Frontiers in Fungal Biology

Fungal genomics has expanded rapidly over the past 30 years, and recently the pace and breath has further quickened for many taxa, although many taxonomic gaps persist. With three decades of rapid growth, fungal genomics now merits a re-examination of its history, progress, and unresolved taxonomic gaps. Here, we review the development of fungal genomics from early efforts such as the Fungal Genome Initiative to current progress driven by third-generation long-read sequencing. We have compiled and summarised publicly available fungal genomes to highlight trends in assembly quality, adoption of long-read technologies, and taxonomic representation. Notably, substantial phylogenetic gaps remain, particularly outside Dikarya, and significant challenges persist for unculturable taxa. This review identifies priorities for the fungal community, including: (1) coordinated efforts to close major taxonomic gaps across the fungal tree of life; (2) improved repository metrics to facilitate identification of high-quality assemblies; and (3) improved and standardised genome annotation which is lacking for most assemblies. Together, these steps will support the development of reliable genomic resources that capture the full breadth of diversity across the fungal kingdom, generating foundational data for comparative genomics, evolutionary biology, functional studies, genetic studies and applied research.

The fungal cell wall is populated by proteins (CWPs), mostly uncharacterized, that show an atypical evolutionary behavior. Most CWPs are glycosylphosphatidylinositol(GPI)-proteins, followed by proteins with internal repeats (PIR), and non-covalently attached proteins that harbor carbohydrate binding domains (CBM). Several structural CWPs are initially bound to the same wall carbohydrates, but either covalently or non-covalently. However, it is not clear whether they work in the same way and if they are subjected to the same evolutionary constraints. In Neurospora crassa, CWPs ACW-1 (NCU08936) and NCW-3 (NCU07817) bind to {beta}-1,3-glucans through a GPI anchor or a predicted CBM-52 domain, respectively. Here, the evolutionary trajectories and functional roles of both CWPs were analyzed. Both proteins localized primarily to distal septa and hyphal wall surfaces. Morphological characterization and stress cell wall assays suggested that both proteins contribute to cell wall integrity, but NCW-3 likely plays a more prominent role. ACW-1 and NCW-3 homologues were predominantly identified in Ascomycota. ACW-1 displayed a broader distribution than NCW-3, whose homologues were largely restricted to Sordariales. Despite these differences, both protein families exhibited similar moderate global conservation and signatures of purifying selection within shared taxa. Nevertheless, a divergence gradient was identified within ACW-1, related to its tandem leucine-rich repeat (LRR) regions. A similar local accumulation of evolutionary change was not observed within NCW-3. These findings suggested that distinct CWP architectures can accommodate different patterns of sequence diversification despite sharing similar global evolutionary change.

Gene-by-environment (GxE) interactions play a major role in shaping both phenotypic and molecular variation, with important implications for human health and disease. In this study, we used the Doxycycline (Dox) regulated, tetracycline-responsive (Tet-Off) promoter system to sequentially reduce or titrate gene expression levels of the essential yeast transcription factor Repressor Activator Protein 1 (RAP1) similar to a hypomorph allele series, across three distinct environments: Yeast Peptone Dextrose (YPD) media, YPD media with Heat Shock (HS), and Yeast Peptone Acetate (YPAC) media. We then performed RNA sequencing (RNA Seq) to assess global transcriptional responses to RAP1 reduction in these different growth environments. Our analysis first focused on the independent effects of varying RAP1 expression levels within and across environments. We then explored GxE interactions, revealing a subset of genes with significant consequences of reduced levels of RAP1 and environment-specific expression patterns. Notably, many genes exhibited opposite effects of RAP1 titration on gene expression when yeast were grown in YPAC media compared to YPD media and/or HS, suggesting environment-dependent regulatory architecture. This design reveals how cells integrate internal transcriptional and regulatory changes with external environmental cues, providing a deeper view of GxE architecture. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified co-regulated gene modules, and by combining this with transcription factor motif enrichment tests, our study identified candidate regulators driving their dynamics. Our findings demonstrate that gene regulatory networks can vary dramatically depending on the environmental context an organism experiences, which can then influence the specific phenotypes produced by a particular genetic perturbation. This illustrates the complexity of genotype-environment interactions and the importance of studying gene function in multiple environments to gain a truly comprehensive understanding of a genes sometimes numerous and diverse functions.

