Update on Nox function, site of action and regulation in Botrytis cinerea
© The Author(s) 2016
Received: 19 August 2016
Accepted: 29 September 2016
Published: 7 October 2016
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.
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.
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.
KeywordsBotrytis cinerea NADPH oxidase ROS ER Regulation
Botrytis cinerea is a filamentous fungus, which is classified as second most devastating plant pathogen  infecting more than 500 economically important hosts . It is the causative agent of the gray mold disease diminishing harvest yields of strawberries, raspberries and grapes . In its lifestyle as well as in the interaction with its host, reactive oxygen species (ROS) play a central role. The molecules are produced in all cells that depend on molecular oxygen. ROS have an ambivalent role since they work as signaling molecules but also interact unspecifically with macromolecules such as DNA and lipids . While plants produce ROS during the so-called “oxidative burst” as first defensive line upon contact with pathogen derived elicitors, B. cinerea produces ROS to trigger plant’s defense reactions, on which it depends to achieve full pathogenicity .
ROS are produced in highly conserved processes; they occur as mere byproducts of the respiratory chain or as results of evolved enzymatic reactions. Specialized enzymes, which are one of the major sources of ROS, are NADPH oxidase (Nox) complexes. Nox complexes are responsible for the transfer of electrons from NADPH as electron donor across membranes to molecular oxygen [27, 51]. Nox are best characterized in mammalian systems, since localization and function of Nox in these systems have been extensively studied in recent years . The catalytic subunit (gp91phox/Nox2) is a transmembrane protein which is stabilized by the likewise membrane standing adaptor protein p22phox. For activation of the multi-enzyme complex cytosolic, regulatory components such as p67phox, p40phox and p47phox are recruited to the catalytic subunits.
Despite of the evolutionary distance, there are quite a few homologies between mammalian and fungal Nox complexes. Besides homologous proteins to the catalytic (gp91phox—NoxA/B or Nox1/2 respectively) and the regulatory subunit (p67phox—NoxR), more recently equivalents of the adaptor protein p22phox (NoxD [25, 44]) and the scaffold protein IQGAP (Iqg1 ) were identified. In fungi, Nox complexes exhibit distinct functions [1, 49]. While the NoxA (Nox1) complex is controlling fruiting body formation [7, 28, 31], formation of sclerotia [14, 40, 42] and conidial anastomosis tubes (CATs) , virulence [23, 42, 53] and cellulose degradation , the NoxB (Nox2) complex is responsible for penetration of host tissue [12, 42], the production of ROS  and ascospore germination [7, 31]. However, the exact composition of the Nox complexes, their site of action as well as the integration in existing signaling pathways are still poorly elucidated. A major contribution for answering those questions might be recent findings that an ER locked allele of NoxA in B. cinerea is capable to partially restore the deletion mutants’ phenotype , revealing that the NoxA complex is active inside and outside of the ER.
For a further functional characterization of the NoxA complex in B. cinerea, complementation studies with an N-terminal truncated version of BcNoxA were conducted. They reveal that the N-terminus of BcNoxA is essential for a functional NoxA complex inside the ER. Moreover, we demonstrate by topology studies with the genetically encoded biosensor roGFP2 that the membrane orientation of the catalytic subunit BcNoxA would allow the production of superoxide inside the ER. In contrast to all expectations, cross-complementation studies give rise to assume that transcriptional regulation of both Nox complexes plays only a minor role during the production of sclerotia and conidial anastomosis tubes (CATs) as well as during pathogenic processes. Finally, we highlight that in contrast to the NoxA complex, the NoxB complex fulfills its predominant role outside the ER. Thus, complementation of the deletion mutant ΔbcnoxB with an ER retained version of the catalytic subunit revealed that BcNoxB is necessary for stress sensitivity inside the ER. Instead, all other processes such as ROS production, pathogenicity and formation of functional appressoria are putatively regulated by BcNoxB outside the ER, most likely at the plasma membrane.
Cultivation of Botrytis cinerea
Botrytis cinerea Pers.:Fr. [Botryotinia fuckeliana (de Bary)Whetzel] B05.10 was isolated from Vitis vinifera  and was used in this study as basis strain and control in all experiments. Further strains are listed in Additional file 1: Table S1.
For cultivation synthetic complete medium (CM)  was used. For transformation, the strains were grown on PDAB medium (Potato dextrose agar [Sigma-Aldrich Chemie, Steinheim, Germany] supplemented with 100 g/l homogenized leaves of French beans (Phaeseolus vulgaris). Plates were grown for 6–8 days at 20 °C under light conditions (12 h light/12 h darkness, full spectrum light) to obtain conidia. Sclerotia production was induced by incubating the strains for 3 weeks at 20 °C in darkness. For DNA preparation, the strains were grown 3–4 days at 18 °C on CM agar overlayed with Cellophane. For stress experiments, the complete medium was supplemented with osmotic, oxidative, cell wall, membrane or ionic stress. Minimal medium was prepared after Czapek Dox (20 g/l sucrose, 3 g/l NaNO3, 1 g/l K2HPO4, 0.5 gl KCl, 0.01 g/l FeSO4·7H2O, 0.5 g/l MgSO4·7H2O, pH 5.2).
