Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi

NADPH oxidases (Nox) are membrane complexes that produce O₂^?. Researches in mammals, plants and fungi highlight the involvement of Nox‐generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91^phox/Nox2 is associated with p22^phox forming the flavocytochrome b₅₅₈ ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22^phox gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22^phox homologue as being mutated in the Podospora anserina mutant IDC⁵⁰⁹. Functional studies show that the fungal p22^phox, PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91^phox homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co‐localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.     

Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina

NADPH oxidases are enzymes that produce reactive oxygen species (ROS) using electrons derived from intracellular NADPH. In plants and mammals, ROS have been proposed to be second messengers that signal defence responses or cell proliferation. By inactivating PaNox1 and PaNox2, two genes encoding NADPH oxidases, we demonstrate the crucial role of these enzymes in the control of two key steps of the filamentous fungus Podospora anserina life cycle. PaNox1 mutants are impaired in the differentiation of fruiting bodies from their progenitor cells, and the deletion of the PaNox2 gene specifically blocks ascospore germination. Furthermore, we show that PaNox1 likely acts upstream of PaASK1, a MAPKKK previously implicated in stationary phase differentiation and cell degeneration. Using nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) assays, we detect a regulated secretion of both superoxide and peroxide during P. anserina vegetative growth. In addition, two oxidative bursts are shown to occur during fruiting body development and ascospore germination. Analysis of mutants establishes that PaNox1, PaNox2, and PaASK1, as well as a still unknown additional source of ROS, modulate these secretions. Altogether, our data point toward a role for NADPH oxidases in signalling fungal developmental transitions with respect to nutrient availability. These enzymes are conserved in other multicellular eukaryotes, suggesting that early eukaryotes were endowed with a redox network used for signalling purposes.     

Overexpression of a human and a fungal ABC transporter similarly suppresses the differentiation defects of a fungal peroxisomal mutant but introduces pleiotropic cellular effects

Among the peroxisome membrane proteins, some are required for peroxisome biogenesis (e.g. PEX2) while others are not, e.g. ABC (ATP-binding cassette) transporters. Unexpectedly, overproduction of the peroxisomal ABC transporter PMP70 was found to be able to restore peroxisome biogenesis in mammalian pex2 mutant cell lines. In the filamentous fungus Podospora anserina, pex2 mutations not only impair peroxisome biogenesis but also cause a precise cell differentiation defect. Here, we show that both defects are partially suppressed by expression of the human cDNA encoding PMP70. In addition, PMP70 expression causes new developmental defects, different from those induced by pex2 mutations. We also show that overexpression of the P. anserina pABC1 gene, which encodes a peroxisomal ABC transporter, leads to similar effects. Taken together, our results demonstrate that: (i) the genetic relationship between PEX2 and PMP70, initially observed in mammals, has been conserved through evolution; (ii) the cell differentiation defect observed in the P. anserina pex2 mutants is indeed linked to impairment in peroxisome biogenesis; and (iii) unexpected detrimental cellular defects result from overproduction of peroxisomal ABC transporters.     

A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina

We present here the properties of a complex III loss-of-function mutant of the filamentous fungus Podospora anserina. The mutation corresponds to a single substitution in the second intron of the gene cyc1 encoding cytochrome c(1), leading to a splicing defect. The cyc1-1 mutant is long-lived, exhibits a defect in ascospore pigmentation, has a reduced growth rate and a reduced ROS production associated with a stabilisation of its mitochondrial DNA. We also show that increased longevity is linked with morphologically modified mitochondria and an increased number of mitochondrial genomes. Overexpression of the alternative oxidase rescues all these phenotypes and restores aging. Interestingly, the absence of complex III in this mutant is not paralleled with a deficiency in complex I activity as reported in mammals although the respiratory chain of P. anserina has recently been demonstrated to be organized according to the "respirasome" model.     

The crucial role of the Pls1 tetraspanin during ascospore germination in Podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a ΔPaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the ΔPaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.     

The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea

The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.     

Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes

Background  

NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.     

IDC1, a pezizomycotina-specific gene that belongs to the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina

Components involved in the activation of the MAPK cascades in filamentous fungi are not well known. Here, we provide evidence that IDC1, a pezizomycotina-specific gene is involved along with the PaNox1 NADPH oxidase in the nuclear localization of the PaMpk1 MAP kinase, a prerequisite for MAPK activity. Mutants of IDC1 display the same phenotypes as mutants in PaNox1 and PaMpk1, i.e., lack of pigment and of aerial hyphae, female sterility, impairment in hyphal interference and inability to develop Crippled Growth cell degeneration. As observed for the PaNox1 mutant, IDC1 mutants are hypostatic to PaMpk1 mutants. IDC1 seems to play a key role in sexual reproduction. Indeed, fertility is diminished in strains with lower level of IDC1. In strains over-expressing IDC1, protoperithecia reach a later stage of development towards perithecia without fertilization; however, upon fertilization maturation of fertile perithecia is diminished and delayed. In addition, heterokaryon construction shows that IDC1 is necessary together with PaNox1 in the perithecial envelope but not in the dikaryon resulting from fertilization.     

Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.

It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.     

