Ctr9 promotes virulence of Candida albicans by regulating methionine metabolism.

Candida albicans, a part of normal flora, is an opportunistic fungal pathogen and causes severe health issues in immunocompromised patients. Its pathogenicity is intricately linked to the transcriptional regulation of its metabolic pathways. Paf1 complex (Paf1C) is a crucial transcriptional regulator that is highly conserved in eukaryotes. The objective of this study was to explore the role of Paf1C in the metabolic pathways and how it influences the pathogenicity of C. albicans. Paf1C knockout mutant strains of C. albicans (ctr9Δ/Δ, leo1Δ/Δ, and cdc73Δ/Δ) were generated using the CRISPR-Cas9 system. To investigate the effect of Paf1C on pathogenicity, macrophage interaction assays and mouse survival tests were conducted. The growth patterns of the Paf1C knockout mutants were analyzed through spotting assays and growth curve measurements. Transcriptome analysis was conducted under yeast conditions (30°C without serum) and hyphal conditions (37°C with 10% FBS), to further elucidate the role of Paf1C in the pathogenicity of C. albicans. CTR9 deletion resulted in the attenuation of C. albicans virulence, in macrophage and mouse models. Furthermore, we confirmed that the reduced virulence of the ctr9Δ/Δ mutant can be attributed to a decrease in C. albicans cell abundance. Moreover, transcriptome analysis revealed that metabolic processes required for cell proliferation are impaired in ctr9Δ/Δ mutant. Notably, CTR9 deletion led to the downregulation of methionine biosynthetic genes and the cAMP-PKA signaling pathway-related hypha essential genes, which are pivotal for virulence. Our results suggest that Ctr9-regulated methionine metabolism is a crucial factor for determining C. albicans pathogenicity.

Antifungal activities of Equol against Candida albicans in vitro and in vivo.

Candida albicans is an opportunistic fungal pathogen that can cause systemic infections in immunocompromised individuals. Morphological transition and biofilm formation are major virulence factors of C. albicans. Moreover, biofilm enhances resistance to antifungal agents. Therefore, it is urgent to identify new and effective compounds to target the biofilm of C. albicans. In the present study, the antifungal activities of equol against C. albicans were investigated. In vitro, the microdilution analysis and spot assay result showed that equol exhibited potent inhibitory activities against C. albicans. Further investigations confirmed that the antifungal effects of equol involved interference with the transition from yeast to hypha and biofilm formation of C. albicans. In addition, transcriptome sequencing and reverse transcription-quantitative PCR (qRT-PCR) analysis showed that equol significantly downregulated the expression of several genes in the Ras1-cAMP-PKA pathway related to hyphae and biofilm formation and significantly upregulated the expression of the negative transcriptional repressors RFG1 and TUP1. Moreover, equol effectively reduced the production of cAMP, a key messenger in the Ras1-cAMP-PKA pathway, while supplementation with cAMP partly rescued the equol-induced defects in hyphal development. Furthermore, in a mouse model of systemic candidiasis (SC), equol treatment significantly decreased the fungal burden (liver, kidneys, and lung) in mice and local tissue damage, while enhancing the production of interleukin-10 (IL-10). Together, these findings confirm that equol is a potentially effective agent for treatment of SC.

Replicating biological 3D root and hyphal networks in transparent glass chips.

Replicating the complex 3D microvascular architectures found in biological systems is a critical challenge in tissue engineering and other fields requiring efficient mass transport. Conventional microfabrication techniques often face limitations in creating extensive hierarchical networks, especially within bulk materials. Here, we report a versatile bioinspired approach to generate optimized 3D microvascular networks within transparent glass matrix by transcribing the natural growth patterns of plants and fungi. Plant seeds or fungal spores are first cultivated on nanoparticle-based culture media. Subsequent heat treatment removes the biological species while sintering the surrounding compound into a solidified chip with replica root/hyphal architectures as open microchannels. A diverse range of architectures, including the hierarchical branching of plant roots and the intricate networks formed by fungal hyphae, can be faithfully replicated. The resultant glass microvascular networks exhibit high chemical and thermal stability, enabling applications under harsh conditions. Fluid flow experiments validate the functionalities of the fabricated channels. By co-cultivating plants and fungi, hierarchical multi-scale architectures mimicking natural vascular systems are achieved. This bioinspired manufacturing technique leverages autonomous biological growth for architectural optimization, offering a complementary approach to existing microfabrication methods. The transparent nature of the glass chips allows for direct optical inspection, potentially facilitating integration with imaging components. This versatile platform holds promise for various engineering applications, such as microreactors, heat exchangers, and advanced filtration systems.

