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Interactions between multiple global change stressors are a defining characteristic of the Anthropocene. Tree-associated pathogens are affecting forested ecosystems worldwide and occur in the context of increased frequency and intensity of extreme climate events such as heat waves, droughts, and floods. The effects of these events, along with subsequent changes in environmental conditions, on remaining and regenerating trees, are not well understood but crucial for the restoration and conservation of forested habitats. In this study, we investigate ash (Fraxinus excelsior) dieback in a temperate broadleaf woodland as a case study to explore the processes influencing non-infected trees during pathogen-induced mortality events. Utilising an experimental setup, we examine tree growth rates at different chronological stages of the disease, including naturally progressing ash dieback (4-5 years since disease outbreak), accelerated ash dieback where ash trees have been girdled (10-15 years), and negligible ash dieback (<20 % ash trees). During a year with typical climatic conditions (2021), soils in accelerated ash dieback plots remained saturated throughout the summer due to insufficient transpiration (57 % higher in the accelerated dieback plots), suggesting a significantly increased risk of summer run-off and floods. However, tree growth rates in these plots were not affected (t-test, t = -0.3 to 1.2, p > 0.05). Conversely, anomalously dry years, such as the 2022 summer drought, saw higher soil moisture in the accelerated ash dieback plots (t-test, t = 4.8, p < 0.01) acting as a buffer, resulting in normal tree growth during drought compared to greatly reduced growth in plots with weaker dieback. These findings emphasise the complex interactions between extreme climate events and pathogen outbreaks. Better understanding of the relationships between pathogens and hydrology on tree growth is imperative and detailed long-term studies on tree growth and hydrology will facilitate and improve mitigation strategies.

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Disease can act as a driving force in shaping genetic makeup across populations, even species, if the impacts influence a particularly sensitive part of their life cycles. White-nose disease is caused by a fungal pathogen infecting bats during hibernation. The mycosis has caused massive population declines of susceptible species in North America, particularly in the genus Myotis. However, Myotis bats appear to tolerate infection in Eurasia, where the fungal pathogen has co-evolved with its bat hosts for an extended period of time. Therefore, with susceptible and tolerant populations, the fungal disease provides a unique opportunity to tease apart factors contributing to tolerance at a genomic level to and gain an understanding of the evolution of non-harmful in host-parasite interactions. To investigate if the fungal disease has caused adaptation on a genomic level in Eurasian bat species, we adopted both whole-genome sequencing approaches and a literature search to compile a set of 300 genes from which to investigate signals of positive selection in genomes of 11 Eurasian bats at the codon-level. Our results indicate significant positive selection in 38 genes, many of which have a marked role in responses to infection. Our findings suggest that white-nose syndrome may have applied a significant selective pressure on Eurasian Myotis-bats in the past, which can contribute their survival in co-existence with the pathogen. Our findings provide an insight on the selective pressure pathogens afflict on their hosts using methodology that can be adapted to other host-pathogen study systems.

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Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host-pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a localizes to the cell surface plasma membrane and can efficiently traffic Cu-ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu-homeostasis pathways in P. destructans.

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The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans.

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The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca2+-signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca2+-concentration ([Ca2+]i) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca2+]i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca2+]i induced by C5a. Moreover, the MR-induced rise in Ca2+-influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.

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Fungal pathogens such as Candida albicans pose a significant threat to human health with limited treatment options available. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leverage a pooled functional genomic screening strategy to identify genes important for fitness of C. albicans in diverse conditions. We identify an essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrate that it encodes subunit 3 of replication factor A (Rfa3). Furthermore, we apply computational analyses to identify functionally coherent gene clusters and predict gene function. Through this approach, we predict the cell-cycle-associated function of C3_06880W, a previously uncharacterized gene required for fitness specifically at elevated temperatures, and follow-up assays confirm that C3_06880W encodes Iml3, a component of the C. albicans kinetochore with roles in virulence in vivo. Overall, this work reveals insights into the vulnerabilities of C. albicans.

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We report an annotated draft genome of Heterobasidion occidentale, a fungus (Basidiomycota, Agaricomycetes) that has pathogenic and saprophytic lifestyles. This fungus belongs to the H. annosum (Fr.) Bref. sensu lato species complex that comprises several root rot pathogens. Heterobasidion occidentale causes annosus root and butt rot primarily in true fir (Abies spp.) and spruce (Picea spp.) species throughout western North America.

