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Production of superoxide and conidia of Coniothyrium minitans CmNox1 and CmNox2 mutants. (a) Detection of superoxide using colony staining with NBT solution (0.05% w/v, 30 min) of cultures of C. minitans wild-type strain ZS-1, CmNox1 deletion mutant ΔCmNox1-6, CmNox2 deletion mutant ΔCmNox2-323 on PDA. (b) Pycnidial and conidial production of C. minitans wild-type strain ZS-1, CmNox1 deletion mutant ΔCmNox1-6, complementary transformant CmNox1-C8, and CmNox2 deletion mutant ΔCmNox2-323 12 days after incubation on PDA. Compared to ZS-1, CmNox1-C8 and ΔCmNox2-323, ΔCmNox1-6 lost the ability to produce pycnidia.
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The NADPH oxidase complex of a sclerotial mycoparasite Coniothyrium minitans, an important biocontrol agent against crop diseases caused by Sclerotinia sclerotiorum, was identified and its functions involved in conidiation and mycoparasitism were studied. Gene knock-out and complementary experiments indicated that CmNox1, but not CmNox2, is necessa...
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... multiple alignment analysis showed that both CmNox1 and CmNox2 contained NOX family signature regions. Phylogenetic analysis of NADPH oxidases in several fungi placed CmNox1 and CmNox2 homologs into two different clades based on the amino acid sequences (Supplementary Figure S1). ...
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... staining with NBT solution showed that superoxide production was decreased significantly in ΔCmNox1-6, compared to ΔCmNox2-323 and the wild-type strain ZS-1 (Fig. 1a). The conidiation of the wild-type strain ZS-1, CmNox1 deletion mutants, CmNox1 complemented mutants, and CmNox2 deletion mutants were determined after incubating for 15 days on PDA (Fig. 1b). CmNox1 deletion mutants completely lost the abil- ity to produce conidia (Table 1). In contrast, the wild-type strain ZS-1, CmNox1 complemented ...
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... staining with NBT solution showed that superoxide production was decreased significantly in ΔCmNox1-6, compared to ΔCmNox2-323 and the wild-type strain ZS-1 (Fig. 1a). The conidiation of the wild-type strain ZS-1, CmNox1 deletion mutants, CmNox1 complemented mutants, and CmNox2 deletion mutants were determined after incubating for 15 days on PDA (Fig. 1b). CmNox1 deletion mutants completely lost the abil- ity to produce conidia (Table 1). In contrast, the wild-type strain ZS-1, CmNox1 complemented mutants and CmNox2 deletion mutants were normal in conidiation under the same condition (Table 1). Unlike strain ZS-1 and ΔCmNox2-323, which could form matured pycnidia and conidia, ΔCmNox1-6 ...
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... strain ZS-1, CmNox1 complemented mutants and CmNox2 deletion mutants were normal in conidiation under the same condition (Table 1). Unlike strain ZS-1 and ΔCmNox2-323, which could form matured pycnidia and conidia, ΔCmNox1-6 could only form a few pycnidial primordia that could not further develop to mature pycnidia, and no conidium was produced (Fig. 1b). These data indicated that CmNox1, but not CmNox2, played significant roles in conidiation and production of super- oxide. The experiments also suggested CmNox1 and CmNox2 are not essential for hyphal growth of C. minitans on ...
Citations
... Moreover, as there were more than 8000 hits in PubMed (https://pubmed.ncbi.nlm.nih.gov/) on "Rac1 GTPase" as of 30 January 2024, we will further concentrate on the shared functions of the two GTPases in these evolutionary distant organisms when associating with and travelling to mitochondria. For a broader overview on other important roles of Rac1 and its fellow Rho-type GTPases in human health and disease, the reader is referred to several excellent reviews [1][2][3][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23], the indicated selection being by no means exhaustive, with apologies to all authors inevitably left unacknowledged. The apology also extends to citations on details of Rac1 regulation, which only refer to a few recent original works that cover previous literature. ...
Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs.