The fungal crop pathogen Blumeria hordei, causal agent of powdery mildew on barley, presents life-history and epidemiological characteristics, as well as and selective pressures due to modern agriculture leading to expected sweepstakes reproduction, that is highly skewed offspring distributions. Using genome-wide polymorphism data and population genomics inferences, we aim to 1) infer the past demographic history and the strength of sweepstakes reproduction in B. hordei, and 2) quantify the contributions of these selective and neutral processes in the genome. An new inference method based on Neural Posterior Estimation and diversity and linkage disequilibrium statistics was developed and tested on simulated and B. hordei genomic data. We confirm that B. hordei exhibits a moderate sweepstakes reproduction (-parameter of 1.6). We highlight that the Site Frequency Spectrum (SFS) appears sensitive to the joint occurrence of sweepstakes and recent demographic changes, which may caution on the reliability of the SFS to infer sweepstakes reproduction. We then scan the genome for selective sweeps, adjusting the significance thresholds of the methods for demographic history and sweepstakes reproduction, thereby yielding a counterintuitive result. When conditioning the significance threshold for sweep detection on simulations under sweepstakes and demography, a very large number of putatively selected regions is found (11.6% of the genome). We suggest that sweepstakes reproduction in B. hordei is due to 1) neutrality (clonal/sexual phases and Boom-and-Bust cycles) generating a genome-wide level of background noise in the coalescent genealogies, and 2) selective sweepstakes due to pervasive positive selection. Our findings have important implications for both population genomic methodology and our understanding of pathogen evolution.

Cell size in a proliferating cell population generally varies over a limited range ([~]2-4-fold). Within such populations, organelle content increases with cell size maintaining a relatively constant organelle density (amount per cell volume). However, cells of different types can differ greatly in cell size as well as in organelle composition. In such cases, it is often unclear to what degree, if any, the differences in organelle composition are due to the difference in cell size. In principle, this issue could be resolved by examining situations where a proliferating population of cells of the same cell type exhibit much greater size variation. Here we characterize how organelle content scales with cell volume in the polymorphic fungus, A. pullulans, whose proliferating cells span a [~]100-fold size range. We find that mitochondria and ER content increases in proportion to cell volume, while this is not the case for vacuoles and peroxisomes. Thus, organelle composition is affected by cell size in this system.

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

Organisms cope with environmental changes by modifying gene expression. To understand how regulatory networks controlling expression plasticity evolve, we analyzed RNAseq data from Saccharomyces cerevisiae, Saccharomyces paradoxus, and their F1 hybrids at multiple timepoints after transferring cells from standard laboratory conditions to five environments (low phosphorus, low nitrogen, hydroxyurea shock, heat stress, and cold stress) and during the diauxic shift. In each of the six datasets, we identified genes that changed expression following the transition to the new environment and used hierarchical clustering to identify genes that increased or decreased in expression. We then compared these classifications between orthologs to identify genes with divergent plasticity. For some genes, plasticity was more extreme in one species than the other, and for others, expression of orthologs changed in opposite directions when acclimating to the same environment. Most cases of plasticity divergence were seen only in one environment and were attributable primarily to trans-regulatory divergence. Using environment-specific regulatory networks inferred from data in Yeastract, we found that divergent plasticity of environment-specific transcription factors generally did not predict divergent plasticity of their target genes. We also found that, as a group, genes with conserved plasticity tended to have more regulatory interactions than genes with divergent plasticity. Interesting patterns of expression divergence were also observed for five transcription factors in the pleiotropic drug resistance network and their target genes that might contribute to phenotypic divergence. Together, these findings show how environment-specific trans-regulatory divergence and combinatorial gene regulation shape the evolution of expression plasticity.