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Pneumocystis species are respiratory fungal pathogens that cause life-threatening opportunistic infections in immunocompromised hosts. Pneumocystis typically evade pulmonary innate immunity but are efficiently eradicated by a functional adaptive immune response. FVB/NJ mice are unique in that they display protective alveolar macrophage-dependent innate immunity against Pneumocystis, and remain resistant to infection even in the absence of CD4+ T lymphocyte function. FVB/NJ alveolar macrophages (AMs) were found to display an M2-biased phenotype at baseline, which was potentiated after stimulation with Pneumocystis, suggesting that macrophage polarization may dictate the outcome of the Pneumocystis-macrophage interaction. To determine whether Stat6, a key global regulator of M2 polarization, was required for FVB/NJ innate immunity, FVB Stat6-/- mice were generated. FVB Stat6-deficient AMs were markedly impaired in their ability to polarize to an M2 phenotype when stimulated with Th2 cytokines. However, FVB Stat6-/- mice remained highly resistant to infection, indicating that Stat6 signaling is dispensable for innate FVB/NJ resistance. Despite the loss of Stat6 signaling, primary AMs from FVB Stat6-/- mice maintained baseline expression of M2 markers, and also strongly upregulated M2-associated genes following direct stimulation with Pneumocystis. Additional FVB/NJ knockout strains were generated, but only FVB MerTK-/- mice showed a marginally increased susceptibility to Pneumocystis infection. Together, these findings demonstrate that effective FVB/NJ innate immunity against Pneumocystis does not require Stat6 signaling and suggest that alternative pathways regulate M2 bias and macrophage-mediated innate resistance in FVB/NJ mice.

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Guava (Psidium guajava L.) is a popular fruit crop that is widely cultivated in Thailand. In November 2023, brown spot disease on guava was observed during postharvest storage at 22 to 31°C and 70 to 75% relative humidity over a period of 3 to 7 days in Fang District, Chiang Mai Province, Thailand. The disease incidence was ~20% of 100 fruits per pallet box. The disease severity on each fruit ranged from 40 to 70% of the surface area affected by lesions. The symptoms appeared as circular to irregular brown to dark brown spots, ranging from 5 to 30 mm in diameter. Fungi were isolated from lesions using a single conidial isolation method (Choi et al. 1999). Two fungal isolates (SDBR-CMU497 and SDBR-CMU498) with similar morphology were obtained. Colonies on potato dextrose agar (PDA) and malt extract agar (MEA) were 65 to 67 and 29 to 38 mm in diameter, respectively after incubation for 1 week at 25°C. Colonies on PDA and MEA were flat, slightly undulate, greenish gray in the center, greyish green at the margin; reverse black. Both isolates produced asexual structures. Pycnidia were black, granular, and grouped. Conidiogenous cells were hyaline, subcylindrical to cylindrical, 8.5 to 17.5 × 3 to 5.5 µm. Conidia were single-celled, hyaline, obovoid to ellipsoid, 5.2 to 9.4 × 3.6 to 7.5 µm (n = 50), smooth-walled, with a single apical appendage. Morphologically, both isolates resembled Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, large subunit (nrLSU), translation elongation factor 1-alpha (tef1-α), actin (act), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using primer pairs ITS5/ITS4, LROR/LRO5, EF1-728F/EF2, ACT-512F/ACT-783R, and GPD1-LM/GPD2-LM, respectively (White et al. 1990; Zhang et al. 2022). Sequences were deposited in GenBank (ITS: PP946770, PP946771; nrLSU: PP948677, PP948678; tef1-α: PP948012, PP948013; act: PP948014, PP948015; GAPDH: PP948016, PP948017). Maximum likelihood phylogenetic analyses of the concatenated five genes identified both isolates as P. capitalensis. Thus, both morphology and molecular data confirmed the fungus as P. capitalensis. To confirm pathogenicity, healthy commercial guava fruits cultivar Kim Ju were surface disinfected by 0.1% NaClO for 3 min, rinsed three times with sterile distilled water, and wounded (Cruz-Lagunas et al. 2023). Conidia were collected from 2-week-old cultures on PDA and suspended in sterile distilled water. Fifteen microliters of a 1 × 106 conidia/ml suspension were dropped onto the wounded fruits. Mock inoculations were used as a control with sterile distilled water. Ten replications were conducted for each treatment and repeated twice. The inoculated fruits were stored in individual sterile plastic boxes at 25°C with 80 to 90% relative humidity. After 7 days, all inoculated fruits exhibited brown to dark brown lesions, while control fruits were asymptomatic. Phyllosticta capitalensis was consistently reisolated from the inoculated tissues on PDA to complete Koch's postulates. Prior to this study, P. capitalensis was known to cause brown or black spot disease on guava fruits cultivated in fields in China (Liao et al. 2020), Egypt (Arafat 2018), and Mexico (Cruz-Lagunas et al. 2023). To our knowledge, this is the first report of P. capitalensis causing postharvest brown spot disease on guava fruit in Thailand. The results will inform epidemiological investigations and future approaches to managing this disease.

