Chasing stress signals - Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea.

Reactive oxygen species (ROS) are important molecules influencing intracellular developmental processes as well as plant pathogen interactions. They are produced at the infection site and affect the intracellular redox homeostasis. However, knowledge of ROS signaling pathways, their connection to other signaling cascades, and tools for the visualization of intra- and extracellular ROS levels and their impact on the redox state are scarce. By using the genetically encoded biosensor roGFP2 we studied for the first time the differences between the redox states of the cytosol, the intermembrane space of mitochondria and the ER in the filamentous fungus Botrytis cinerea. We showed that the ratio of oxidized to reduced glutathione inside of the cellular compartments differ and that the addition of hydrogen peroxide (H2O2), calcium chloride (CaCl2) and the fluorescent dye calcofluor white (CFW) have a direct impact on the cellular redox states. Dependent on the type of stress agents applied, the redox states were affected in the different cellular compartments in a temporally shifted manner. By integrating the biosensor in deletion mutants of bcnoxA, bcnoxB, bctrx1 and bcltf1 we further elucidated the putative roles of the different proteins in distinct stress-response pathways. We showed that the redox states of ΔbcnoxA and ΔbcnoxB display a wild-type pattern upon exposure to H2O2, but appear to be strongly affected by CaCl2 and CFW. Moreover, we demonstrated the involvement of the light-responsive transcription factor BcLtf1 in the maintenance of the redox state in the intermembrane space of the mitochondria. Finally, we report that CaCl2 as well as cell wall stress-inducing agents stimulate ROS production and that ΔbcnoxB produces significantly less ROS than the wild type and ΔbcnoxA.

Update on Nox function, site of action and regulation in Botrytis cinerea.

BACKGROUND: The production of reactive oxygen species (ROS) and a balanced redox homeostasis are essential parameters, which control the infection process of the plant pathogen Botrytis cinerea. The necrotrophic fungus is able to cope with the plants' oxidative burst and even produces its own ROS to overcome the plants' defense barrier. Major enzyme complexes, which are responsible for the production of superoxide, are NADPH oxidase (Nox) complexes. They play a central role in various growth, differentiation and pathogenic processes. However, information about their regulation and the integration in the complex signaling network of filamentous fungi is still scarce. RESULTS: In this work, we give an update on Nox structure, function, site of action and regulation. We show that functionality of the catalytic Nox-subunits seems to be independent from their transcriptional regulation and that the membrane orientation of BcNoxA would allow electron transport inside the ER. Following previous studies, which provided evidence for distinct functions of the NoxA complex inside the ER, we highlight in this work that the N-terminus of BcNoxA is essential for these functions. Finally, we elucidate the role of BcNoxD and BcNoxB inside the ER by complementing the deletion mutants with ER bound alleles. CONCLUSIONS: This study provides a deeper analysis of the Nox complexes in B. cinerea. Besides new insights in the overall regulation of the complexes, we provide further evidence that the NoxA complex has a predominant role inside the ER, while the NoxB complex is mainly important outside the ER, likely at the plasma membrane. By considering all other putative Nox complex members, we propose a putative model, which describes the distinct complex pattern upon certain differentiation processes.

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.

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 IDC(509). 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.

Functional analysis of BcBem1 and its interaction partners in Botrytis cinerea: impact on differentiation and virulence.

In phytopathogenic fungi the establishment and maintenance of polarity is not only essential for vegetative growth and differentiation, but also for penetration and colonization of host tissues. We investigated orthologs of members of the yeast polarity complex in the grey mould fungus Botrytis cinerea: the scaffold proteins Bem1 and Far1, the GEF (guanine nucleotide exchange factor) Cdc24, and the formin Bni1 (named Sep1 in B. cinerea). BcBem1 does not play an important role in regular hyphal growth, but has significant impact on spore formation and germination, on the establishment of conidial anastomosis tubes (CATs) and on virulence. As in other fungi, BcBem1 interacts with the GEF BcCdc24 and the formin BcSep1, indicating that in B. cinerea the apical complex has a similar structure as in yeast. A functional analysis of BcCdc24 suggests that it is essential for growth, since it was not possible to obtain homokaryotic deletion mutants. Heterokaryons of Δcdc24 (supposed to exhibit reduced bccdc24 transcript levels) already show a strong phenotype: an inability to penetrate the host tissue, a significantly reduced growth rate and malformation of conidia, which tend to burst as observed for Δbcbem1. Also the formin BcSep1 has significant impact on hyphal growth and development, whereas the role of the putative ortholog of the yeast scaffold protein Far1 remains open: Δbcfar1 mutants have no obvious phenotypes.

Correction: The NADPH Oxidase Complexes in Botrytis cinerea: Evidence for a Close Association with the ER and the Tetraspanin Pls1.

[This corrects the article on p. e55879 in vol. 8.].

The NADPH oxidase complexes in Botrytis cinerea: evidence for a close association with the ER and the tetraspanin Pls1.

NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ΔbcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination.

Reactive oxygen species generation in fungal development and pathogenesis.

Reactive oxygen species (ROS) generated by NADPH-dependent oxidases (Nox) have been shown to function as signaling molecules and to be essential for many differentiation processes in mammals and plants. There is growing evidence that ROS are important for many aspects of fungal life including vegetative hyphal growth, differentiation of conidial anastomosis tubes, fruiting body and infection structure formation, and for induction of apoptosis. Recent results from studies in fungal saprophytic and pathogenic model systems have shed new light on the role of Nox in cytoskeleton organization, the structure of Nox complexes and links to components of the apical complex, and the localization of Nox to the endoplasmic reticulum.

Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity.

In many filamentous ascomycete species, the early steps of colony development include fusion between germinating vegetative spores (conidial germlings). Often these fusion events are mediated by specialized hyphal structures, so-called conidial anastomosis tubes (CATs). Here, we show that germling fusion in the grey mould Botrytis cinerea is mediated by hyphal structures possessing the typical features of CATs. Formation of these structures is delayed when spores are germinating on complex media compared to growth on poor substrates. Fusion frequency is also influenced by the growth conditions of the precultures from which spores were obtained. During germination on hydrophobic plant surfaces, which induce pathogenic development, CAT formation is significantly suppressed. Screening of existing B. cinerea gene knockout mutants identified strains lacking the NADPH oxidase BcNoxA or the potential Nox regulator BcNoxR as fusion deficient, suggesting a potential role of reactive oxygen species (ROS) signalling in CAT formation and fusion.