Relative importance of an arbuscular mycorrhizal fungus (Rhizophagus intraradices) and root hairs in plant drought tolerance

Both arbuscular mycorrhizal (AM) fungi and root hairs play important roles in plant uptake of water and mineral nutrients. To reveal the relative importance of mycorrhiza and root hairs in plant water relations, a bald root barley (brb) mutant and its wild type (wt) were grown with or without inoculation of the AM fungus Rhizophagus intraradices under well-watered or drought conditions, and plant physiological traits relevant to drought stress resistance were recorded. The experimental results indicated that the AM fungus could almost compensate for the absence of root hairs under drought-stressed conditions. Moreover, phosphorus (P) concentration, leaf water potential, photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency were significantly increased by R. intraradices but not by root hairs, except for shoot P concentration and photosynthetic rate under the drought condition. Root hairs even significantly decreased root P concentration under drought stresses. These results confirm that AM fungi can enhance plant drought tolerance by improvement of P uptake and plant water relations, which subsequently promote plant photosynthetic performance and growth, while root hairs presumably contribute to the improvement of plant growth and photosynthetic capacity through an increase in shoot P concentration.     

Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous fungus Podospora anserina: transformation of protoplasts

From a genomic library of the filamentous fungus Podospora anserina, we have cloned a 4.9-kb fragment which complements an Escherichia coli mutant strain deficient for orotidylic acid pyrophosphorylase (pyrE gene). The recombinant plasmid pPAura5 also transforms to prototrophy a mutant strain of P. anserina carrying a mutation in the ura5 gene and lacking OMPppase activity.     

Glycolipid intermembrane transfer is accelerated by HET-C2, a filamentous fungus gene product involved in the cell-cell incompatibility response

Among filamentous fungi capable of mycelial growth, het genes play crucial roles by regulating heterokaryon formation between different individuals. When fusion occurs between fungal mycelia that differ genetically at their het loci, the resulting heterokaryotic cells are quickly destroyed. It is unclear how het gene products of Podospora anserina trigger heterokaryon incompatibility. One unexplored possibility is that glycosphingolipids play a role because the het-c2 gene encodes a protein that displays 32% sequence identity and an additional 30% similarity to the mammalian glycolipid transfer protein. Here, P. anserina protoplasts containing wild-type het-c2 genes were shown to have greater glycosphingolipid transfer activity than protoplasts with disrupted het-c2 genes, a condition previously linked to altered cell compatibility following hyphal fusion. The observed glycolipid transfer activity could not be accounted for by nonspecific lipid transfer protein activity. Direct assessment showed that purified, recombinant HET-C2 accelerates the intermembrane transfer of glycolipid in vitro, but that the HET-C2 activity is mitigated much less by negatively charged membranes than the mammalian glycolipid transfer protein. The findings are discussed within the context of HET-C2 being a member of an emerging family of ancestral sphingolipid transfer proteins that play important roles in cell proliferation and accelerated death.     

BcNoxD,a putative ER protein,is a new component of the NADPH oxidase complex in Botrytis cinerea

NADPH oxidases (Nox) are major enzymatic producer of reactive oxygen species (ROS). In fungi these multi‐enzyme complexes are involved in sexual differentiation and pathogenicity. However, in contrast to mammalian systems, the composition and recruitment of the fungal Nox complexes are unresolved. Here we introduce a new Nox component, the membrane protein NoxD in the grey mold fungus Botrytis cinerea. It has high homology to the ER protein Pro41 from Sordaria macrospora, similar functions to the catalytic Nox subunit BcNoxA in differentiation and pathogenicity, and shows similarities to phagocytic p22phox. BcNoxA and BcNoxD interact with each other. Both proteins are involved in pathogenicity, fusion of conidial anastomosis tubes (CAT) and formation of sclerotia and conidia. These data support our earlier view based on localization studies, for an ER‐related function of the Nox complex. We present the first evidence that some functions of the BcNoxA complex are indeed linked to the ER, while others clearly require export from the ER.     

Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.     

Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity

Prions have been described in mammals and fungi. The [Het-s] infectious genetic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. This protein is involved in the control of a cell death reaction termed heterokaryon incompatibility. The infectious form of HET-s corresponds to a self-perpetuating amyloid. The purpose of the present paper is to describe the techniques that can be used to analyse [Het-s] prion propagation in vivo and HET-s amyloid aggregation in vitro. In addition, we report several methods that can be used to infect Podospora with recombinant HET-s amyloid.     

Orchestration of morphogenesis in filamentous fungi: conserved roles for Ras signaling networks

Filamentous fungi undergo complex developmental programs including conidial germination, polarized morphogenesis, and differentiation of sexual and asexual structures. For many fungi, the coordinated completion of development is required for pathogenicity, as specialized morphological structures must be produced by the invading fungus. Ras proteins are highly conserved GTPase signal transducers and function as major regulators of growth and development in eukaryotes. Filamentous fungi typically express two Ras homologs, comprising distinct groups of Ras1-like and Ras2-like proteins based on sequence homology. Recent evidence suggests shared roles for both Ras1 and Ras2 homologs, but also supports the existence of unique functions in the areas of stress response and virulence. This review focuses on the roles played by both Ras protein groups during growth, development, and pathogenicity of a diverse array of filamentous fungi.