Fungal Cell Wall Composition Research Articles

Cannabinoids: A new natural agent to control postharvest decay development

Pathogenic fungi are a major cause of postharvest loss of fresh produce. While chemical fungicides effectively control postharvest diseases their use raises concerns and alternatives are required. Natural compounds with antifungal properties and natural defense response elicitors are explored. Among those compounds, phytocannabinoids are secondary metabolites generated from Cannabis sativa that are of particular interest. Among the non-psychotropic cannabinoids are cannabidiol (CBD) and cannabigerol (CBG). The current study investigates the effect of CBD and CBG’s effect on postharvest fungal pathogens and the fruit defense response. CBD and CBG inhibit the growth of major postharvest pathogens such as Botrytis cinerea, Penicillium expansum, and Colletotrichum gloeosporioides. Moreover, CBD and CBG significantly decreased gray mold development caused by B. cinerea on grapes. Studying their mode of action indicated that both CBD and CBG affected B. cinerea fungal cell wall composition and cell-wall-related remodeling genes, as well as cell membrane fluidity and permeability. CBD treatment causes hyperpolarization of the fungal mitochondria membrane, which results in increased intracellular ROS levels. Evaluation of the effect of CBD and CBG on the fruit defense response displayed a priming effect by up-regulating pathogenic related proteins and induced salicylic acid and flavonols production resulting in inhibition of decay development. Overall, the results of the current study reveal the direct and indirect ability of natural phytocannabinoids to control fungal decay development and reduce food loss. This novel eco-friendly postharvest treatment has the potential to replace chemically hazardous fungicides.

A comparative study between Fusarium solani and Neocosmospora vasinfecta revealed differential profile of fructooligosaccharide production.

Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the food's nutritional and technological properties. The rhizosphere of plants that accumulate fructopolysaccharides as inulin has been revealed as a source of filamentous fungi. These fungi can produce FOS either by inulin hydrolysis or by biosynthesis from sucrose, including unusual FOS with enhanced prebiotic properties. Here, we investigated the ability of Fusarium solani and Neocosmospora vasinfecta to produce FOS from different carbon sources. Fusarium solani and N. vasinfecta grew preferentially in inulin instead of sucrose, resulting in the FOS production as the result of endo-inulinase activities. N. vasinfecta was also able to produce the FOS 1-kestose and 6-kestose from sucrose, indicating transfructosylating activity, absent in F. solani. Moreover, the results showed how these carbon sources affected fungal cell wall composition and the expression of genes encoding for β-1,3-glucan synthase and chitin synthase. Inulin and fructose promoted changes in fungal macroscopic characteristics partially explained by alterations in cell wall composition. However, these alterations were not directly correlated with the expression of genes related to cell wall synthesis. Altogether, the results pointed to the potential of both F. solani and N. vasinfecta to produce FOS at specific profiles.

The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogen Zymoseptoria tritici.

Summary Zymoseptoria tritici is a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections, the fungus shows a markedly different gene expression pattern. However, the underlying mechanisms causing differential gene expression during host and non‐host interactions are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection programme in compatible hosts. MoCOD1, a member of the fungal Zn(II)2Cys6 transcription factor family, has been shown to directly affect pathogenicity in the rice blast pathogen Magnaporthe oryzae. Here, we analyse the role of the putative transcription factor Zt107320, a homologue of MoCOD1, during infection of compatible and incompatible hosts by Z. tritici. We show for the first time that Zt107320 is differentially expressed in host versus non‐host infections and that lower expression corresponds to an incompatible infection of non‐hosts. Applying reverse genetics approaches, we further show that Zt107320 regulates the dimorphic switch as well as the growth rate of Z. tritici and affects fungal cell wall composition in vitro. Moreover, ∆Zt107320 mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 is involved in the regulation of growth processes and pathogenicity during infection.

The LaeA orthologue in Epichloë festucae is required for symbiotic interaction with Lolium perenne

LaeA is a conserved global regulator of secondary metabolism and development in fungi. It is often required for successful pathogenic interactions. In this study, the laeA homologue in the fungal grass endophyte E. festucae was deleted and functionally characterised in vitro and its role in the mutualistic E. festucae interaction with Lolium perenne (perennial ryegrass) was determined. We showed that laeA in E. festucae is required for normal hyphal morphology, resistance to oxidative stress, and conidiation under nutrient-limited in vitro conditions. In planta studies revealed that laeA is expressed in a tissue-specific manner and is required to form a compatible plant interaction, with the majority of seedlings inoculated with a laeA deletion mutant either dying or being uninfected. In mature infected plants no difference was observed in the number or morphology of endophytic hyphae. However, the number of epiphyllous hyphae were greatly increased. Comparative transcriptomics analyses suggested roles for plant cell wall degradation, fungal cell wall composition, secondary metabolism and small-secreted proteins in Epichloë foliar symbiosis.

MaPmt4, a protein O-mannosyltransferase, contributes to cell wall integrity, stress tolerance and virulence in Metarhizium acridum.