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Recently Candida auris has emerged as a multi-resistant fungal pathogen, with a significant clinical impact, and is able to persist for a long time on human skin and hospital environments. It is a critical issue on the WHO fungal priority list and therefore it is fundamental to reinforce hospital surveillance protocols to limit nosocomial outbreaks. The purpose of this study was to apply Fourier transform infrared spectroscopy (FT-IR) to investigate the phylogenetic relationships among isolated strains from a C. auris outbreak at the University Intensive Care Unit of a Tertiary University hospital in Turin (Italy). To calculate a clustering cut-off, intra- and inter-isolate, distance values were analysed. The data showed the presence of a major Alfa cluster and a minor Beta cluster with a defined C. auris clustering cut-off. The results were validated by an external C. auris strain and Principal Component and Linear Discriminant Analyses. The application of FT-IR technology allowed to obtain important information about the phylogenetic relationships between the analysed strains, defining for the first time a "not WGS-based" clustering cut-off with a statistical-mathematical approach. FT-IR could represent a valid alternative to molecular methods for the rapid and cost-saving typing of C. auris strains with important clinical implications.

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Plant immune homeostasis is achieved through a balanced immune activation and suppression, enabling effective defense while averting autoimmunity. In Arabidopsis, disrupting a mitogen-activated protein (MAP) kinase cascade triggers nucleotide-binding leucine-rich-repeat (NLR) SUPPRESSOR OF mkk1/2 2 (SUMM2)-mediated autoimmunity. Through an RNAi screen, we identify PUB5, a putative plant U-box E3 ligase, as a critical regulator of SUMM2-mediated autoimmunity. In contrast to typical E3 ligases, PUB5 stabilizes CRCK3, a calmodulin-binding receptor-like cytoplasmic kinase involved in SUMM2 activation. A closely related E3 ligase, PUB44, functions oppositely with PUB5 to degrade CRCK3 through monoubiquitylation and internalization. Furthermore, CRCK3, highly expressed in roots and conserved across plant species, confers resistance to Fusarium oxysporum, a devastating soil-borne fungal pathogen, in both Arabidopsis and cotton. These findings demonstrate the antagonistic role of an E3 ligase pair in fine-tuning kinase proteostasis for the regulation of NLR-mediated autoimmunity and highlight the function of autoimmune activators in governing plant root immunity against fungal pathogens.

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The spread and adaptation of fungal plant pathogens in agroecosystems are facilitated by environmental homogeneity. Metagenomic sequencing of infected tissues allowed us to monitor eco-evolutionary dynamics and interactions between host, pathogen and plant microbiome. Exserohilum turcicum, the causal agent of northern corn leaf blight (NCLB) in maize, is distributed in multiple clonal lineages throughout Europe. To characterize regional pathogen diversity, we conducted metagenomic DNA sequencing on 241 infected leaf samples from the highly susceptible Swiss maize landrace Rheintaler Ribelmais, collected over 3 years (2016-2018) from an average of 14 agricultural farms within the Swiss Rhine Valley. All major European clonal lineages of E. turcicum were identified. Lineages differ by their mating types which indicates potential for sexual recombination and rapid evolution of new pathogen strains, although we found no evidence of recent recombination. The associated eukaryotic and prokaryotic leaf microbiome exhibited variation in taxonomic diversity between years and locations and is likely influenced by local weather conditions. A network analysis revealed distinct clusters of eukaryotic and prokaryotic taxa that correlates with the frequency of E. turcicum sequencing reads, suggesting causal interactions. Notably, the yeast genus Metschnikowia exhibited a strongly negative association with E. turcicum, supporting its known potential as biological control agent against fungal pathogens. Our findings show that metagenomic sequencing is a useful tool for analysing the role of environmental factors and potential pathogen-microbiome interactions in shaping pathogen dynamics and evolution, suggesting their potential for effective pathogen management strategies.