Colletotrichum siamense is a predominant causal agent of anthracnose in rubber tree and numerous economically important crops, causing severe yield losses worldwide. Conidial germination represents a critical early step for successful infection, while the high-osmolarity glycerol (HOG) MAPK pathway and ergosterol biosynthesis individually govern fungal development, stress adaptation and fungicide responses. However, the molecular crosstalk between these two modules remains largely elusive in phytopathogenic fungi. Here, we identified CsErg5B, a sterol C-22 desaturase homolog, as a direct target of the HOG- regulated transcription factor CsAtf1 in C. siamense. CsErg5B was indispensable for ergosterol biosynthesis, conidial germination, appressorium formation, and full virulence. The {Delta}CsErg5B mutant showed increased conidiation but severely impaired germination, and exhibited elevated resistance to fludioxonil while hypersensitivity to azole fungicides. Epistasis analysis using the {Delta}CsErg5B/{Delta}CsCyp51G1 double mutant - where CsCyp51G1 serves as another downstream target of CsAtf1 - revealed that CsErg5B functions as the predominant downstream effector of CsAtf1 in modulating conidial development and fludioxonil sensitivity. Furthermore, overexpression of CsErg5B significantly rescued the defects in conidial germination and fludioxonil sensitivity in both {Delta}CsAtf1 and {Delta}CsPbs2 mutants. Taken together, our findings uncover a HOG MAPK - CsAtf1 - CsErg5B regulatory axis that connects HOG MAPK signaling to ergosterol homeostasis, thereby governing conidial germination and fungicide sensitivity in C. siamense. This study provides novel insights into the regulatory network underlying fungal development and fungicide response, and offers promising molecular targets for the integrated management of plant anthracnose.

Genetically identical cells grown in the same environment show variation in gene expression known as expression noise. Expression noise can be heritable and impact fitness, making it subject to natural selection. Increasing expression noise for the Saccharomyces cerevisiae TDH3 gene was shown to be beneficial in glucose-based media when mean TDH3 expression was far from the fitness optimum but deleterious when it was close to this optimum. Here, we show that growth on different carbon sources alters the effects of new mutations on TDH3 expression noise and examine the fitness effects of changing expression noise. In galactose-based media, we observed the same relationship between expression noise and fitness seen in glucose-based media, but in glycerol- and ethanol-based media, we observed the opposite relationship or no significant relationship, respectively. Using simulations of single-cell organisms, we found that these differences were most likely explained by environment-specific relationships between gene expression and fitness. We also found that, far from the optimum, the fitness effects of noise were greatest when expression was highly heritable between mother and daughter cells. The empirical observations and simulations reported in this study show how environments influence both the production of expression noise and its impacts on fitness.

Yeast responding to acute stress reallocate cellular resources, in part via the Environmental Stress Response (ESR) that induces stress-defense genes while repressing ribosome-biogenesis and growth genes. The purpose and regulation of coordinated induction and repression is incompletely understood, but both responses are influenced by ESR transcription factors Msn2 and Msn4 (Msn2/4). Here we used single-cell microscopy and transcriptomic analysis to investigate the role of upstream regulator Pde2 in ESR regulation and post-stress fitness. Loss of PDE2 weakened and shortened Msn2 activation following salt stress and produced muted induction of Msn2/4 targets, similar to a msn2{triangleup}msn4{triangleup} strain. In contrast, Pde2 had at most a minor impact on ESR repressor Dot6, yet was important for repression of its targets beyond Msn2/4 influence. Consistent with our recent resource-reallocation model, pde2{triangleup} cells had normal or faster post-stress growth rates, despite weaker activation of the ESR. We discuss implications for ESR regulation and function.

Bacterial spot is a consistent threat to global tomato and pepper productions; however, Ohios fresh market production currently lacks the updated surveillance data necessary to provide accurate management solutions. While traditional diagnostics focus on identification of a single causal agent, shotgun metagenomic sequencing (MGS) offers a comprehensive view of the infection court. An assignment-first MGS workflow was developed and validated in this study, utilizing Kraken2 databases to extract Xanthomonas species associated with bacterial spot and to characterize the microbial communities of bacterial spot in Ohio production systems. Through in silico spiking experiments, thresholds were established for bacterial spot identification. Species and pathovar identification via average nucleotide identity (ANI) remained accurate at abundance as low as 0.1%. A minimum of 2% Xanthomonas reads were required for high genome completeness (BUSCO >90%) and 3% for reliable type III secretion system (T3SS) effector profiling. Analysis of 63 samples from fresh-market production fields identified Xanthomonas hortorum pv. gardneri, Xanthomonas euvesicatoria pv. euvesicatoria, and Xanthomonas arboricola residing in symptomatic samples, alongside other taxa including Pseudomonas and Stenotrophomonas. Phylogenetic comparisons of metagenome-assembled genomes (MAGs) were comparable to whole genome sequences (WGS) from the same samples, supporting the reliability of culture-independent diagnostics. These results provide a robust framework for utilizing metagenomics as a diagnostic tool, expanding our knowledge of bacterial spot population structure in Ohio, and uncovering the bacterial communities associated with bacterial spot.