... Numerous genes have been identified to be involved in C. minitans conidiation and mycoparasitism (Rogers et al., 2008;Wei et al., 2013Wei et al., , 2016Yang et al., 2016). Among them, several previously reported signalling pathways, including those associated with mitochondrial structure and function, ROS accumulation and peroxisomes, are upstream of the intrinsic apoptotic pathway (Albert & Sellem, 2002;Jiang & Wang, 2004;Osiewacz & Borghouts, 2000;Scheckhuber et al., 2007;Sharon et al., 2009;Smith et al., 2010;Tanaka et al., 2019). ...
... Among them, several previously reported signalling pathways, including those associated with mitochondrial structure and function, ROS accumulation and peroxisomes, are upstream of the intrinsic apoptotic pathway (Albert & Sellem, 2002;Jiang & Wang, 2004;Osiewacz & Borghouts, 2000;Scheckhuber et al., 2007;Sharon et al., 2009;Smith et al., 2010;Tanaka et al., 2019). Certain genes, such as CmNox1, CmPEX6, CmAim24 and PIF1, play essential roles in ROS generation, oxidant production, mitochondrial morphology regulation and maintaining mitochondrial stability (Rogers et al., 2008;Wei et al., 2013Wei et al., , 2016Yang et al., 2020), indicating their critical involvement in regulating conidiation and mycoparasitism through the mitochondriondependent apoptosis pathway. ...
Apoptosis‐like programmed cell death is associated with fungal development, ageing, pathogenicity and stress responses. Here, to explore the potential of Botrytis cinerea type II inhibitor of apoptosis (IAP) BcBIR1 in elevating the biocontrol efficacy of Coniothyrium minitans, the BcBIR1 gene was heterologously expressed in C. minitans. Results indicated that the strains expressing BcBIR1 had higher rates of conidiation, mycelial growth and biomass growth than the wild‐type strain. Moreover, BcBIR1 was found to inhibit apoptosis, indicating its role as an IAP in C. minitans. Under various abiotic stresses, the growth rates of BcBIR1‐expressing strains were significantly higher than that of the wild‐type strain. Moreover, the conidial survival rate of the BcBIR1‐expressing strains treated with ultraviolet irradiation was enhanced. In antifungal activity assay, the culture filtrates of BcBIR1‐expressing strains displayed a stronger inhibitory effect on B. cinerea and Sclerotinia sclerotiorum than the wild‐type strain. The study also found that BcBIR1 expression increased the mycoparasitism against the sclerotia, but not the hyphae of S. sclerotiorum. Taken together, these results suggest that BcBIR1 enhances vegetative growth, conidiation, anti‐apoptosis activity, abiotic stress resistance, antifungal activity and mycoparasitism in C. minitans. As an IAP, BcBIR1 may improve the control capacity of C. minitans against S. sclerotiorum.
... Serine/threonine-protein kinase SKY1(MSTRG.7638) has a much higher expression level in IL and MF compared to that in ST. MAP kinases belong to the family of serine/threonine protein kinases and are activated by a MAPKKK-MAPKK-MAP kinase cascade, which plays critical roles in pathogenicity and in fungal development [38]. It was suggested that the ROS signal and MAPK cascade might be cross-linked and crucial for the fungal pathogenicity and sexual development in O. sinensis. ...
Ophiocordyceps sinensis, an ascomycete caterpillar fungus, has been used as a Traditional Chinese Medicine owing to its bioactive properties. However, until now the bio-active peptides have not been identified in this fungus. Here, the raw RNA sequences of three crucial growth stages of the artificially cultivated O. sinensis and the wild-grown mature fruit-body were aligned to the genome of O. sinensis. Both homology-based prediction and de novo-based prediction methods were used to identify 8541 putative antioxidant peptides (pAOPs). The expression profiles of the cultivated mature fruiting body were similar to those found in the wild specimens. The differential expression of 1008 pAOPs matched genes had the highest difference between ST and MF, suggesting that the pAOPs were primarily induced and play important roles in the process of the fruit-body maturation. Gene ontology analysis showed that most of pAOPs matched genes were enriched in terms of ‘cell redox homeostasis’, ‘response to oxidative stresses’, ‘catalase activity’, and ‘ integral component of cell membrane’. A total of 1655 pAOPs was identified in our protein-seqs, and some crucial pAOPs were selected, including catalase, peroxiredoxin, and SOD [Cu–Zn]. Our findings offer the first identification of the active peptide ingredients in O. sinensis, facilitating the discovery of anti-infectious bio-activity and the understanding of the roles of AOPs in fungal pathogenicity and the high-altitude adaptation in this medicinal fungus.
