Fungal Reactive Oxygen Species Research Articles

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

A Putative NADPH Oxidase Gene in Unicellular Pathogenic Candida glabrata Is Required for Fungal ROS Production and Oxidative Stress Response.

Most previous studies on fungal NADPH oxidases (Nox) focused on multicellular fungi and highlighted the important roles of Nox-derived reactive oxygen species (ROS) in cellular differentiation and signaling communication. However, there are few reports about Nox in unicellular fungi. A novel NOX ortholog, CAGL0K05863g (named CgNOX1), in Candida glabrata was investigated in this study. Deletion of CgNOX1 led to a decrease in both intracellular and extracellular ROS production. In addition, the Cgnox1∆ mutant exhibited hypersensitivity to hydrogen peroxide and menadione. Also, the wild-type strain showed higher levels of both CgNOX1 mRNA expression and ROS production under oxidative stress. Moreover, the absence of CgNOX1 resulted in impaired ferric reductase activity. Although there was no effect on in vitro biofilm formation, the CgNOX1 mutant did not produce hepatic apoptosis, which might be mediated by fungal Nox-derived ROS during co-incubation. Together, these results indicated that the novel NOX gene plays important roles in unicellular pathogenic C. glabrata and its interaction with host cells.

Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity.

Aspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk for IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with chronic granulomatous disease (CGD). However, treatments for Aspergillus infections remain limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization classified A. fumigatus as a critical priority fungal pathogen. Our cell death research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

Design, synthesis, and biological evaluation of selenium-containing small molecule compounds based on the dual mechanism of fungal CYP51 inhibition and fungal ROS generation

Herein, we modified our previously published miconazole analogue containing selenium A03 of fungal CYP51 (belongs to the cytochrome P450 monooxygenase (CYP) superfamily) inhibitors to obtain novel analogues with inhibitory effects on multiple fungal pathogens, but also with a novel mechanism of action such as inducing fungal ROS (reactive oxygen species) generation. Firstly, three novel series of selenium-containing azole derivatives were prepared to determine their antifungal activity against common test fungal strains and fluconazole-resistant strains. Among all the novel derivatives, compounds B06, B11 and B14 with excellent antifungal activity (0.03-4 µg/mL) can inhibit the fluconazole-resistant Candida Strain 17#, CaR, 632, 901 and 904 in the range of MIC (minimal inhibitory concentrations) values (0.25–16 µg/mL). Antifungal mechanism studies revealed representative compounds not only had dose-dependent inhibitory activity of CYP51 but also down-regulated the expression of resistance-associated genes. Interestingly, representative compound B11 could also promote the generation of ROS. Therefore, the MIC values of Candida albicans (SC5314) inhibited by B11 and C06 in the presence of different concentrations of antioxidant NAC (N-acetyl-L-cysteine) were measured to further verify the synergistic antifungal effect between the mechanism promoting fungal ROS production and CYP51 inhibitory activity. Meanwhile, the results of cytotoxic evaluation showed that representative compounds were at the same level as that of miconazole with IC50 of 8.61∼29.91 µM, suggesting that the toxicity of the target compounds did not increase after the introduction of selenium atoms. Moreover, the in vitro stability assays also indicated our developed compounds possessed better liver microsomal metabolic stabilities (T1/2=14.9–20.1 min) compared with miconazole (T1/2=8.3 min). And representative compound B11 also displayed stability in rat plasma (T1/2>360 min). In addition, the docking results showed that the binding modes of target compounds B06, B11 and C06 to Candida albicans CYP51 were almost the same as those of miconazole. Overall, the selenium-containing small molecule compounds with dual mechanism can be further developed for the potential treatment of invasive fungal infections.

Sclerotinia sclerotiorum utilizes host-derived copper for ROS detoxification and infection.

Necrotrophic plant pathogen induces host reactive oxygen species (ROS) production, which leads to necrosis in the host, allowing the pathogen to absorb nutrients from the dead tissues. Sclerotinia sclerotiorum is a typical necrotrophic pathogen that causes Sclerotinia stem rot in more than 400 species, resulting in serious economic losses. Here, we found that three S. sclerotiorum genes involved in copper ion import/transport, SsCTR1, SsCCS and SsATX1, were significantly up-regulated during infection of Brassica oleracea. Function analysis revealed that these genes involved in fungal ROS detoxification and virulence. On the host side, four genes putatively involved in copper ion homeostasis, BolCCS, BolCCH, BolMT2A and BolDRT112, were significantly down-regulated in susceptible B. oleracea, but stably expressed in resistant B. oleracea during infection. Their homologs were found to promote resistance to S. sclerotiorum and increase antioxidant activity in Arabidopsis thaliana. Furthermore, copper concentration analysis indicated that copper flow from healthy area into the necrotic area during infection. A model was proposed that S. sclerotiorum utilizes host copper to detoxify ROS in its cells, whereas the resistant hosts may restrict the supply of essential copper nutrients to S. sclerotiorum by maintaining copper ion homeostasis during infection.

Optimization of the HyPer sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant-pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H2 O2 ) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H2 O2 dynamics in real time during important stages of the plant infection process.

Appressorium‐localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host‐specific, toxin‐producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant-pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.