Generation of mutant strains
After transforming S. cerevisiae FY834, positive transformants were selected on SD medium lacking uracil since the strain is auxotroph for this amino acid. Total DNA was isolated and transformed in E. coli. Re-isolation was done by the Nucleo spin® plasmid easypure kit (Macherey–Nagel, Düren, Germany) and correct assembly was tested by sequencing.
Botrytis cinerea was transformed with 40–60 µg of plasmid DNA as described previously . Selection was done via hygromycin (70 μg/ml of hygromycin B [Invitrogen, San Diego, USA] or nourseothricin (50 μg/ml of nourseothricin [Werner-Bioagents, Jena, Germany]). Positive transformants were purified by single spore isolation: Conidia were spread on the appropriate selective medium; afterwards, germinated conidia were picked and cultivated on new selective plates. Genomic DNA isolation was performed according to Cenis . Correct and ectopic integration were checked by diagnostic PCR and Southern blot (primer 19–28 and 35–40).
Growth and pathogenicity assays
For characterization of the different mutant strains various growth, differentiation and pathogenicity assays were performed. For testing defects in pathogenicity, French bean plants (Phaseolus vulgaris) were infected according to Klimpel et al.  with agar plugs or conidia from freshly sporulated PDAB plates. Germination on glass surfaces was tested as described by Doehlemann et al. . Penetration ability was checked on onion epidermal layers. Conidia were washed and placed on epidermal layers of onions. Before microscopy, the hyphae growing on top of the layers were stained with lactophenol blue. For observation of infection cushions (ICs) the epidermal layers were incubated with agar plugs overnight . For visualization of conidial anastomosis tubes (CATs) media and strains were prepared following .
Quantification of conidia
For quantification of conidia a defined amount of spores (0.5 × 105) were plated on CM plates. After 14–21 days of incubation under daytime rhythm, the spores were washed down with 10 µl H2O, followed by a second wash step (5 ml) to harvest the remaining spores. Dilutions were prepared and spores were quantified in several replicates using a Thoma chamber.
Quantification of ROS levels was accomplished by applying the Total ROS/superoxide detection kit (Enzo Lifescience, Lausen, Switzerland). The detection mix was prepared as described in the instructor’s manual. For visualization, conidia were harvested from a freshly grown PDAB plate and 100 µl (105 conidia/ml) were grown overnight in a 96-well plate (Microplate pureGradeTM 96 well PS, transp. Bottom, black, Brand GmbH & Co KG, Germany). Just before measurement the detection mix was added (1/10 vol). Pictures were taken using the wavelengths 490/525 (Ex/Em). For every well 3 × 3 reads were done with a gain that was set to 200.
Light microscopy imaging was performed using the Axio Imager 2 and the Axiovert (Zeiss, Jena, Germany). Visualization of infection cushions was done with the 20× objective lens, while germinated conidia were analyzed with 40× or 63× magnification. ER staining was accomplished using the ER-Tracker™ Blue-White DPX (Life Technologies, Germany) in McIlvaine standard buffer . The samples were observed via the filter set 49 DAPI shift free (excitation G 365, beam splitter FT 395, emission BP 445/50). GFP fluorescence was detected with filter set 38 (excitation BP 470/40, beam splitter FT 495, emission BP 525/50). Images were captured with a Zeiss AxioCamMRm camera and further processed using the AxiovisionRel 4.8 software package.
Confocal laser scanning microscopy (CLSM) imaging and ratiometric analysis
roGFP2 measurements were done using an inverted microscope (Leica DMIRE2) equipped with a Leica TCS SP2 scan head (Leica Microsystems, Wetzlar, Germany). Conidia were prepared as described before . Results were obtained by using the excitation wavelengths 395 (first track) and 488 (second track) as well as a 505–530 bandpass filter for collecting images. Z-stacks were displayed as average projections via the CLSM software. Further evaluation was done with the Image J program (v.1.44f; http://rsb.info.nih.gov/ij/) as it was shown before .
For measuring ROS levels, a Tecan Safire reader was used. Conidia were harvested and placed into a 96-well plate Microplate pureGradeTM 96 well PS, transp. Bottom, black, Brand GmbH & Co KG, Germany). After growth overnight (105 conidia/ml) the detection mix was added and fluorescence was measured with 3 × 3 reads/well and excitation/emission as described above. The gain was set to 200.