... However, its importance and regulatory functions vary among different fungal pathogens. For example, deletion of ChMK1 results in hypersensitivity to CFW and CR in C. higginsianum (Wei et al. 2016), but the Cfpmk1 mutant has increased tolerance against CR and SDS in C. fructicola . Whereas the CcPmk1 mutant is hypersensitive to cell wall stress and cell wall lytic enzymes in Cytospora chrysosperma (Yu et al. 2019), the chk1 mutant has the hyphal autolysis and aerial hyphal growth defects in C. heterostrophus (Lev et al. 1999). ...
Like other eukaryotes, fungi use MAP kinase (MAPK) pathways to mediate cellular changes responding to external stimuli. In the past two decades, three well-conserved MAP kinase pathways have been characterized in various plant pathogenic fungi for regulating responses and adaptations to a variety of biotic and abiotic stresses encountered during plant infection or survival in nature. The invasive growth (IG) pathway is homologous to the yeast pheromone response and filamentation pathways. In plant pathogens, the IG pathway often is essential for pathogenesis by regulating infection-related morphogenesis, such as appressorium formation, penetration, and invasive growth. The cell wall integrity (CWI) pathway also is important for plant infection although the infection processes it regulates vary among fungal pathogens. Besides its universal function in cell wall integrity, it often plays a minor role in responses to oxidative and cell wall stresses. Both the IG and CWI pathways are involved in regulating known virulence factors as well as effector genes during plant infection and mediating defenses against mycoviruses, bacteria, and other fungi. In contrast, the high osmolarity growth (HOG) pathway is dispensable for virulence in some fungi although it is essential for plant infection in others. It regulates osmoregulation in hyphae and is dispensable for appressorium turgor generation. The HOG pathway also plays a major role for responding to oxidative, heat, and other environmental stresses and is overstimulated by phenylpyrrole fungicides. Moreover, these three MAPK pathways crosstalk and coordinately regulate responses to various biotic and abiotic stresses. The IG and CWI pathways, particularly the latter, also are involved in responding to abiotic stresses to various degrees in different fungal pathogens, and the HOG pathway also plays a role in interactions with other microbes or fungi. Furthermore, some infection processes or stress responses are co-regulated by MAPK pathways with cAMP or Ca ²⁺ /CaM signaling. Overall, functions of individual MAP kinase pathways in pathogenesis and stress responses have been well characterized in a number of fungal pathogens, showing the conserved genetic elements with diverged functions, likely by rewiring transcriptional regulatory networks. In the near future, applications of genomics and proteomics approaches will likely lead to better understanding of crosstalk among the MAPKs and with other signaling pathways as well as roles of MAPKs in defense against other microbes (biotic interactions).
... From the MC to ST stages, the host Hepialus larvae acts as a growth container for O. sinensis mycelium, splitting and proliferating continuously in it 31 . The increase in body filling leads to increased pressure in the surrounding cells, which induces autophosphorylation of sln1 , then subsequently transmitting the stress signal from sln1 to ssk1 through ypd1, thereby activating MAPK 22,32,33 . On the other hand, oxidative stress can activate different signal transduction pathways, either via the localization of specific regulators to the nucleus upon stress, followed by subsequent activation of detoxification genes expressions, or phosphorylation-driven intervention of the MAPK Table 1. ...