[Aspergillus Cell Surface Structural Analysis and Its Applications to Industrial and Medical Use].

The hyphal surface of cells of filamentous fungi is covered with cell wall, which is mainly composed of polysaccharides. Since the cell wall is the first structure to come in contact with the infection host, the environment, and the fungus itself, the elucidation of the cell wall structure and biogenesis is essential for understanding fungal ecology. Among filamentous fungi, the genus Aspergillus is an important group in the industrial, food, and medical fields. It is known that Aspergillus species form hyphal pellets in shake liquid culture. The authors previously found the role of α-1,3-glucan in hyphal aggregation in Aspergillus species. In addition, extracellular polysaccharide galactosaminogalactan contributed to hyphal aggregation as well, and dual disruption of biosynthesis genes of α-1,3-glucan and galactosaminogalactan resulted in complete hyphal dispersion in shake liquid culture. The characteristic of mycelia to form pellets under liquid culture conditions was the main reason why the growth measurement methods used for unicellular organisms could not be applied. We reported that hyphal growth of the dual disruption mutant could be measured by optical density. A real-time plate reader could be used to determine the growth curve of the mycelial growth of the dual disruption mutant. This measurement approach not only provides basic microbiological insights in filamentous fungi, but also has the potential to be applied to high-throughput screening of anti-Aspergillus drugs.

Glucocorticoids Suppress NF-κB-Mediated Neutrophil Control of Aspergillus fumigatus Hyphal Growth.

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. In this article, we used a zebrafish larva Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection, and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. Dexamethasone partially suppresses NF-κB activation at the infection site by inducing the transcription of IκB via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKKγ to prevent NF-κB activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKKγ crispant larvae further increases invasive hyphal growth and host mortality, suggesting that dexamethasone may suppress other pathways in addition to NF-κB to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-κB-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae.

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Biochemical Characterization of Recombinant Enterococcus faecalis EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against Candida albicans.

Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV88-His6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV88-His6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV88-His6, we revealed that EntV88-His6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV88-His6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro. The combination of EntV88-His6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Inhibitory effects and mode of antifungal action of isobavachalcone on Candida albicans growth and virulence factors.

The fungus Candida albicans causes various kinds of human infections, including oral thrush, vulvovaginitis and life-endangering bloodstream infections, the incidence of which are rising. Worsening this, the clinical antifungals are limited to a few, highlighting the necessity to develop novel antifungal therapies. In this study, the antifungal activities of isobavachalcone against C. albicans SC5314 and nine C. albicans clinical isolates were tested. The effects of isobavachalcone (IBC) on C. albicans virulence factors, such as hyphal formation, adhesion, biofilm formation and extracellular phospholipase production, as well as the underlying mechanism, were also evaluated. Antifungal susceptibility test revealed that IBC has significant anti-Candida activities, with both MIC and MFC being 4-5 µg/mL against all strains tested. Hyphal formation in RPMI-1640, Spider and GlcNAc medium, adhesion to abiotic polystyrene surfaces and surfaces of A549 cells, could be inhibited by IBC. Most important, IBC could inhibit the C. albicans biofilm formation and development. PI staining tests showed that IBC could increase the cell membrane permeability, suggesting the damages to the fungal cell membrane. IBC was further demonstrated to induce excessive ROS production in C. albicans planktonic cells and its mature biofilms, as revealed by DCFH fluorescence detection through flowcytometry and relative fluorescence intensity analysis (with a microplate reader). The roles of ROS in the antifungal activity of IBC were further confirmed through antioxidant rescue assays in MIC and biofilm formation tests. Compared to its antifungal activity, the cytotoxicity against mammalian cells was low, indicating its potential in developing antifungal therapies.

Insight into the nuclear distribution patterns of conidia and the asexual life cycle of Polyporus umbellatus.