Nucleotide and protein sequences of B. cinerea strain B05.10 were obtained by the database Ensembl (http://fungi.ensembl.org/Botrytis_cinerea/Info/Index). For the analysis of the sequences, different programs were used: Signal peptides were predicted by SOSUIsignal , subcellular localization patterns of proteins were predicted by ProtComp v.9.0 (http://linux1.softberry.com/berry.phtml?topic=protcompan&group=help&subgroup=proloc), transmembrane regions were predicted by using the programs TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and SACS MEMSAT2  as well as PRED-TMR .
In B. cinerea, two Nox complexes are responsible for important vegetative and pathogenic growth and differentiation processes [43, 44]. The most recent studies focused on the identification of new members of both Nox complexes [32, 43, 44]. However, information about Nox complex regulation, site of action and direct linkages to existing signaling pathways remained scarce.
Transcriptional regulation of BcNoxA/B is not important for their distinct function
To elucidate whether transcriptional regulation of both catalytic subunits does have any influence on their function, hybrid constructs were generated. The coding region of bcnoxA was cloned downstream of the bcnoxB promoter with (C1) or without (C2) the N-terminal elongation of bcnoxB, while the coding region of bcnoxB was fused to the promoter region of bcnoxA (C3, Fig. 1).
All constructs were transformed in the deletion mutants of bcnoxA or bcnoxB, respectively and integrated into the bcniaD or bcniiA locus (see “Materials and methods”, Additional file 1: Table S1 as well as . Since BcNoxA is involved in formation and fusion of conidial anastomosis tubes (CATs—specialized hyphae that facilitate the exchange of cellular material), infection cushions (IC—bulbous aggregates that facilitate the penetration of plant tissue) and sclerotia as well as affects the colonization of plant tissue, the ΔbcnoxA transformants were investigated with special focus on these phenotypes. Only the construct C2 was able to fully complement the phenotype of ΔbcnoxA (Fig. 1a–g) whereas the fusion protein C1 only partially restored the wild type phenotype. Thus, mutants containing the C1 construct displayed wild type like pathogenicity (Fig. 1a, b) and a restored formation of infection cushions (Fig. 1g), but produced a minor amount of CATs (Fig. 1c), sclerotia (Fig. 1d) and conidiospores (Fig. 1e). Mutants with an integrated C3 construct displayed the deletion mutants’ phenotype.
Since BcNoxB affects the production of functional appressoria, the penetration of plant tissue, sensitivity against H2O2 as well as the production of ROS and conidiospores, all transformants (ΔbcnoxB background) were analyzed with special focus on these phenotypes.
While mutants containing the constructs C1 or C2 resembled the phenotype of ΔbcnoxB, mutants expressing the C3 construct appeared to be wild type like (Additional file 4: Figure S4). Thus, the construct C3 mediates the restoration of full pathogenicity (Additional file 4: Figure S4A), the formation of functional appressoria (Additional file 4: Figure S4B), conidiation (Additional file 4: Figure S4C) as well as stress resistance (Additional file 4: Figure S4D) and ROS production (Additional file 4: Figure S4E).
In conclusion, the catalytic subunits of the Nox complexes fail to replace each other, despite of their structural similarities. Moreover, the N-terminal elongation (Additional file 3: Figure S3B) of BcNoxB seems to compromise the folding of BcNoxA since the construct C1 is not sufficient to fully restore the phenotype of ΔbcnoxA. Finally, unexpectedly the transcriptional regulation (exchange of promoters) appears to be not important for the distinct functions of Nox complexes.
Membrane orientation of BcNoxA would allow electron transport into the ER
After generating the strains B05.10_roGFP2_NoxA and B05.10_NoxA_roGFP2, the correct integration of the fusion constructs (see “Materials and methods”, Additional file 1: Table S1) was checked via diagnostic PCR. The functionality of both constructs was verified before, by complementing ΔbcnoxA. Both proteins (roGFP2_NoxA/NoxA_roGFP2) appeared to be localized to the plasma membrane, perinuclear regions as well as filamentous ER structures (Fig. 2c); confirming the localization pattern observed for BcNoxA_GFP . Fluorometric measurements with both roGFP2 versions (located at the N- or C-terminus of BcNoxA) resulted in the report of a typically cytoplasmic redox state (Fig. 2b), suggesting that both protein ends are located inside the cytosol. In both cases the untreated 395/488ratio (wild type: 0.22) displays values of 0.23 (NoxA_roGFP2) and 0.21 (roGFP2_NoxA). Upon exposure to H2O2, the 395/488ratio increases up to its maximum after 8 min post induction in the cytosolic wild type control as well as in the strains NoxA_roGFP2 and roGFP2_NoxA. A similar behavior upon H2O2 induction was observed in previous studies [20, 33].
These results suggest that the membrane orientation of BcNoxA would allow the transport of electrons from the cytosol across the ER- or plasma membrane. In the end, this ROS production inside of the ER as well as into the extracellular space would be possible.