Aphanomyces euteiches, the causative agent of Aphanomyces root rot (ARR), is of major concern for pea and other legume crops globally. This oomycete pathogen causes substantial decreases in crop yields, is unaffected by most fungicides, and persists in the soil for many years via its resilient oospores. Given the significance of pea crops in sustainable agriculture, namely the ability to fix nitrogen and act as a sustainable protein source, solutions to ARR are of high importance. We used RNA-seq in a novel strain of Pseudomonas donghuensis to identify two biosynthetic gene clusters under GacA/S control that are involved in producing bioactive molecules capable of inhibiting A. euteiches. Based on similarity to other reported clusters in Pseudomonas, the first is predicted to encode for a pseudoiodinine compound, while the second is predicted to produce the siderophore 7-hydroxytropolone. Individual knockouts of each cluster showed loss of inhibitory action of P. donghuensis NRC29 against A, euteiches in vivo. This is the first report highlighting the potential of P. donghuensis and the products of the two identified biosynthetic pathways as biocontrol agents for A. euteiches. Further investigations into the efficacy of P. donghuensis NRC29 and its metabolites in inhibiting A. euteiches in field trials will be of high value in developing sustainable strategies for ARR mitigation. ImportanceModern fungicidal treatments for control of root rot in pulse crops are ineffective for control of A. euteiches, leaving limited strategies for management of A. euteiches infected fields. We describe a novel P. donghuensis strain with potential for biocontrol against this persistent pathogen. Given the economic value of peas and other pulses globally, further work into harnessing the bioactive metabolites produced by this strain into a practical in-field treatment will be valuable.

Magnaporthe oryzae, the rice blast fungus, plays a role as a model organism for molecular plant-microbe interaction research. Studies on the pathogenic mechanism of this fungus revealed many genes involved in signaling pathways. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological processes during the pathogenesis of M. oryzae. Identifying the genome-wide protein-protein interaction (PPI) network is one of the omics-level approaches, which helps to understand signaling and regulatory pathways. However, existing biological network resources of M. oryzae are not sufficient to decipher pathogenesis mechanisms due to the abundance of false positives/negatives. In this study, a reliable PPI network database of M. oryzae, MagNet, was constructed with three methods, including homology-based Interolog search, co-expression network construction, and domain-domain interaction (DDI)-based prediction. With three approaches altogether, the pan-network with 5,600,976 interactions was generated, including 217,531 highly confident interactions supported by all three methods. Experimental data on M. oryzae PPIs supported that our PPI network can predict PPIs with higher accuracy compared to the previously constructed databases. MagNet would provide integrated biological network data, which can help to understand the molecular mechanisms of the rice blast fungus. The PPI data can be accessed via https:/magnet.scnu.ac.kr.

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

O_LIHost specialization is a major driver of genetic structure in fungal plant pathogens, but it remains unclear whether specialization on different cereal hosts prevents sexual recombination when mating-type compatibility is retained. We addressed this question in stripe rust, caused by Puccinia striiformis, by crossing wheat-adapted P. striiformis f. sp. tritici and barley-adapted P. striiformis f. sp. hordei, two divergent host-adapted forms that share common barberry (Berberis vulgaris) as a sexual host. C_LIO_LIControlled reciprocal crosses on barberry produced 18 aeciospore-derived progeny, demonstrating that wheat- and barley-adapted Puccinia striiformis can undergo sexual recombination despite strong host specialization during asexual infection. Chromosome-scale parental assemblies placed the homeodomain (HD) mating-type locus, containing bW-HD1 and bE-HD2, on chromosome 2 and the pheromone receptor (PR) mating-type locus, containing STE3 and mfa genes, on chromosome 6. HD restriction genotyping showed biparental inheritance in all progeny, with each progeny carrying one HD haplotype from each parent. Together with conservation of PR-associated coding sequences and amplification of STE3-associated markers in progeny, these results are consistent with retention of tetrapolar mating across the two host-adapted lineages. C_LIO_LIHost interaction phenotypes were assessed across wheat and barley differentials, near-isogenic lines and wild relatives. The parental isolates retained contrasting wheat- and barley-restricted profiles, whereas progeny did not reproduce either parental virulence profile, but instead showed recombinant infection patterns, including compatibility with both wheat and barley genotypes. C_LIO_LIThese findings indicate that host specialization in Puccinia striiformis does not necessarily prevent sexual compatibility on a shared alternate host. Together with retention of tetrapolar mating, alternate-host sexual reproduction may provide a route for genetic exchange between host-specialized pathogen populations, enabling recombination to generate new combinations of host-interaction traits when divergent pathogen lineages mate on a shared alternate host. C_LI