OCCURRENCE, DISTRIBUTION AND BALANCE OF ROS DURING NITROGEN DEPRIVATION AND EARLY DEVELOPMENT IN COLLETOTRICHUM ACUTATUM INFECTING STRAWBERRY

A nonpathogenic mutant of Colletotrichum acutatum exhibited extended germ tube growth prior to appressoria formation on solid surface and strawberry leaves in the absence of an external nitrogen source, but grew necrotrophically when inoculated directly onto wounded sites. The deduced amino acid sequence of the impaired gene product was characterized as a transcriptional regulator of nitrogen metabolism. Inoculation of leaves with wildtype or the mutant in the presence of a preferred nitrogen source resulted in massive epiphytic hyphal production, appressoria formation and rapid symptom development. Proteins patterns of different developmental stages and growth conditions revealed variation in abundance of proteins belonging to the ROS (Reactive oxygen species) scavenger machinery. CuZn superoxide dismutase and glutathione peroxidase were up-regulated at the appressoria formation stage and nitrogen limiting conditions, compared to growth in complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressoria formation stage. Fungal ROS was detected within germinating conidia during strawberry host pre-penetration, penetration and colonization stages. Application of an exogenous antioxidant quenched ROS production and reduced appressoria formation frequency. This study indicates that developmental processes occurring under nutritional deprivation in C. acutatum infecting strawberry are facilitated by metabolic shifts which may be mediated by fungal ROS production.

Mechanisms modulating fungal attack in post-harvest pathogen interactions and their control

As biotrophs, insidious fungal infections by post-harvest pathogens remain quiescent during fruit growth, but at a particular phase, during ripening and senescence, the pathogens transform to necrotrophs and elicit the typical decay symptoms. Exposure of unripe hosts to pathogens initiates defensive signal-transduction cascades that limit fungal growth and development. Exposure to the same pathogens during ripening and storage activates a substantially different signalling cascade that facilitates fungal colonization. This review will focus on modulation of post-harvest host-pathogen interactions by pH and reactive oxygen species (ROS). Modulation of host pH in response to a host signal is bidirectional and includes either alkalinisation by ammonification of the host tissue, or acidification by secretion of organic acids. These changes sensitise the host and activate the transcription and secretion of fungal hydrolases that promote maceration of the host tissue. This sensitisation is further enhanced at various stages by the accumulation of host or fungal ROS that can further weaken host tissue and amplify fungal development. Several specific examples of coordinated responses that conform with this scheme are described, followed by discussion of the means to exploit these mechanisms to establish new approaches to post-harvest disease control.

The small GTPase Rac and the p21‐activated kinase Cla4 in Claviceps purpurea: interaction and impact on polarity, development and pathogenicity

Claviceps purpurea, the ergot fungus, is a highly specialized pathogen of grasses; its colonization of host ovarian tissue requires an extended period of strictly polarized, oriented growth towards the vascular tissue. To understand this process, we study the role of signalling factors affecting polarity and differentiation. We showed that the small GTPase Cdc42 is involved in polarity, sporulation and in planta growth in C. purpurea. Here we present evidence that the GTPase Rac has an even stronger and, in some aspects, inverse impact on growth and development: Deltarac mutants form coralline-like colonies, show hyper-branching, loss of polarity, sporulation and ability to penetrate. Functional analyses and yeast two-hybrid studies prove that the p21-activated kinase Cla4 is a major downstream partner of Rac. Phosphorylation assays of MAP kinases and expression studies of genes encoding reactive oxygen species (ROS)-scavenging and -generating enzymes indicate a function of Rac and Cla4 in fungal ROS homoeostasis which could contribute to their drastic impact on differentiation.

Differential protein expression in Colletotrichum acutatum: changes associated with reactive oxygen species and nitrogen starvation implicated in pathogenicity on strawberry

The cellular outcome of changes in nitrogen availability in the context of development and early stages of pathogenicity was studied by quantitative analysis of two-dimensional gel electrophoresis of Colletotrichum acutatum infecting strawberry. Significant alterations occurred in the abundance of proteins synthesized during appressorium formation under nitrogen-limiting conditions compared with a complete nutrient supply. Proteins that were up- or down-regulated were involved in energy metabolism, nitrogen and amino acid metabolism, protein synthesis and degradation, response to stress and reactive oxygen scavenging. Members belonging to the reactive oxygen species (ROS) scavenger machinery, superoxide dismutase and glutathione peroxidase, were up-regulated at the appressorium formation stage, as well as under nitrogen-limiting conditions relative to growth with a complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressorium formation stage. Fungal ROS were detected within germinating conidia during host pre-penetration, penetration and colonization stages, accompanied by plant ROS, which were abundant in the apoplastic space. Application of exogenous antioxidants quenched ROS production and reduced the frequency of appressorium formation. Up-regulation in metabolic activity was detected during appressorium formation and nutrient deficiency compared with growth under complete nutrient supply. Enhanced levels of proteins related to the glyoxylate cycle and lipid metabolism (malate dehydrogenase, formate dehydrogenase and acetyl-CoA acetyltransferase) were observed at the appressorium formation stage, in contrast to down-regulation of isocitrate dehydrogenase. The present study demonstrates that appressoria formation processes, occurring under nutritional deprivation, are accompanied by metabolic shifts, and that ROS production is an early fungal response that may modulate initial stages of pathogen development.

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus-plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloë festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae-perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus-plant interaction.