The N-terminus of BcNoxA is essential for its ER functions
Since it was shown that an ER-hooked allele of BcNoxA is only partially able to complement the deletion mutant’s phenotype , we analyzed the importance of the predicted N-terminal signal peptide of BcNoxA. For this, an allele, which encodes for a truncated version of BcNoxA (loss of the first 41 aa—Additional file 3: Figure S3A) was transformed into the ΔbcnoxA mutant.
Microscopic analyses show that the localization pattern changes in comparison to the non-truncated version. While BcNoxA_GFP localizes to filamentous ER structures and the nuclear envelope (Fig. 3G (a)) as reported , the truncated BcNoxA_GFP is found in a faint network of filamentous structures (Fig. 3G (b)), septa (Fig. 3G (c)) and was only rarely observed in the nuclear envelope (Fig. 3G (a)). Summarized, the N-terminus of BcNoxA seems to be essential for the proper localization of the protein as well as for its functions inside of the ER.
ER-hooked alleles of BcNoxD and BcNoxA are involved in identical processes
BcNoxD, a putative ER membrane protein was shown to be a new member of the NoxA complex in B. cinerea . Deletion mutants of bcnoxD resemble the phenotype of ΔbcnoxA and both proteins directly interact in yeast-2-hybrid and co-IP assays. To investigate whether BcNoxD has also distinct functions inside and outside of the ER as it was previously confirmed for BcNoxA, an ER retention signal was fused to the 3′ end of the bcnoxD coding region (BcNoxD_HDEL).
The ER-hooked allele of BcNoxD is able to restore the formation of IC’s and the colonization defect of its deletion mutant (Additional file 5: Figure S5A/B/G). Moreover, BcNoxD_HDEL restores the resistance against oxidative stress (Additional file 5: Figure S5F) which was also demonstrated for the ER-hooked allele of BcNoxA (Additional file 6: Figure S6). In contrast, the mutant strain is still impaired in the formation of CATs, sclerotia and conidiospores (Additional file 5: Figure S5C/D/E). Summarized, both strains ΔbcnoxA:bcnoxA_HDEL and ΔbcnoxD:bcnoxD_HDEL display an identic functional pattern, i.e. the native proteins have identical distinct functions within and outside the ER.
BcNoxB has a role inside and outside the ER
Since previous studies suggested a distinct function of the NoxA complex inside the ER, further studies were conducted to elucidate the role of the NoxB complex inside and outside the ER. As previously done for BcNoxA and BcNoxD, the ER retention signal “HDEL” was fused to the 3′ end of the bcnoxB coding region. The construct was transformed into ΔbcnoxB and integrated into the bcniaD locus .
In conclusion, the NoxB complex of B. cinerea seems to have a role inside and outside the ER, most likely at the plasma membrane. While stress sensitivity and conidiospore production are regulated by BcNoxB inside of the ER, the protein affects ROS production and pathogenicity outside the ER.
The production of ROS and the cellular redox homeostasis are essential for the plant pathogen B. cinerea and its interaction with the host. By producing superoxide, Nox complexes directly contribute to elevated levels of ROS and directly influence the redox balance of the fungus . Since the link between Nox-function and differentiation processes of filamentous fungi were identified , many studies aim to uncover Nox-function, composition, regulation and the integration in the complex signaling network.
In this study, we elucidate the orientation of BcNoxA in the lipid bilayer by the genetically encoded biosensor roGFP2. The biosensor, which was fused to the N- and C-terminus end of bcnoxA, can be used as a tool to study membrane topologies and for answering the question, whether the orientation of the catalytic subunit would allow ROS production e.g. inside the ER. CLSM analyses reveal that both ends of the protein are located inside the cytosol (Fig. 2). Thus, the biosensor mirrored a greatly diminished 395/488ratio (indicating a compartment with reduced conditions) and, therefore, resembled results of previous studies that proposed the cytosol of B. cinerea as compartment with the most reduced conditions [20, 33]. Since the NADPH binding domain is predicted to be at the very end of the C-terminus of BcNoxA, the orientation would allow the transfer of electrons from the cytosol across membranes e.g. into the ER lumen (Fig. 2a). The activity of the NoxA complex would lead to an oxidation of the cytosol and the reduction of molecular oxygen on the other site of the lipid bilayer. A similar mechanism was observed previously in the mammalian system, where the dual oxidase 2 (Duox2—belonging to the Nox family) produces superoxide inside the ER in a calcium dependent manner . Moreover, this suggested pattern was recently confirmed by ratiometric measurements using the genetically encoded biosensor roGFP2 in the wild type and deletion mutants of bcnoxA in B. cinerea .
