The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

Utilization of CRISPR-Cas genome editing technology in filamentous fungi: function and advancement potentiality.

Filamentous fungi play a crucial role in environmental pollution control, protein secretion, and the production of active secondary metabolites. The evolution of gene editing technology has significantly improved the study of filamentous fungi, which in the past was laborious and time-consuming. But recently, CRISPR-Cas systems, which utilize small guide RNA (sgRNA) to mediate clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), have demonstrated considerable promise in research and application for filamentous fungi. The principle, function, and classification of CRISPR-Cas, along with its application strategies and research progress in filamentous fungi, will all be covered in the review. Additionally, we will go over general matters to take into account when editing a genome with the CRISPR-Cas system, including the creation of vectors, different transformation methodologies, multiple editing approaches, CRISPR-mediated transcriptional activation (CRISPRa) or interference (CRISPRi), base editors (BEs), and Prime editors (PEs).

Exploring Non-Thermal Plasma and UV Radiation as Biofilm Control Strategies against Foodborne Filamentous Fungal Contaminants.

In recent years, non-thermal plasma (NTP) has emerged as a promising tool for decontamination and disinfection within the food industry. Given the increasing resistance of microbial biofilms to conventional disinfectants and their adverse environmental effects, this method has significant potential for eliminating biofilm formation or mitigating the metabolic activity of grown biofilms. A comparative study was conducted evaluating the efficacy of UV radiation and NTP in eradicating mature biofilms of four common foodborne filamentous fungal contaminants: Alternaria alternata, Aspergillus niger, Fusarium culmorum, and Fusarium graminearum. The findings reveal that while UV radiation exhibits variable efficacy depending on the duration of exposure and fungal species, NTP induces substantial morphological alterations in biofilms, disrupting hyphae, and reducing extracellular polymeric substance production, particularly in A. alternata and F. culmorum. Notably, scanning electron microscopy analysis demonstrates significant disruption of the hyphae in NTP-treated biofilms, indicating its ability to penetrate the biofilm matrix, which is a promising outcome for biofilm eradication strategies. The use of NTP could offer a more environmentally friendly and potentially more effective alternative to traditional disinfection methods.

Differential expression of immune-related biomarkers in primary cultures from Atlantic salmon (Salmo salar) exposed to processed Paecilomyces variotii with or without inactivated Moritella viscosa.

Paecilomyces variotii (a filamentous fungus), is a promising novel protein source in fish feeds due to its high nutritional value. Also, P. variotii has Microbial-Associated Molecular Patterns (MAMPs) such as glucans and nucleic acids that could modulate the host's immune response. To understand the potential bioactive properties of this fungus in Atlantic salmon (Salmo salar), our study was conducted to evaluate the gene expression of immune-related biomarkers (e.g., cytokines, effector molecules and receptors) on primary cultures from salmon head kidney (HKLs) and spleen leukocytes (SLs) exposed to either UV inactivated or fractions from P. variotii with or without inactivated Moritella viscosa (a skin pathogen in salmonids). Moreover, the effect of the fermentation conditions and down-stream processing on the physical ultrastructure and cell wall glucan content of P. variotii was characterized. The results showed that drying had a significant effect on the cell wall ultrastructure of the fungi and the choice of fermentation has a significant effect on the quantity of ß-glucans in P. variotii. Furthermore, stimulating Atlantic salmon HKLs and SLs with P. variotii and its fractions induced gene expression related to pro-inflammatory (tnfα, il1ß) and antimicrobial response (cath2) in HKLs, while response in SLs was related to both pro-inflammatory and regulatory response (tnfα, il6 and il10). Similarly, the stimulation with inactivated M. viscosa alone led to an up-regulation of genes related to pro-inflammatory (tnfα, il1ß, il6) antimicrobial response (cath2), intra-cellular signalling and recognition of M. viscosa (sclra, sclrb) and a suppression of regulatory response (il10) in both HKLs and SLs. Interestingly, the co-stimulation of cells with P. variotii and M. viscosa induced immune homeostasis (il6, tgfß) and antimicrobial response (cath2) in SLs at 48h. Thus, P. variotii induces immune activation and cellular communication in Atlantic salmon HKLs and SLs and modulates M. viscosa induced pro-inflammatory responses in SLs. Taken together, the results from physical and chemical characterization of the fungi, along with the differential gene expression of key immune biomarkers, provides a theoretical basis for designing feeding trials and optimize diets with P. variotii as a functional novel feed ingredient for Atlantic salmon.