In eukaryotic cells, protein O-glycosylation is an essential protein modification. Analysis of the Metarhizium acridum genome database revealed a total of three O-glycoside mannosyltransferase homologs (Pmt1, Pmt2 and Pmt4), closely related to Saccharomyces cerevisiae Pmt1, Pmt2, and Pmt4. In this study, the functions of MaPmt4, encoding a protein O-mannosyltransferase in M. acridum, were characterized using disruption and complementation strategies. Disruption of MaPmt4 delayed the conidial germination and reduced the fungal tolerances to heat shock and UV-B irradiation, but did not affect conidial yield. Inactivation of MaPmt4 displayed increased sensitivity to cell wall-perturbing agents, formed thinner cell walls, and changed composition of fungal cell wall, demonstrating that MaPmt4 was also important to maintain fungal cell wall integrity. Bioassays by topical inoculation and intrahemocoel injection showed that the MaPmt4 deletion mutant exhibited greatly reduced virulence. The subsequent examination revealed that the inactivation of MaPmt4 impaired appressorium formation, decreased fungal growth in locust hemolymph in vitro, and boosted insect immune responses, the latter in part potentially owing to the changes in conidial surface structures, and thus attenuated the virulence of MaPmt4 deletion mutant. Furthermore, the results of comparative proteomics showed that MaPmt4 played important roles in fungal cell wall integrity, stress tolerances, and virulence via broad genetic pathways.

Lung eosinophil recruitment in response to Aspergillus fumigatus is correlated with fungal cell wall composition and requires γδ T cells

The differential recognition of fungal cell wall polysaccharides that program innate and adaptive immunity to the human opportunistic fungal pathogen Aspergillus fumigatus has been a focus of considerable interest. In a mouse model of fungal conidia aspiration, decreased relative levels of cell wall core carbohydrates β-1,3-glucan to chitin in A. fumigatus isolates and mutant strains were correlated with increased airway eosinophil recruitment. In addition, an increase in fungal surface chitin exposure induced by the β-1,3-glucan synthesis-targeting drug caspofungin was associated with increased murine airway eosinophil recruitment after a single challenge of conidia. The response to increased A. fumigatus chitin was associated with increased transcription of IL-17A after a single aspiration, although this cytokine was not required for eosinophil recruitment. Rather, both RAG1 and γδ T cells were required, suggesting that this subset of innate-like lymphocytes may be an important regulator of potentially detrimental type 2 immune responses to fungal inhalation and infection.

The fungal-specific β-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants.

β-glucans are well-known modulators of the immune system in mammals but little is known about β-glucan triggered immunity in planta. Here we show by isothermal titration calorimetry, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy that the FGB1 gene from the root endophyte Piriformospora indica encodes for a secreted fungal-specific β-glucan-binding lectin with dual function. This lectin has the potential to both alter fungal cell wall composition and properties, and to efficiently suppress β-glucan-triggered immunity in different plant hosts, such as Arabidopsis, barley and Nicotiana benthamiana. Our results hint at the existence of fungal effectors that deregulate innate sensing of β-glucan in plants.

Extracellular Vesicle-Associated Transitory Cell Wall Components and Their Impact on the Interaction of Fungi with Host Cells.

Classic cell wall components of fungi comprise the polysaccharides glucans and chitin, in association with glycoproteins and pigments. During the last decade, however, system biology approaches clearly demonstrated that the composition of fungal cell walls include atypical molecules historically associated with intracellular or membrane locations. Elucidation of mechanisms by which many fungal molecules are exported to the extracellular space suggested that these atypical components are transitorily located to the cell wall. The presence of extracellular vesicles (EVs) at the fungal cell wall and in culture supernatants of distinct pathogenic species suggested a highly functional mechanism of molecular export in these organisms. Thus, the passage of EVs through fungal cell walls suggests remarkable molecular diversity and, consequently, a potentially variable influence on the host antifungal response. On the basis of information derived from the proteomic characterization of fungal EVs from the yeasts Cryptoccocus neoformans and Candida albicans and the dimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis, our manuscript is focused on the clear view that the fungal cell wall is much more complex than previously thought.

Lectin-microarray technique for glycomic profiling of fungal cell surfaces.

Lectin microarrays are rows of lectins with different carbohydrate-binding specificities spotted on surfaces of glass slides. Lectin microarray technique enables glycomic analyses of carbohydrate composition of fungal cell walls. We will describe an application of the technique in analyzing cell surface glycome of yeast-form fungal cells in the living state. The analysis reveals genus- and species-dependent complex cell surface carbohydrate structures of fungi, and enabled us, therefore, to suggest that cell walls of yeast cells, which have been considered to have relatively simple structures, actually have a more complex structure containing galactose and fucose. This shows that the technique can be used to find new insights into the study of phylogenetic relations and into the classification of cells in the fungal kingdom based on cell wall glycome.