For further investigations of the coherence between Nox structure and function, we modified the N-terminus of BcNoxA by removing the putative signal peptide (Fig. 3/Additional file 3: Figure S3A). In mammals, a direct link between N-terminus and localization as well as type of ROS was already illustrated . In the fungal system, the truncation of the N-terminal region affected both, localization and function of BcNoxA. In contrast to the wild type version of BcNoxA, the protein was rarely observed in the nuclear envelope, but seemingly more often in filamentous structures of the ER (Fig. 3G (b)). Complementation studies revealed that the truncated version of BcNoxA is able to complement the deletion mutants’ phenotype only partially. Thus, the formation of sclerotia and CATs was restored (Fig. 3C, F), whereas IC formation and pathogenicity were still disturbed (Fig. 3A, B, H). Interestingly, the complemented phenotypes were inverse to the ones of the ER hooked allele (Fig. 5) . However it remains elusive, whether the inverse phenotype is due to the diverging localization of the protein or due the missing interaction to other Nox complex members, both attributable to the missing signal peptide. Nevertheless, these data substantiate that the the NoxA complex has distinct functions in different cellular compartments and suggest that the N-terminal region of BcNoxA is essential for its function inside the ER. This hypothesis is further supported by cross complementation studies, which aimed to uncover the importance of the transcriptional regulation of bcnoxA/bcnoxB for their protein function. After complementing ΔbcnoxA with the respective constructs (C1–C3, Fig. 1) our data indicate that only bcnoxA under the control of P bcnoxB (C2) is sufficient to complement all phenotypes of the deletion mutant. Surprisingly, the N-terminal elongation of bcnoxB in construct C1 prevents full complementation of the respective deletion mutant. The complemented strain resembled the phenotype of ΔbcnoxA::bcnoxA_HDEL. This underlines the importance of the N-terminal region for the proper localization of BcNoxA and coincides with the previous reported results in mammalian systems .
Even more captivating are the cross complementation studies after considering the results of the respective ΔbcnoxB complementation. Our final data reveal that only the construct containing the full-length version of bcnoxB is sufficient to restore the wild type phenotype (Additional files 4: Figure S4, 5: Figure S5). In conclusion, the results strongly suggest that (1) the catalytic subunits are not able to replace each other, (2) the N-terminal elongation of BcNoxB is not able to transfer characteristics of BcNoxB onto BcNoxA and most important (3) the transcriptional regulation is not important for BcNoxA/B function since the successful cross complementation was promoter independent. Especially the irrelevance of the transcriptional regulation for pathogenic and vegetative differentiation processes is striking. Thus, previous studies in mammals revealed that the expression pattern of the different Nox isoforms is essential for their function and that distinct signaling pathways regulate the expression of the different isoforms (reviewed in [26, 42]). Nevertheless, in B. cinerea, post-translational modifications and mechanisms that directly affect the respective proteins may be more important than the transcriptional regulation. Thus, (calcium-dependent) phosphorylation events, glycosylation, nitrosylation or direct redox dependent regulation of cysteine residues might affect Nox complex activity as seen in plant and mammalian cells (summarized in ; reviewed in [16, 46, 47].
By considering all phenotypes of putative Nox complex members, we propose a model of NoxA complex compositions dependent on the site of action and the respective differentiation process (Fig. 6a). Thus, the NoxA complex might regulate pathogenic processes such as colonization of plant tissue and infection cushion formation as well as vegetative processes such as stress resistance inside the ER (Additional file 6: Figure S6A/Fig. 6). In this situation, the NoxA complex consists of the membrane standing subunits BcNoxA and BcNoxD, which are putatively linked via the scaffold protein BcIqg1 to its cytosolic and regulatory subunits BcRac as well as BcNoxR. Since the deletion mutant of the second scaffold protein BcBem1 displays also defects in the colonization of plant tissue  and in the formation of infection cushions (Additional file 6: Figure S6B) an association to the NoxA complex is likely. The putative association is supported by Takemoto et al. (2011), who linked Bem1 to Nox complexes in Epichloe festucae . On the other side, the unaffected stress resistance of Δbcbem1, its lacking Nox complex interaction partner in B. cinerea as well as its localization in the cytosol and septa  cast some doubts about its membership in the NoxA complex. Outside the ER, the NoxA complex might be similar composed during the formation of CATs and conidiospores, but slightly different during sclerotia development. Δbcbem1 and Δbciqg1 were shown to produce higher amounts of sclerotia than the wild type [15, 32]. This might hint to a negative regulation mechanism either, or to a different composition of the NoxA complex with respect to this differentiation process.
To unravel the site of action and regulation processes of the NoxB complex in B. cinerea, a similar complementation approach with an ER locked allele was conducted. In contrast to BcNoxA, the second catalytic subunits has its main functions outside the ER. Thus, the formation of functional appressoria and the related penetration of plant tissue as well as the production of ROS were not complemented by ΔbcnoxB::bcnoxB_HDEL (Figs. 4, 5). Since previous studies already indicated that the second catalytic subunit has a predominant role in the ROS production , it is reasonable that those massive amounts most likely are generated at the plasma membrane towards the extracellular space. Here, ROS may act against the host defense barrier or may be transported back inside the fungal hyphae via evolved transport systems, such as aquaporins . The latter hypothesis is supported by previous findings that superoxide accumulates inside the hyphal tip during the formation of penetration structures . A direct production e.g. inside the ER/cytosol may lead to an intoxication and cell death.