Autophagy is a critical cellular process regulated by Atg proteins, yet its modulation by redox-active compounds and iron remains incompletely understood. Here, we investigated the effects of dithiothreitol (DTT) and iron on autophagy and on AaAtg4 protease activity in the plant-pathogenic fungus Alternaria alternata. Using GFP-tagged AaAtg8, fluorescence microscopy and proteolysis assays revealed that DTT markedly enhanced autophagic vacuole formation and GFP release, indicating increased autophagic flux. Western blot analyses confirmed that DTT promoted AaAtg8 lipidation, while co-treatment with hydrogen peroxide (H2O2) suppressed this modification. AaAtg4 was constitutively active and could process AaAtg8 regardless of DTT supplementation, whereas moderate DTT concentrations elevated AaAtg4 protein abundance and phosphorylation. Bimolecular fluorescence complementation assays demonstrated that DTT, but not iron, facilitated AaAtg4-AaAtg8 interactions and vacuolar localization, whereas H2O2 counteracted these effects. Notably, combined DTT and H2O2 sustained autophagy at a low but stable level, suggesting a redox balance in autophagic regulation. Iron supplementation selectively destabilized AaAtg8 and modulated AaAtg4 phosphorylation in a concentration-dependent manner, without altering autophagy or protease activity. Collectively, these findings demonstrate that DTT enhances autophagy primarily by promoting AaAtg8 lipidation, AaAtg4 phosphorylation, and AaAtg4-AaAtg8 complex formation, while exerting minimal influence on AaAtg4 protease activity. In contrast, ion regulates autophagy flux through its effects on AaAtg4 phosphorylation and AaAtg8 stability, without significantly altering AaAtg4 protease activity, AaAtg8 lipidation, or AaAtg4-AaAtg8 interactions. Together, this work underscores the intricate interplay between redox signaling, nutrient cues, and autophagy regulation in A. alternata. IMPORTANCEThis study provides critical new insights into how redox-active compounds and iron modulate autophagy in the plant-pathogenic fungus Alternaria alternata, a pathogen of agricultural relevance. By dissecting the distinct roles of DTT, hydrogen peroxide, and iron in regulating AaAtg8 lipidation, AaAtg4 phosphorylation, and AaAtg4-AaAtg8 interactions, our findings reveal that autophagy is not simply a constitutive process but is finely tuned by redox balance and nutrient cues. This work advances the fundamental understanding of autophagy regulation in filamentous fungi, highlights the interplay between oxidative stress and protease activity, and establishes a framework for exploring how environmental factors shape fungal pathogenicity. Ultimately, these insights may inform novel strategies to mitigate crop fungal diseases by targeting autophagic pathways.