Characterization of potassium-solubilizing fungi, Mortierella spp., isolated from a poplar plantation rhizosphere soil.

Potassium-solubilizing microorganisms are capable of secreting acidic chemicals that dissolve and release potassium from soil minerals, thus facilitating potassium uptake by plants. In this study, three potassium-dissolving filamentous fungi were isolated from the rhizosphere soil of a poplar plantation in Jiangsu Province, China. Phylogenetic analyses based on ITS, 18 S, and 28 S showed that these three isolates were most similar to Mortierella. These strains also possessed spherical or ellipsoidal spores, produced sporangia at the hyphal tip, and formed petal-like colonies on PDA media resembling those of Mortierella species. These findings, along with further phenotypic observations, suggest that these isolates were Mortierella species. In addition, the potassium-dissolution experiment showed that strain 2K4 had a relatively high potassium-solubilizing capacity among these isolated fungi. By investigating the influences of different nutrient conditions (carbon source, nitrogen source, and inorganic salt) and initial pH values on the potassium-dissolving ability, the optimal potassium-solubilization conditions of the isolate were determined. When potassium feldspar powder was used as an insoluble potassium source, isolate 2K4 exhibited a significantly better polysaccharide aggregation ability on the formed mycelium-potassium feldspar complex. The composition and content of organic acids secreted by strain 2K4 were further detected, and the potassium-dissolution mechanism of the Mortierella species and its growth promotion effect were discussed, using maize as an example.

Human Vγ9Vδ2 T cells exhibit antifungal activity against Aspergillus fumigatus and other filamentous fungi.

Invasive aspergillosis (IA) and mucormycosis are life-threatening diseases, especially among immunocompromised patients. Drug-resistant Aspergillus fumigatus strains have been isolated worldwide, which can pose a serious clinical problem. As IA mainly occurs in patients with compromised immune systems, the ideal therapeutic approach should aim to bolster the immune system. In this study, we focused on Vγ9Vδ2 T cells that exhibit immune effector functions and examined the possibility of harnessing this unconventional T cell subset as a novel therapeutic modality for IA. A potent antifungal effect was observed when A. fumigatus (Af293) hyphae were challenged by Vγ9Vδ2 T cells derived from peripheral blood. In addition, Vγ9Vδ2 T cells exhibited antifungal activity against hyphae of all Aspergillus spp., Cunninghamella bertholletiae, and Rhizopus microsporus but not against their conidia. Furthermore, Vγ9Vδ2 T cells also exhibited antifungal activity against azole-resistant A. fumigatus, indicating that Vγ9Vδ2 T cells could be used for treating drug-resistant A. fumigatus. The antifungal activity of Vγ9Vδ2 T cells depended on cell-to-cell contact with A. fumigatus hyphae, and degranulation characterized by CD107a mobilization seems essential for this activity against A. fumigatus. Vγ9Vδ2 T cells could be developed as a novel modality for treating IA or mucormycosis. IMPORTANCE: Invasive aspergillosis (IA) and mucormycosis are often resistant to treatment with conventional antifungal agents and have a high mortality rate. Additionally, effective antifungal treatment is hindered by drug toxicity, given that both fungal and human cells are eukaryotic, and antifungal agents are also likely to act on human cells, resulting in adverse effects. Therefore, the development of novel therapeutic agents specifically targeting fungi is challenging. This study demonstrated the antifungal activity of Vγ9Vδ2 T cells against various Aspergillus spp. and several Mucorales in vitro and discussed the mechanism underlying their antifungal activity. We indicate that adoptive immunotherapy using Vγ9Vδ2 T cells may offer a new therapeutic approach to IA.