Strikingly, only the stress resistance is regulated by both Nox complexes inside the ER, suggesting that both complexes contribute to the sensing machinery of oxidative (Fig. 4; Additional file 5: Figure S5A), osmotic and cell wall stress (data not shown) when hooked to the ER membrane. In contrast, BcNoxB inside the ER affects conidiation, while NoxA contributes to this differentiation process outside the ER. A similar interplay between both Nox complexes was recently observed upon calcium stress exposure .
As previously discussed for the NoxA complex, also the composition of the NoxB complex slightly differs dependent on the site of action as well as on the differentiation process (Fig. 6b). Inside the ER, the catalytic subunit seems to work as single membrane standing protein since its putative adaptor protein BcPls1 neither affects conidiation nor contributes to stress resistance of B. cinerea . In contrast, all other processes, which are putatively regulated by BcNoxB outside the ER, might be affected by a complex consisting of the catalytic subunit (BcNoxB), the tetraspanine BcPls1, the regulator BcNoxR as well as the cytosolic members BcRac and BcBem1 (Fig. 6b). However, the link between BcPls1 and BcNoxB appears to be weak and might be restricted to specific time points and differentiation processes, which is also supported by the lacking physical interaction of both proteins in yeast based interaction assays as well as in co-IP and pulldown experiments (data not shown). The contribution of BcBem1 to the development of functional appressoria needs to be further elucidated, since Δbcbem1 is able to form functional penetration structures . However, all subunits display penetration defects on bean plants as well as a reduced amount of secreted ROS (Additional file 6: Figure S6C). Processes such as sclerotia formation are not regulated by the NoxB complex (Additional file 6: Figure S6D).
In conclusion, we can use our obtained data to propose a putative model for both Nox complexes dependent on their site of action and their function. Strikingly, both complexes have distinct functions inside and outside the ER and as previously seen in the mammalian system, the compositions might differ dependent on the respective differentiation process. According to bioinformatic analyses, we confirmed that the orientation of the catalytic subunit BcNoxA would allow the production of ROS inside the ER lumen. Remarkable is, that against previous observations and assumptions, the transcriptional regulation of both Nox complexes seems to be unimportant for their distinct functions. Nevertheless, studies are needed to further elucidate the integration of both complexes in the up- and downstream wiring as well as the exact composition and connections of the catalytic subunits with its regulator as soon as the respective scaffold and adaptor proteins are missing.
RM: conceptualization and methodology; US: generation and analysis of mutants resulting in Fig. 1 and Additional file 4: Figure S4; JB: Contribution to the strain generation in Fig. 4 and generation/analysis of strains for co-IP experiments; RM: generation and analysis of all other mutants, writing of the manuscript. RM, US, JB, PT: review and editing. All authors read and approved the final manuscript.
We thank Bettina Richter for excellent technical assistance, Kim Cohrs and Julia Schumacher for discussion and the DFG (Tu 50/19) for funding.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this work are included in this article and its supplementary files.
Consent for publication
Not applicable—Our manuscript does not contain any individual person’s data!
This work was funded by the DFG (Tu50/19).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Aguirre J, Rios-Momberg M, Hewitt D, Hansberg W. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol. 2005;13(0966-842X (Print); 0966-842X (Linking)):111–8.