Ophiocordyceps sinensis has been a source of valuable materials in traditional Asian medicine for over two thousand years. With recent global warming and overharvest, however, the availability of these wild fungi has decreased dramatically. While fruiting body of O. sinensis has been artificially cultivated, the molecular mechanisms that govern the induction of fruiting body at the transcriptional and post-transcriptional levels are unclear. In this study, we carried out both mRNA and small RNA sequencing to identify crucial genes and miRNA-like RNAs (milRNAs) involved in the development of fruiting body. A total of 2875 differentially expressed genes (DEGs), and 71 differentially expressed milRNAs (DEMs) were identified among the mycoparasite complex, the sclerotium (ST) and the fruiting body stage. Functional enrichment and Gene Set Enrichment Analysis indicated that the ST had increased oxidative stress and energy metabolism and that mitogen-activated protein kinase signaling might induce the formation of fruiting body. Integrated analysis of DEGs and DEMs revealed that n_os_milR16, n_os_milR21, n_os_milR34, and n_os_milR90 could be candidate milRNAs that regulate the induction of fruiting body. This study provides transcriptome-wide insight into the molecular basis of fruiting body formation in O. Sinensis and identifies potential candidate genes for improving induction rate.
... Filamentous fungi also use NOX adaptors Bem (equivalent to animal p40 and/or p47) and NoxD (p22) (7,8). ROS from Rac activated fungal NOX is known to signal polarized cell growth across diverse classes of filamentous fungi (8)(9)(10)(11)(12)(13)(14). ...
Across eukaryotes, Rho GTPases such as Rac and Cdc42 play important roles in establishing cell polarity, which is a key feature of cell growth. In mammals and filamentous fungi, Rac targets large protein complexes containing NADPH oxidases (NOX) that produce reactive oxygen species (ROS). In comparison, Rho GTPases of unicellular eukaryotes were believed to signal cell polarity without ROS, and it was unclear whether Rho GTPases were required for ROS production in these organisms. We document here the first example of Rho GTPase-mediated post-transcriptional control of ROS in a unicellular microbe. Specifically, Cdc42 is required for ROS production by the NOX Fre8 of the opportunistic fungal pathogen Candida albicans. During morphogenesis to a hyphal form, a filamentous growth state, C. albicans FRE8 mRNA is induced which leads to a burst in ROS. Fre8-ROS is also induced during morphogenesis when FRE8 is driven by an ectopic promoter; hence Fre8 ROS production is additionally controlled at the post-transcriptional level. Using fluorescently tagged Fre8, we observe that the majority of the protein is associated with the vacuolar system. Interestingly, much of Fre8 in the vacuolar system appears inactive, and Fre8-induced ROS is only produced at sites near the hyphal tip, where Cdc42 is also localized during morphogenesis. We observe that Cdc42 is necessary to activate Fre8-mediated ROS production during morphogenesis. Cdc42 regulation of Fre8 occurs without the large NOX protein complexes typical of higher eukaryotes and therefore represents a novel form of ROS control by Rho GTPases.
... Conversely, deletion of a chaperon encoding gene, crhsp70, caused morphological changes and reduction of mycoparasitism in this fungus (Sun et al., 2019). Targeted gene KO demonstrated the role of an oxidase gene in the mycoparasitism exerted by Paraconiothyrium minitans (Wei et al., 2016). Homologous recombination combined with Agrobacterium tumefaciensmediated transformation (ATMT), a "silver bullet in a golden age of functional genomics" (Idnurm et al., 2017), was used for functional genetic studies of some regulatory genes of T. atroviride (Zeilinger 2004). ...
Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of a wide range of plant pathogens. Deciphering the molecular mechanisms of the mycoparasitic process may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and the development of a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.
... In M. oryzae, NOX1 is also involved in cell wall organization, and Δnox1 mutants are resistant to cell wall-perturbing agents, such as calcofluor white (Egan et al., 2007). This role may be conserved in fungi, because in the mycoparasitic fungus Coniothyrium minitans, for example, CmNOX1 and CmSLT2 (an orthologue of M. oryzae MPS1), regulate localization of the cell wall integrity-associated mitogen-activated protein kinase (MAPK) and mediate changes in gene expression associated with cell integrity (Wei et al., 2016). ...
Chitosan is a partially deacetylated linear polysaccharide composed of β‐1,4‐linked units of d‐glucosamine and N‐acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood.
Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium‐mediated plant infection. Chitosan inhibits septin‐mediated F‐actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell.
Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase‐dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C‐dependent cell wall integrity pathway, the Mps1 mitogen‐activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)‐sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan.
Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin‐mediated plant infection.
... From the MC to ST stages, the host Hepialus larvae act as a growth container for O. sinensis fungi, and splits with the continued proliferation of mycelium 31 . The increase in body lling leads to increased pressure in the surrounding cells, which induces autophosphorylation of Sln1 HK, which subsequently transmits the stress signal from Sln1 to Ssk1 through Ypd1, thereby activating MAPK 22,32,33 . On the other hand, oxidative stress can activate different signal transduction pathways, either via the localization of speci c regulators to the nucleus upon stress, followed by subsequent activation of detoxi cation genes expressions, or phosphorylation-driven intervention of the MAPK pathway 20,34,35 . ...
Ophiocordyceps sinensis has been a source of valuable materials in traditional Asian medicine for over two thousand years. With recent global warming and overharvest, however, the availability of these wild fungi has decreased dramatically. While fruiting bodies of O. sinensis have been artificially cultivated, the molecular mechanisms that govern the induction of fruiting bodies at the transcriptional and post-transcriptional levels are unclear. In this study, we carried out both mRNA and small RNA sequencing to identify crucial genes and miRNA-like RNAs (milRNAs) involved in the development of fruiting bodies. A total of 2875 differentially expressed genes (DEGs) and 71 differentially expressed milRNAs (DEMs) were identified among the mycoparasite complex (MC), the sclerotium (ST) and the fruiting body (FB) stage. Functional enrichment and Gene Set Enrichment Analysis, analyses indicated that the ST had increased oxidative stress and energy metabolism, and that mitogen-activated protein kinase signaling might induce the formation of fruiting bodies. Integrated analysis of DEGs and DEMs revealed that n_os_milR16, n_os_milR21, n_os_milR34, and n_os_milR90 could be candidate milRNAs that regulate the induction of fruiting bodies. This study provides transcriptome-wide insight into the molecular basis of fruiting body formation in O. Sinensis, and identifies potential candidate genes for improving induction rate.
... In the vascular wilt fungus F. oxysporum, NOX-mediated H 2 O 2 production, as a signal, is located upstream of the Ste2dependent phosphorylation of Mpk1 (Nordzieke et al. 2019). In the mycoparasite Coniothyrium minitans, NOX-derived ROS affect CmSlt2 localization (Wei et al. 2016). Therefore, we speculated that ClNOX2 might posttranscriptionally regulate ClM1. ...
The role of NADPH oxidases (NOXs) in pathogenesis and development in the Curvularia leaf spot agent Curvularia lunata remains poorly understood. In this study, we identified C. lunata ClNOX2, which localized to the plasma membrane and was responsible for reactive oxygen species (ROS) generation. Scavenging the ROS production inhibited the conidial germination and appressorial formation. The ClNOX2 and ClBRN1 deletion mutants were defective in 1,8-dihydroxynaphthalene (DHN) melanin accumulation, appressorial formation, and cellulase synthesis and exhibited lower virulence. However, disruption of the ClNOX2 and ClBRN1 genes facilitated hyphal growth, enhanced stress adaptation to cell-wall-disrupting agents, and promoted developmental processes such as conidiation, conidial germination, and pseudothecium and ascus formation. Interestingly, loss of ClM1, the cell wall integrity (CWI) mitogen-activated protein kinase gene in C. lunata, led to morphology and pathogenicity phenotypes similar to ClNOX2 and ClBRN1 deletion mutants such as abnormal conidia, fewer appressoria, less melanin, increased hyphal growth, and enhanced tolerance to Congo red (CR). These results indicated that the ClNOX2 gene plays an important role in C. lunata development and virulence via regulating intracellular DHN melanin biosynthesis. Quantitative reverse-transcription PCR revealed that the ClNOX2-related ROS signaling pathway and ClM1-mediated CWI signaling pathway are cross-linked in regulating DHN melanin biosynthesis. Our findings provide new insights into how ClNOX2 participates in pathogenesis and development in hemibiotrophic plant fungal pathogens.
[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