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At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

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Animal models are frequently used as surrogates to understand human disease. In the fungal pathogen Cryptococcus species complex, several variations of a mouse model of disease were developed that recapitulate different aspects of human disease. These mouse models have been implemented using various inbred and outbred mouse backgrounds, many of which have genetic differences that can influence host response and disease outcome. In this review, we will discuss the most commonly used inbred mouse backgrounds in C. neoformans infection models.

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The rise of Candida auris, a multidrug-resistant fungal pathogen, across more than 40 countries, has signaled an alarming threat to global health due to its significant resistance to existing antifungal therapies. Characterized by its rapid spread and robust drug resistance, C. auris presents a critical challenge in managing infections, particularly in healthcare settings. With research on its biological traits and genetic basis of virulence and resistance still in the early stages, there is a pressing need for a concerted effort to understand and counteract this pathogen. This review synthesizes current knowledge on the epidemiology, biology, genetic manipulation, pathogenicity, diagnostics, and resistance mechanisms of C. auris, and discusses future directions in research and therapeutic development. By exploring the complexities surrounding C. auris, we aim to underscore the importance of advancing research to devise effective control and treatment strategies.

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Candida albicans is a major fungal pathogen of humans that can cause serious systemic infections in vulnerable immunocompromised populations. One of its virulence attributes is its capacity to transition between yeast and filamentous morphologies, but our understanding of this process remains incomplete. Here, we analyzed data from a functional genomic screen performed with the C. albicans Gene Replacement And Conditional Expression collection to identify genes crucial for morphogenesis in host-relevant conditions. Through manual scoring of microscopy images coupled with analysis of each image using a deep learning-based method termed Candescence, we identified 307 genes important for filamentation in tissue culture medium at 37°C with 5% CO2. One such factor was orf19.5963, which is predicted to encode the prenyltransferase Nus1 based on sequence homology to Saccharomyces cerevisiae. We further showed that Nus1 and its predicted interacting partner Rer2 are important for filamentation in multiple liquid filament-inducing conditions as well as for wrinkly colony formation on solid agar. Finally, we highlight that Nus1 and Rer2 likely govern C. albicans morphogenesis due to their importance in intracellular trafficking, as well as maintaining lipid homeostasis. Overall, this work identifies Nus1 and Rer2 as important regulators of C. albicans filamentation and highlights the power of functional genomic screens in advancing our understanding of gene function in human fungal pathogens.

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Fusarium oxysporum is a cross-kingdom pathogen. While some strains cause disseminated fusariosis and blinding corneal infections in humans, others are responsible for devastating vascular wilt diseases in plants. To better understand the distinct adaptations of F. oxysporum to animal or plant hosts, we conducted a comparative phenotypic and genetic analysis of two strains: MRL8996 (isolated from a keratitis patient) and Fol4287 (isolated from a wilted tomato [Solanum lycopersicum]). In vivo infection of mouse corneas and tomato plants revealed that, while both strains cause symptoms in both hosts, MRL8996 caused more severe corneal ulceration and perforation in mice, whereas Fol4287 induced more pronounced wilting symptoms in tomato. In vitro assays using abiotic stress treatments revealed that the human pathogen MRL8996 was better adapted to elevated temperatures, whereas the plant pathogen Fol4287 was more tolerant of osmotic and cell wall stresses. Both strains displayed broad resistance to antifungal treatment, with MRL8996 exhibiting the paradoxical effect of increased tolerance to higher concentrations of the antifungal caspofungin. We identified a set of accessory chromosomes (ACs) and protein-encoding genes with distinct transposon profiles and functions, respectively, between MRL8996 and Fol4287. Interestingly, ACs from both genomes also encode proteins with shared functions, such as chromatin remodeling and post-translational protein modifications. Our phenotypic assays and comparative genomics analyses lay the foundation for future studies correlating genotype with phenotype and for developing targeted antifungals for agricultural and clinical uses.

