recurrent loss of these symbionts in galling species reinforces evidence that galls are nutrient sinks and, combined with the rapidity of the evolutionary timeline, highlights the dynamic role of insect-symbiont relationships during the diversification of feeding ecologies. We propose new Candidatus names for the novel Morganella-like and Dickeya-like symbionts.

BACKGROUND: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. RESULTS: Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. CONCLUSIONS: We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

Endofungal Mycetohabitans (formerly Burkholderia) 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 from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-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 two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain 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 nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. 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.

We conducted a research campaign in a neotropical rainforest in Costa Rica throughout the drought phase of an El-Nino Southern Oscillation event to determine microbial community dynamics and soil C fluxes. Our study included nests of the leafcutter ant Atta cephalotes, as soil disturbances made by these ecosystem engineers may influence microbial drought response. Drought decreased the diversity of microbes and the abundance of core microbiome taxa, including Verrucomicrobial bacteria and Sordariomycete fungi. Despite initial responses of decreasing diversity and altered composition, 6 months post-drought the microbiomes were similar to pre-drought conditions, demonstrating the resilience of soil microbial communities to drought events. A. cephalotes nests altered fungal composition in the surrounding soil, and reduced both fungal mortality and growth of Acidobacteria post-drought. Drought increased CH4 consumption in soils due to lower soil moisture, and A. cephalotes nests decrease the variability of CH4 emissions in some soil types. CH4 emissions were tracked by the abundance of methanotrophic bacteria and fungal composition. These results characterize the microbiome of tropical soils across both time and space during drought and provide evidence for the importance of leafcutter ant nests in shaping soil microbiomes and enhancing microbial resilience during climatic perturbations.