Enzymes with Lactonase Activity against Fungal Quorum Molecules as Effective Antifungals.

Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-ß-lactamase NDM-1, and organophosphate hydrolase His6-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His6-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed.

The serine/threonine protein kinase MpSTE1 directly governs hyphal branching in Monascus spp.

Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)-assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites. KEY POINTS: • MpSTE1 is the first characterized regulator for tightly regulating hyphal branching • Overexpression of mpSTE1 significantly improves secondary metabolite production • A high-throughput image analysis tool was developed for counting hyphal branching.

Biodegradation capabilities of filamentous fungi in high-concentration heavy crude oil environments.

In this comprehensive study, we delved into the capabilities of five fungal strains: Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum, Penicillium glabrum, and Penicillium rubens (the latter isolated from heavy crude oil [HCO]) in metabolizing HCO as a carbon source. Employing a meticulously designed experimental approach, conducted at room temperature (25 °C), we systematically explored various culture media and incubation periods. The results unveiled the exceptional resilience of all these fungi to HCO, with A. flavus standing out as the top performer. Notably, A. flavus exhibited robust growth, achieving a remarkable 59.1% expansion across the medium's surface, accompanied by distinctive macroscopic traits, including a cottony appearance and vibrant coloration. In an effort to further scrutinize its biotransformation prowess, we conducted experiments in a liquid medium, quantifying CO2 production through gas chromatography, which reached its zenith at day 30, signifying substantial bioconversion with a 38% increase in CO2 production. Additionally, we monitored changes in surface tension using the Du Noüy ring method, revealing a reduction in aqueous phase tension from 72.3 to 47 mN/m. This compelling evidence confirms that A. flavus adeptly metabolizes HCO to fuel its growth, while concurrently generating valuable biosurfactants. These findings underscore the immense biotechnological potential of A. flavus in addressing challenges related to HCO, thereby offering promising prospects for bioremediation and crude oil bioupgrading endeavors.

Polarity-dependent expression and localization of secretory glucoamylase mRNA in filamentous fungal cells.

In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we analyzed mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae, by the MS2 system, in which mRNA can be visualized in living cells. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion, in polarity-dependent expression and localization manners. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that although glaA mRNA localized to stress granules (SGs) and processing bodies (PBs) under high temperature, glaA mRNA was not seen under ER stress, suggesting that there are different regulatory mechanisms of glaA mRNA by SG and PB under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi.

Fungal Colonization of the Airways of Patients with Cystic Fibrosis: the Role of the Environmental Reservoirs.

Filamentous fungi frequently colonize the airways of patients with cystic fibrosis and may cause severe diseases, such as the allergic bronchopulmonary aspergillosis. The most common filamentous fungi capable to chronically colonize the respiratory tract of the patients are Aspergillus fumigatus and Scedosporium species. Defining the treatment strategy may be challenging, the number of available drugs being limited and some of the causative agents being multiresistant microorganisms. The knowledge of the fungal niches in the outdoor and indoor environment is needed for understanding the origin of the contamination of the patients. In light of the abundance of some of the causative molds in compost, agricultural and flower fields, occupational activities related to such environments should be discouraged for patients with cystic fibrosis (CF). In addition, the microbiological monitoring of their indoor environment, including analysis of air and dust on surfaces, is essential to propose preventive measures aiming to reduce the exposure to environmental molds. Nevertheless, some specific niches were also identified in the indoor environment, in relation with humidity which favors the growth of thermotolerant molds. Potted plants were reported as indoor reservoirs for Scedosporium species. Likewise, Exophiala dermatitidis may be spread in the kitchen via dishwashers. However, genotype studies are still required to establish the link between dishwashers and colonization of the airways of CF patients by this black yeast. Moreover, as nothing is known regarding the other filamentous fungi associated with CF, further studies should be conducted to identify other potential specific niches in the habitat.