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Starting in the fall of 2019, mortality, blight symptoms, and signs of white fungal mycelia were observed on external host tissues of non-native landscape trees as well as numerous native trees, understory shrubs, and vines throughout northern and central Florida, USA. We determined that the fungus is an undescribed species of Basidiomycota based on morphological characteristics and DNA sequence analysis. Phylogenetic analyses of the internal transcribed spacer (ITS), large subunit (LSU), and translation elongation factor 1-alpha (tef1) regions revealed that this novel plant pathogen is an undescribed taxon of the genus Parvodontia (Cystostereaceae, Agaricales). We propose the name Parvodontia relampaga sp. nov. which describes its unique morphological features and phylogenetic placement. We confirmed the pathogenicity of P. relampaga in greenhouse inoculations on host plants from which strains of this novel pathogen were isolated, including the non-native gymnosperm Afrocarpus falcatus, the non-native and commercially important Ligustrum japonicum, and the native tree Quercus hemisphaerica. P. relampaga was also detected on a total of 27 different species of woody host plants, including such economically and ecologically important hosts as Fraxinus, Ilex, Magnolia, Persea, Prunus, Salix, Vitis, and Vaccinium. For this new plant disease, we propose the name "relampago blight," which refers to the lightning-like rhizomorph growth (relámpago means 'lightning' in Spanish). This study presents a newly discovered fungal taxon with a wide host range on both angiosperms and gymnosperms that may be an emerging pathogen of concern in Florida and the Gulf Coast region.

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Successful host tissue colonization is crucial for fungal pathogens to cause mycosis and complete the infection cycle, in which fungal cells undergo a series of morphological transition-included cellular events to combat with hosts. However, many transcription factors (TFs) and their mediated networks regulating fungal pathogen colonization of host tissue are not well characterized. Here, a TF (BbHCR1)-mediated regulatory network was identified in an insect pathogenic fungus, Beauveria bassiana, that controlled insect hemocoel colonization. BbHCR1 was highly expressed in fungal cells after reaching insect hemocoel and controlled the yeast (in vivo blastospores)-to-hyphal morphological switch, evasion of immune defense response, and fungal virulence. Comparative analysis of RNA sequencing and chromatin immunoprecipitation sequencing identified a core set of BbHCR1 target genes during hemocoel colonization, in which abaA and brlA were targeted to limit the rapid switch from blastospores to hyphae and fungal virulence. Two targets encoding hypothetical proteins, HP1 and HP2, were activated and repressed by BbHCR1, respectively, which acted as a virulence factor and repressor, respectively, suggesting that BbHCR1 activated virulence factors but repressed virulence repressors during the colonization of insect hemocoel. BbHCR1 tuned the expression of two dominant hemocoel colonization-involved metabolite biosynthetic gene clusters, which linked its regulatory role in evasion of immune response. Those functions of BbHCR1 were found to be collaboratively regulated by Fus3- and Hog1-MAP kinases via phosphorylation. These findings have drawn a regulatory network in which Fus3- and Hog1-MAP kinases phosphorylate BbHCR1, which in turn controls the colonization of insect body cavities by regulating fungal morphological transition and virulence-implicated genes.IMPORTANCEFungal pathogens adopt a series of tactics for successful colonization in host tissues, which include morphological transition and the generation of toxic and immunosuppressive molecules. However, many transcription factors (TFs) and their linked pathways that regulate tissue colonization are not well characterized. Here, we identified a TF (BbHCR1)-mediated regulatory network that controls the insect fungal pathogen, Beauveria bassiana, colonization of insect hemocoel. During these processes, BbHCR1 targeted the fungal central development pathway for the control of yeast (blastospores)-to-hyphae morphological transition, activated virulence factors, repressed virulence repressors, and tuned the expression of two dominant hemocoel colonization-involved immunosuppressive and immunostimulatory metabolite biosynthetic gene clusters. The BbHCR1 regulatory function was governed by Fus3- and Hog1-MAP kinases. These findings led to a new regulatory network composed of Fus3- and Hog1-MAP kinases and BbHCR1 that control insect body cavity colonization by regulating fungal morphological transition and virulence-implicated genes.

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The ability of C. neoformans to survive and replicate within host phagocytes enables it to evade the immune system and allows for persistence of the infection. As such, measuring fungal burden of C. neoformans strains-and indeed how drug treatments can influence fungal burden-provides important information about C. neoformans pathogenesis. In this chapter, we describe two methods that may be used to appraise fungal burden: a standard end-point colony-formation assay for calculating the average number of yeast per host cell and a fluorescence microscopy-based method that may be used to measure changes in fungal burden in individual living macrophages in real time.

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