- Ameziane-El-Hassani R, Morand S, Boucher JL, Frapart YM, Apostolou D, Agnandji D, Gnidehou S, Ohayon R, Noël-Hudson MS, Francon J, Lalaoui K, Virion A, Dupuy C. Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity. J Biol Chem. 2005;280:30046–54.View ArticlePubMedGoogle Scholar
- An B, Li B, Li H, Zhang Z, Qin G, Tian S. Aquaporin8 regulates cellular development and reactive oxygen species production, a critical component of virulence in Botrytis cinerea. New Phytol. 2016;209(4):1668–80.View ArticlePubMedGoogle Scholar
- Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev. 1998;78(2):547–81.PubMedGoogle Scholar
- Brun S, Malagnac F, Bidard F, Lalucque H, Silar P. Functions and regulation of the Nox family in the filamentous fungus Podospora anserina: a new role in cellulose degradation. Mol Microbiol. 2009;74(2):480–96.View ArticlePubMedGoogle Scholar
- Buettner P, Koch F, Voigt K, Quidde T, Risch S, Blaich R, Bruckner B, Tudzynski P. Variations in ploidy among isolates of Botrytis cinerea: implications for genetic and molecular analyses. Curr Genet. 1994;25(5):445–50.View ArticleGoogle Scholar
- Cano-Dominguez N, Alvarez-Delfin K, Hansberg W, Aguirre J. NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa. Eukaryot Cell. 2008;7(8):1352–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Cenis JL. Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Res. 1992;20(9):2380.View ArticlePubMedPubMed CentralGoogle Scholar
- Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, Weiss RL, Borkovich KA, Dunlap JC. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. PNAS USA. 2006;103(27):10352–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD. The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol. 2012;13(4):414–30.View ArticlePubMedGoogle Scholar
- Doehlemann G, Berndt P, Hahn M. Different signalling pathways involving a Gα protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Mol Microbiol. 2006;59(3):821–35.View ArticlePubMedGoogle Scholar
- Egan MJ, Wang ZY, Jones MA, Smirnoff N, Talbot NJ. Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. PNAS USA. 2007;104(28):11772–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Elad Y, Pertot I, Cores-Prado AM, Stewart A. Plant hosts of Botrytis spp. In: Fillinger S, Elad Y, editors. Botrytis—the fungus, the pathogen and its management in agricultural systems. Berlin: Springer; 2016.Google Scholar
- Giesbert S, Schuerg T, Scheele S, Tudzynski P. The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea. Mol Plant Pathol. 2008;9(3):317–27.View ArticlePubMedGoogle Scholar
- Giesbert S, Siegmund U, Schumacher J, Kokkelink L, Tudzynski P. Functional analysis of BcBem1 and its interaction partners in Botrytis cinerea: impact on differentiation and virulence. PLoS One. 2014;9(5):e95172.View ArticlePubMedPubMed CentralGoogle Scholar
- Goerlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res. 2000;87:26–32.View ArticleGoogle Scholar
- Gomi M, Akazawa F, Mitaku S. SOSUIsignal: software system for prediction of signal peptide and membrane protein. Genome Inform. 2000;11:414–5.Google Scholar
- Govrin EM, Levine A. The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr Biol. 2000;10(13):751–7.View ArticlePubMedGoogle Scholar
- Gronover CS, Kasulke D, Tudzynski P, Tudzynski B. The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea. Mol Plant Microbe Interact. 2001;11(0894-0282 (Print); 0894-0282 (Linking)):1293–302.
- Heller J, Meyer AJ, Tudzynski P. Redox-sensitive GFP2: use of the genetically encoded biosensor of the redox status in the filamentous fungus Botrytis cinerea. Mol Plant Pathol. 2012;8(1364-3703 (Electronic); 1364-3703 (Linking)):935–47.
- Helmcke I, Heumueller S, Tikkanen R, Schröder K, Brandes RP. Identification of structural elements in Nox1 and Nox4 controlling localization and activity. Antioxid Redox Signal. 2009;11(6):1279–87.View ArticlePubMedGoogle Scholar
- Jones D, Taylor W, Thornton J. A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry (NY). 1994;33(10):3038–49.View ArticleGoogle Scholar
- Kim HJ, Chen C, Kabbage M, Dickman MB. Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases. Appl Environ Microbiol. 2011;77(21):7721–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Klimpel A, Gronover CS, Williamson B, Stewart JA, Tudzynski B. The adenylate cyclase (BAC) in Botrytis cinerea is required for full pathogenicity. Mol Plant Pathol. 2002;3(1364-3703 (Electronic); 1364-3703 (Linking)):439–50
- Lacaze I, Lalucque H, Siegmund U, Silar P, Brun S. Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi. Mol Microbiol. 2015;95(6):1006–24.View ArticlePubMedGoogle Scholar
- Lambeth JD. Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med. 2007;43(3):332–47.View ArticlePubMedPubMed CentralGoogle Scholar
- Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 2004;4(3):181–9.View ArticlePubMedGoogle Scholar
- Lara-Ortiz T, Riveros-Rosas H, Aguirre J. Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans. Mol Microbiol. 2003;50(4):1241–55.View ArticlePubMedGoogle Scholar
- Laurindo FR, Araujo TL, Abrahao TB. Nox NADPH oxidases and the endoplasmic reticulum. Antioxid Redox Signal. 2014;20(17):2755–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Li X, Zhang H, Tian L, Huang L, Liu S, Li D, Song F. Tomato SlRbohB, a member of the NADPH oxidase family, is required for disease resistance against Botrytis cinerea and tolerance to drought stress. Front Plant Sci. 2015;6:463.PubMedPubMed CentralGoogle Scholar
- Malagnac F, Lalucque H, Lepere G, Silar P. Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina. Fungal Genet Biol. 2004;41(11):982–97.View ArticlePubMedGoogle Scholar
- Marschall R, Tudzynski P. BcIqg1, a fungal IQGAP homolog, interacts with NADPH oxidase, MAP kinase and calcium signaling proteins and regulates virulence and development in Botrytis cinerea. Mol Microbiol. 2016;101(2):281–98.View ArticlePubMedGoogle Scholar
- Marschall R, Schumacher J, Siegmund U, Tudzynski P. 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. Fungal Genet Biol. 2016;90:12–22.View ArticlePubMedGoogle Scholar
- Marschall R, Tudzynski P. A new and reliable method for live imaging and quantification of reactive oxygen species in Botrytis cinerea: technological advancement. Fungal Genet Biol. 2014;71:68–75.View ArticlePubMedGoogle Scholar
- McIlvaine TC. A buffer solution for colorimetric comparison. J Biol Chem. 1921;49:183–6.Google Scholar
- Pasquier C, Promponas VJ, Palaios GA, Hamodrakas JS, Hamodrakas SJ. A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm. Protein Eng. 1999;12:381–5.View ArticlePubMedGoogle Scholar
- Pontecorvo G, Roper JA, Hemmons LM, MacDonald KD, Bufton AW. The genetics of Aspergillus nidulans. Adv Genet. 1953;5:141–238.PubMedGoogle Scholar
- Roca MG, Weichert M, Siegmund U, Tudzynski P, Fleissner A. Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity. Fungal Biol. 2012;116(1878-6146 (Print)):379–87.