Cumulative expression of heterologous XlnR regulatory modules and AraRA731V in Penicillium oxalicum enhances saccharification efficiency of corn stover and corn fiber.

Penicillium oxalicum engineered strain DB2 and its mutant strains with multiple regulatory modules were constructed. Mutant strain RE-4-2 with two regulatory modules showed a significant increase in the reducing sugar released from corn stover and corn fiber as well as in the conversion of cellulose than DB2. RE-5-2 with three regulatory modules showed a further increase in reducing sugar released from corn stover and the conversion of cellulose on the basis of RE-4-2. RE-4-2-AraRA731V constructed by overexpressing AraRA731V in RE-4-2 showed an increase of 7.2 times and 1.2 times in arabinofuranosidase and xylosidase activities, respectively. Reducing sugar yield and cellulose conversion of corn stover and corn fiber by RE-4-2-AraRA731V were further increased.

Molecular identification and lipolytic potential of filamentous fungi isolated from residual cooking oil.

Filamentous fungi, microorganisms that develop and are located in different habitats, are considered important producers of enzymes and metabolites with potential for the biotechnology industry. The objective of this work was to isolate and identify filamentous fungi that grow in used oil. Two fungal species were characterised through their morphology and molecular identification. The DNA of each extracted strain was amplified by PCR using primers ITS1 and ITS4, obtaining sequences that were later in GenBank (NCBI). A white coloured strain (HB) with a cottony, white, hyaline morphology and irregular borders was observed; so too, a brown colony (HC) with a sandy surface, a well-defined border of beige colour in early growth until it became a dark brown colour. The identity result by homology of the sequences in the BLASTn database was 100% and 99.55%, indicating that they correspond to Cladosporiumtenuissimum and Fomitopsismeliae, respectively. Finally, the results in lipolytic activity show greater potential for Fomitopsismeliae with 0.61 U/l in residual oil. Thus, it is important to highlight the potential of this type of waste to favour the prospection of microorganisms for a sustainable alternative for future studies of biological conversion.

C-terminus of serine–arginine protein kinase-like protein, SrpkF, is involved in conidiophore formation and hyphal growth under salt stress in Aspergillus aculeatus / El extremo C de la proteína similar a la serina-arginina proteína quinasa, SrpkF, participa en la formación de conidióforos y el crecimiento de hifas bajo estrés salino en Aspergillus aculeatus

The serine–arginine protein kinase–like protein, SrpkF, was identified as a regulator for the cellulose-responsive induction of cellulase genes in Aspergillus aculeatus. To analyze various aspects of SrpkF function, we examined the growth of the control strain (MR12); C-terminus deletion mutant, which produced SrpkF1–327 (ΔCsrpkF); whole gene-deletion mutant of srpkF (ΔsrpkF), srpkF overexpressing strain (OEsprkF); and the complemented strain (srpkF+) under various stress conditions. All test strains grew normally on minimal medium under control, high salt (1.5 M KCl), and high osmolality (2.0 M sorbitol and 1.0 M sucrose). However, only ΔCsrpkF showed reduced conidiation on 1.0 M NaCl media. Conidiation of ΔCsrpkF on 1.0 M NaCl media was reduced to 12% compared with that of srpkF+. Further, when OEsprkF and ΔCsrpkF were pre-cultured under salt stress conditions, germination under salt stress conditions was enhanced in both strains. By contrast, deletion of srpkF did not affect hyphal growth and conidiation under the same conditions. We then quantified the transcripts of the regulators involved in the central asexual conidiation pathway in A. aculeatus. The findings revealed that the expression of brlA, abaA, wetA, and vosA was reduced in ΔCsrpkF under salt stress. These data suggest that in A. aculeatus, SrpkF regulates conidiophore development. The C-terminus of SrpkF seems to be important for regulating SrpkF function in response to culture conditions such as salt stress.(AU)

A bird's-eye view of the endoplasmic reticulum in filamentous fungi.

SUMMARYThe endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca2+ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.