P. umbellatus sclerotium is a traditional Chinese medicine that is widely utilized in China, Korea, Japan, and other countries due to its diverse medicinal activities, such as diuretic, antitumor, anticancer, and immune system enhancement effects. Conidia, which are common asexual spores in various fungi, are not universally present in Polyporus species. In this study, the asexual life cycle of P. umbellatus was elucidated. Conidia, i.e. arthorconidia, were produced by both dikaryotic and monokaryotic strains. In the dikaryotic strain, binucleate, uninucleate, and nuclei-free conidia were identified with proportions of 67.9 %, 12.4 %, and 19.7 %, respectively. Conversely, the monokaryotic strain did not produce binucleate conidia. This discrepancy suggests that binucleate spores are heterokaryons, while uninucleate spores are homokaryons. Clamp connections were observed in dikaryotic hyphae, but were absent in monokaryotic hyphae. Monokaryotic strains were obtained from conidia of the dikaryotic strain. Additionally, mating types were determined through pairing tests, and successful crossbreeding occurred between monokaryotic strains derived from conidia and basidiospores from different strains. This study introduced the first crossbreeding strategy for P. umbellatus.

A Brg1-Rme1 circuit in Candida albicans hyphal gene regulation.

Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1, established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1Δ/Δ mutation is most prominent under static or "biofilm-like" growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1: hypha-associated gene expression levels are substantially reduced in a brg1Δ/Δ mutant and partially restored in a brg1Δ/Δ rme1Δ/Δ double mutant. An rme1Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions.IMPORTANCECandida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1, whose activity is most prominent under biofilm-like growth conditions.

Fleagrass (Adenosma buchneroides Bonati) Acts as a Fungicide Against Candida albicans by Damaging Its Cell Wall.

Fleagrass, a herb known for its pleasant aroma, is widely used as a mosquito repellent, antibacterial agent, and for treating colds, reducing swelling, and alleviating pain. The antifungal effects of the essential oils of fleagrass and carvacrol against Candida albicans were investigated by evaluating the growth and the mycelial and biofilm development of C. albicans. Transmission electron microscopy was used to evaluate the integrity of the cell membrane and cell wall of C. albicans. Fleagrass exhibited high fungicidal activity against C. albicans at concentrations of 0.5% v/v (via the Ras1/cAMP/PKA pathway). Furthermore, transmission electron microscopy revealed damage to the cell wall and membrane after treatment with the essential oil, which was further confirmed by the increased levels of ß-1,3-glucan and chitin in the cell wall. This study showed that fleagrass exerts good fungicidal and hyphal growth inhibition activity against C. albicans by disrupting its cell wall, and thus, fleagrass may be a potential antifungal drug.

Myo5B plays a significant role in the hyphal growth and virulence of the human pathogenic fungus Mucor lusitanicus.

Mucormycosis is an emerging and deadly invasive fungal infection caused by fungi belonging to the Mucorales order. We investigated the myosin superfamily, which encompasses diverse actin-based motor proteins with various cellular functions. Specifically, the role of the Myo5B (ID 179665) protein from the myosin class V family in Mucor lusitanicus was explored by generating silencing phenotypes and null mutants corresponding to the myo5B gene. Silencing fungal transformants exhibited a markedly reduced growth rate and a nearly complete absence of sporulation compared to the wild-type strain. The myo5BΔ null mutant strain displayed atypical characteristics, including abnormally short septa and inflated hyphae. Notably, there were a majority of small yeast-like cells instead of filamentous hyphae in the mutant. These yeast-like cells cannot germinate normally, resulting in a loss of polarity. In vivo virulence assays conducted in the Galleria mellonella invertebrate model revealed that the myo5BΔ mutant strain was avirulent. These findings shed light on the crucial contributions of the Myo5B protein to the dimorphism and pathogenicity of M. lusitanicus. Therefore, the myosin V family is a potential target for future therapeutic interventions aimed at treating mucormycosis.

Fungal colonization and penetration of mortar as a suitable simulant for concrete: Implications for fungal biodeterioration in the built environment.

A range of fungal species showed variable abilities to colonize and penetrate a mortar substrate. Calcium biomineralization was a common feature with calcium-containing crystals deposited in the microenvironment or encrusting hyphae, regardless of the specific mortar composition. Several species caused significant damage to the mortar surface, exhibiting burrowing and penetration, surface etching, and biomineralization. In some cases, extensive biomineralization of hyphae, probably by carbonatization, resulted in the formation of crystalline tubes after hyphal degradation on mortar blocks, including those amended with Co or Sr carbonate. Ca was the only metal detected in the biomineralized formations with Co or Sr undetectable. Aspergillus niger, Stemphylium sp. and Paecilomyces sp. could penetrate mortar with differential responses depending on the porosity. Fluorescent staining of thin sections recorded penetration depths of ∼530 um for A. niger and ∼620 um for Stemphylium sp. Penetration depth varied inversely with porosity and greater penetration depths were achieved in mortar with a lower porosity (lower water/cement ratio). These results have provided further understanding of biodeteriorative fungal interactions with cementitious substrates that can clearly affect structural integrity. The potential significance of fungal colonization and such biodeteriorative phenomena should not be overlooked in built environment contexts, including radionuclide storage and surface decontamination.