- Schumacher J. Tools for Botrytis cinerea: new expression vectors make the gray mold fungus more accessible to cell biology approaches. Fungal Genet Biol. 2012;6(1096-0937 (Electronic); 1087-1845 (Linking)):483–497
- Schuermann J, Buttermann D, Herrmann A, Giesbert S, Tudzynski P. Molecular characterization of the NADPH oxidase complex in the ergot fungus Claviceps purpurea: CpNox2 and CpPls1 are important for a balanced host–pathogen interaction. Mol Plant-Microbe Interact. 2013;26(10):1151–64.View ArticleGoogle Scholar
- Schwarzlaender M, Fricker MD, Mueller C, Marty L, Brach T, Novák J, Sweetlove L, Hell R, Meyer A. Confocal imaging of glutathione redox potential in living plant cells. J Microsc. 2008;231:299–316.View ArticleGoogle Scholar
- Segmueller N, Kokkelink L, Giesbert S, Odinius D, van Kan J, Tudzynski P. NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea. Mol Plant Microbe Interact. 2008;21(6):808–19.View ArticleGoogle Scholar
- Siegmund U, Heller J, van Kan JA, Tudzynski P. The NADPH oxidase complexes in Botrytis cinerea: evidence for a close association with the ER and the tetraspanin Pls1. PLoS One. 2013;8(2):e55879.View ArticlePubMedPubMed CentralGoogle Scholar
- Siegmund U, Marschall R, Tudzynski P. BcNoxD, a putative ER protein, is a new component of the NADPH oxidase complex in Botrytis cinerea. Mol Microbiol. 2015;95(6):988–1005.View ArticlePubMedGoogle Scholar
- Siegmund U, Viefhues A. Reactive oxygen species in the Botrytis—host interaction. In: Fillinger S, Elad Y, editors. Botrytis—the fungus, the pathogen and its management in agricultural systems. Berlin: Springer; 2016.Google Scholar
- Skryhan K, Cuesta-Seijo JA, Nielsen MM, Marri L, Mellor SB, Glaring MA, Jensen PE, Palcic MM, Blennow A. The role of cysteine residues in redox regulation and protein stability of Arabidopsis thaliana starch synthase 1. PLoS One. 2015;10(9):e0136997.View ArticlePubMedPubMed CentralGoogle Scholar
- Steinhorst L, Kudla J. Signaling in cells and organisms—calcium holds the line. Curr Opin Plant Biol. 2014;22:14–21.View ArticlePubMedGoogle Scholar
- Takemoto D, Kamakura S, Saikia S, Becker Y, Wrenn R, Tanaka A, Sumimoto H, Scott B. Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex. PNAS USA. 2011;108(7):2861–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Takemoto D, Tanaka A, Scott B. NADPH oxidases in fungi: diverse roles of reactive oxygen species in fungal cellular differentiation. Fungal Genet Biol. 2007;44(11):1065–76.View ArticlePubMedGoogle Scholar
- Tsachaki M, Birk J, Egert A, Odermatt A. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions. Biochim Biophys Acta (BBA) Mol Cell Res. 2015;1853(7):1672–82.View ArticleGoogle Scholar
- Tudzynski P, Heller J, Siegmund U. Reactive oxygen species generation in fungal development and pathogenesis. Curr opin Microbiol. 2012;15(6):653–9.View ArticlePubMedGoogle Scholar
- Williamson B, Tudzynski B, Tudzynski P, van Kan JA. Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol. 2007;8(5):561–80.View ArticlePubMedGoogle Scholar
- Yang SL, Chung K. The NADPH oxidase-mediated production of hydrogen peroxide (H2O2) and resistance to oxidative stress in the necrotrophic pathogen Alternaria alternata of citrus. Mol Plant Pathol. 2012;13(8):900–14.View ArticlePubMedGoogle Scholar