In vivo assembly of genetic constructs in filamentous fungus Talaromyces cellulolyticus.

In the filamentous fungus Talaromyces cellulolyticus, similar to other filamentous fungi, non-homologous recombination predominates over homologous recombination. For instance, to achieve an acceptable integration frequency of a genetic construct into a target site on the intact chromosome, the flanking sequences directing this integration should be approximately 2.5 kb in length. However, despite the requirement of long flanks for integration into the intact chromosome, we found that homologous recombination between linear DNA fragments in T. cellulolyticus effectively occurs when these fragments overlap by just 50 bp. This allows for the assembly of full-sized genetic constructs in vivo from relatively small blocks, eliminating the need for in vitro assembly, similar to the approach previously developed for the yeast Saccharomyces cerevisiae. To validate this possibility, we replaced the native promoter of the target gene by transforming the recipient strain with five DNA fragments: two flanks for recombination with the target locus, two parts of the marker gene, and a donor promoter. This discovery significantly expedites the genetic engineering of T. cellulolyticus and potentially other fungi.

Methods for the detection of intracellular calcium in filamentous fungi.

Calcium (Ca2+), a critical secondary messenger, is also known as the molecule of life and death. The cell responds to a minute change in Ca2+ concentration and tightly maintains Ca2+ homeostasis. Therefore, determining the cell Ca2+ level is critical to understand Ca2+ distribution in the cell and various cell processes. Many techniques have been developed to measure Ca2+ in the cell. We review here different methods used to detect and measure Ca2+ in filamentous fungi. Ca2+-sensitive fluorescent chlortetracycline hydrochloride (CTC), Ca2+-selective microelectrode, Ca2+ isotopes, aequorins, and RGECOs are commonly used to measure the Ca2+ level in filamentous fungi. The use of CTC was one of the earliest methods, developed in 1988, to measure the Ca2+ gradient in the filamentous fungus Neurospora crassa. Subsequently, Ca2+-specific microelectrodes were developed later in the 1990s to identify Ca2+ ion flux variations, and to measure Ca2+ concentration. Another method for quantifying Ca2+ is by using radio-labeled Ca2+ as a tracer. The usage of 45Ca to measure Ca2+ in Saccharomyces cerevisiae was reported previously and the same methodology was also used to detect Ca2+ in N. crassa recently. Subsequently, genetically engineered Ca2+ indicators (GECIs) like aequorins and RGECOs have been developed as Ca2+ indicators to detect and visualize Ca2+ inside the cell. In this review, we summarize various methodologies used to detect and measure Ca2+ in filamentous fungi with their advantages and limitations. •Chlortetracycline (CTC) fluorescence assay is used for visualizing Ca2+ level, whereas microelectrodes technique is used to determine Ca2+ flux in the cell.•Radioactive 45Ca is useful for quantification of Ca2+ in the cellular compartments.•Genetically modified calcium indicators (GECIs) are used to study Ca2+ dynamics in the cell.

Mycological Methods for Routine Air Sampling and Interpretation of Results in Operating Theaters.

Many infectious diseases are transmitted via the air and are, therefore, particularly difficult to combat. These infections include various invasive mycoses caused by molds. The usual route of infection is the inhalation of conidia. In hospitals, infection can also occur through the deposition of conidia in otherwise sterile anatomical sites during surgical and other invasive procedures. Therefore, knowledge of airborne mold concentrations can lead to measures to protect patients from fungal infections. The literature on this topic contains insufficient and sometimes ambiguous information. This is evidenced by the fact that there are no international recommendations or guidelines defining the methodology of air sampling and the interpretation of the results obtained. Surgical departments, intensive care units and medical mycology laboratories are, therefore, left to their own devices, leading to significant differences in the implementation of mycological surveillance in hospitals. The aim of this mini-review is to provide an overview of the current methods of air sampling and interpretation of results used in medical mycology laboratories.

An Efficient Homologous Recombination-Based In Situ Protein-Labeling Method in Verticillium dahliae.

Accurate determination of protein localization, levels, or protein-protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.