Regulatory complexity of cellular differentiation in Candida albicans revealed through systematic screening of protein kinase mutants.

A recent study in mBio reports the construction and preliminary screening of a library containing mutants of 99 of the 119 predicted protein kinases in Candida albicans (the majority of the remaining 20 are probably essential) (J. Kramara, M.-J. Kim, T. L. Ollinger, L. C. Ristow, et al., mBio e01249-24, 2024, https://doi.org/10.1128/mbio.01249-24). Using a quantitative competition assay in 10 conditions that represent nutritional, osmotic, cell wall, and pH stresses that are considered to model various aspects of the host environment allowed them to phenotypically cluster kinases, which highlight both the integration and specialization of signaling pathways, suggesting novel functions for many kinases. In addition, they tackle two complex and partially overlapping differentiation events, hyphal morphogenesis and biofilm formation. They find that a remarkable 88% of the viable kinase mutants in C. albicans affect hyphal growth, illustrating how integrated morphogenesis is in the overall biology of this organism, and begin to dissect the regulatory relationships that control this key virulence trait.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

The spliceosome impacts morphogenesis in the human fungal pathogen Candida albicans.

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.

Mechanical strategies supporting growth and size diversity in Filamentous Fungi.

The stereotypical tip growth of filamentous fungi supports their lifestyles and functions. It relies on the polarized remodeling and expansion of a protective elastic cell wall (CW) driven by large cytoplasmic turgor pressure. Remarkably, hyphal filament diameters and cell elongation rates can vary extensively among different fungi. To date, however, how fungal cell mechanics may be adapted to support these morphological diversities while ensuring surface integrity remains unknown. Here, we combined super-resolution imaging and deflation assays to measure local CW thickness, elasticity and turgor in a set of fungal species spread on the evolutionary tree that spans a large range in cell size and growth speeds. While CW elasticity exhibited dispersed values, presumably reflecting differences in CW composition, both thickness and turgor scaled in dose-dependence with cell diameter and growth speeds. Notably, larger cells exhibited thinner lateral CWs, and faster cells thinner apical CWs. Counterintuitively, turgor pressure was also inversely scaled with cell diameter and tip growth speed, challenging the idea that turgor is the primary factor dictating tip elongation rates. We propose that fast-growing cells with rapid CW turnover have evolved strategies based on a less turgid cytoplasm and thin walls to safeguard surface integrity and survival.

Hyphae of the fungus Aspergillus nidulans demonstrate chemotropism to nutrients and pH.

The importance of fungi in ecological systems and pathogenicity hinges on their ability to search for nutrients, substrates, and hosts. Despite this, the question of whether fungal hyphae exhibit chemotropism toward them remains largely unresolved and requires close examination at the cellular level. Here, we designed a microfluidic device to assess hyphal chemotropism of Aspergillus nidulans in response to carbon and nitrogen sources, as well as pH. Within this device, hyphae could determine their growth direction in a two-layer flow with distinct compositions that were adjacent but non-mixing. Under conditions with and without a carbon source, hyphae changed growth direction to remain in the presence of a carbon source, but it was still difficult to distinguish between differences in growth and chemotropism. Although nitrogen sources such as ammonia and nitrate are important for growth, the hyphae indicated negative chemotropism to avoid them depending on the specific transporters. This fungus grows equally well at the colony level in the pH range of 4 to 9, but the hyphae exhibited chemotropism to acidic pH. The proton pump PmaA is vital for the chemotropism to acid pH, while the master regulatory for pH adaptation PacC is not involved, suggesting that chemotropism and adaptive growth via gene expression regulation are distinct regulatory mechanisms. Despite various plasma membrane transporters are distributed across membranes except at the hyphal tip, the control of growth direction occurs at the tip. Finally, we explored the mechanisms linking these two phenomena, tip growth and chemotropism.

Biocontrol potential and growth-promoting effect of endophytic fungus Talaromyces muroii SD1-4 against potato leaf spot disease caused by Alternaria alternata.

BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.