Stage-specific innate immune recognition ofAspergillus fumigatusand modulation by echinocandin drugs

The pulmonary innate immune system clears inhaled Aspergillus fumigatus conidia (spores) from terminal airways. Failure to control conidial germination in immune compromised hosts can result in hyphal tissue invasion and fatal disease. Insight into the molecular recognition of A. fumigatus by host leukocytes indicates that the innate immune system exploits obligate changes in fungal cell wall composition that occur at the first stage of germination, conidial swelling. Germinating spores activate at least two host signal transduction pathways. Surface exposure of fungal beta-(1,3) glucan, a polysaccharide constituent of the fungal cell wall, triggers dectin-1 signaling by host phagocytes. Spore germination leads to the induction of Toll-like receptor (TLR) signaling as well. This stage-specific recognition mechanism focuses host antifungal responses on cells with the potential for tissue invasion and may serve to limit potentially deleterious effects of inflammation in space and time. Fungal beta-(1,3) glucan not only activates host innate immune responses but also represents the target of echinocandin drugs. The activity of echinocandin drugs has largely been understood on the basis of pharmacologic growth inhibition of yeast and moulds, resulting in lysis of yeast cells and stunting of dysmorphic hyphae. The recognition that fungal beta-1,3 glucan activates dectin-1 signaling suggests that echinocandin drugs may exert immune modulatory effects by altering innate immune responses to drug-treated fungal cells, a view supported by recent data from studies on C. albicans, A. fumigatus, and non-Aspergillus moulds.

Use of Molecular Cytology to Study the Structure and Biology of Phytopathogenic and Mycorrhizal Fungi

Molecular cytology, that is, thein situlocalization of selected molecules by labeling with lectins, enzymes, and antibodies, has made a major contribution to our understanding of the structure and biology of fungi and is increasingly becoming an integral part of molecular, genetic, and biochemical studies. The review presented in this article concentrates on recent advances in the application of molecular cytology in investigations of the structure and biology of phytopathogenic and mycorrhizal fungi and of the molecular basis of their infection of host plants. The review examines details of the structure and molecular composition of fungal cell walls revealed by lectin, enzyme, and antibody labeling. Molecular composition is shown to vary according to taxonomic relationships and as a reflection of differences in cell type, location within the cell, and within thickness of the wall. Sites of synthesis and secretion of wall components are also detected through the labeling of selected molecules.In situlabeling of cytoskeletal elements, microtubules and actin microfilaments, has provided much information on the role of these elements in tip growth, organelle distribution, and spore development. Molecular cytology, particularly through the generation of monoclonal antibodies, has also revealed new and exciting information on specialized infection structures formed by fungi in order to infect host plants. The sites of storage and secretion of adhesives and degradative enzymes have been documented, as have surface specializations that may be associated with avoidance of detection by the host. In addition,in situlabeling with enzymes and antibodies has aided studies of the host defense response, including mechanisms of detection of fungal elicitor molecules, changes in wall composition, and the secretion of antifungal compounds. With the increasing production of monoclonal antibodies to fungal molecules, molecular cytology promises to continue to make an important contribution to our understanding of fungal cell structure and function in the future.

Degradation of fungal cell walls taking into consideration the polysaccharide composition

Differences in polysaccharide composition of various fungal cell walls were indicated by their susceptibility to enzymatic digestion. This information was used to optimize the enzymatic extraction of intracellular enzymes or the preparation of fungal protoplasts in high yield. Bacterial glucanase and chitinase specially purified were used for this study. Mycelium of Aspergillus niger grown on uric acid was treated with mixtures of glucanase and chitinase. Cell wall breakdown products were analysed and the ratio of chitin to glucan was estimated to be 1:1.4. A. niger protoplast formation was optimized using this information. When the mixture of chitinase to glucanase was 1:1.4, similar to the fungal cell wall composition, a 95% yield of protoplasts was obtained after 30 min and their mean size was 7 μm. However, a ratio of 1.5 to 1 (chitinase to glucanase) was needed for the maximum extraction of uricase. Yield was 10.5 μ g −1 cells after 1.5 h incubation at 28°C. Glucanase alone resulted in a maximum yield of 1.9 μ g −1 while chitinase alone yielded 6.0 μ g −1 under the same conditions.

The Occurrence of 1,3-β-Glucanase in Healthy andVerticillium-infected, Resistant and Susceptible Tomato Plants

The Occurrence of 1,3-β-Glucanase in Healthy and<i>Verticillium</i>-infected, Resistant and Susceptible Tomato Plants

An Assessment of Infrared Spectra as Indicators of Fungal Cell Wall Composition

Infrared spectroscopy is assessed as a technique for identifying polymers derived from fungal cell walls, both as isolated materials and in mixtures with one another. The technique is then applied to a study of the composition of fungal cell walls and the conclusion reached that infrared spectra provide a rapid and valuable indication of the major components of such walls. They can also be used to follow the effect of chemical treatments designed to separate major wall components.