Brown rot wood-degrading fungi release carbon (C) from deadwood but leave behind a large fraction of C sequestered in lignin residues or as fungal metabolites. The strength of sequestration in these C residuals remains unclear, but proteobacteria-dominated bacterial communities have been implicated in metabolizing C from decay residues, possibly erasing the C sequestration potential assumed for brown rot. Here, we paired a model brown rot fungus (Rhodonia placenta) with a model Alphaproteobacterium (Rhodopseudomonas palustris) to track fungal release and bacterial utilization of C derived from decaying wood. We found that fungal decay products generated by R. placenta could be used by R. palustris for growth, and later decay stages contained more usable substrates than early stages. High performance liquid chromatography with mass spectrometry identified a range of aromatic and non-aromatic compounds in the fungal-decayed wood, but after 95 days of bacterial growth, R. palustris preferentially consumed non-aromatic acids over aromatic lignin monomers. Genes involved with aromatic compound degradation were unimportant for bacterial growth, and RNA sequencing revealed that aromatic compound degradation genes were repressed on decayed wood extract. Randomly barcoded transposon sequencing failed to identify a solitary catabolic pathway used by R. palustris, suggestive of substrate co-utilization, and surprisingly, showed that genes involved with copper toxicity were essential. Finally, we found that genes involved with biosynthesis of certain cofactors and amino acids were no longer essential on decayed wood extract, suggesting these nutrients were readily accessible. This study helps lay the foundation to understand potential bacterial-fungal interactions in decayed wood. Graphical abstractTo explore how brown rot fungi support and compete with bacterial partners in the wood decay environment, the model brown rot fungus Rhodonia placenta was used to degrade aspen wafers which were then infused into bacterial growth medium. By leveraging the range of molecular biology tools available for the model Alphaproteobacterium Rhodopseudomonas palustris, we discovered that R. palustris preferentially consumes short organic acids instead of aromatic lignin monomers which it would otherwise consume if provided in isolation. Additionally, R. palustris scavenged certain amino acids (AAs) and enzyme cofactors including methionine, biotin, and PLP from the decayed wood extract, highlighting these as key shared resources for bacterial-fungal partnerships. We found that R. placenta increased the concentration of certain metals (Cu and Al) inducing a metal stress response in R. palustris, indicating that metal toxicity could be an important mode of competition between fungi and bacteria in the wood decay environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/723453v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16f31fcorg.highwire.dtl.DTLVardef@13a9b34org.highwire.dtl.DTLVardef@a37dcforg.highwire.dtl.DTLVardef@198bf1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.

The biological conservation between fungi and mammals due to a common ancestor has made development of selective antifungal drugs a difficult challenge. Further complicating this situation is the selection of antifungal drug-resistant organisms during drug treatment. The pathogenic yeast Nakaseomyces glabratus (called here Candida glabrata) presents an especially challenging organism due to its tendency to frequently lose susceptibility to the major antifungal drug class the azoles. Additionally, C. glabrata develops resistance to echinocandin drugs, a second, more recently described antifungal agent at 10 times the rate of other organisms. Previous work has established that the sterol responsive transcriptional regulator Upc2A is a key determinant of azole susceptibility in C. glabrata and plays a role in echinocandin resistance. We used a biochemical approach to identify proteins that co-purified with Upc2A and identified the Ypk2 AGC kinase as an interacting protein. Strains lacking YPK2 exhibited increased susceptibility to fluconazole and the echinocandin caspofungin. A ypk2{Delta} strain failed to normally induce transcription of several ERG genes but exhibited normal induction of the CDR1 ATP-binding cassette transporter gene. Isogenic ypk2{Delta} strains were also highly susceptible to the three major classes of antifungal drugs, indicating that this kinase behaves as a multidrug susceptibility factor. RNA-seq analyses indicated that the transcriptional response to exposure is different for each drug and each response is differentially altered upon loss of Ypk2. Our data indicate that Ypk2 plays an important role in coordinating gene expression that impacts susceptibility to all major antifungal drug classes.

Escherichia coli couples the initiation of DNA replication with cell size by modulating the activity of the replication initiator protein DnaA. The activity of DnaA is regulated by both its interconversion between an active and inactive form and its titration on binding sites on the chromosome. Whereas its interconversion has been thoroughly studied, the extent to which DnaA titration can control replication initiation is poorly understood. Here, we describe the control of E. coli DNA replication via titration by modulating the expression of an always-active DnaA variant in four growth conditions. While we obtained stable cell cycles during slow growth, faster growth associated with overlapping replication forks led to replicative instability and DNA damage. Overall, our results provide insights into the limits of titration-based systems in the control of genome replication and their potential role in the evolutionary trajectory of E. coli. Finally, this study provides design principles for a simplified, titration-only regulatory mechanism for DNA replication in synthetic cells.