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

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

Role of reactive oxygen species in fungal cellular differentiations

Regulated synthesis of reactive oxygen species (ROS) by specific fungal NADPH oxidases (Noxs) plays a key role in fungal cellular differentiation and development. Fungi have up to three different Nox isoforms, NoxA, B and C. The NoxA isoform has a key role in triggering the development of fruiting bodies in several sexual species whereas NoxB plays a key role in ascospore germination. The function of NoxC remains unknown. Both NoxA and NoxB are required for the development of fungal infection structures by some plant pathogens. ROS production by NoxA is critical for maintaining a fungal-plant symbiosis. Localised synthesis of ROS is also important in establishing and maintaining polarised hyphal growth. Activation of NoxA/NoxB requires the regulatory subunit, NoxR, and the small GTPase RacA. The BemA scaffold protein may also be involved in the assembly of the Nox complex. By analogy with mammalian systems MAP and PAK kinases may regulate fungal Nox activation. How fungal cells sense and respond to ROS associated with cellular differentiations remains to be discovered.     

Conservation of fungal and animal nicotinamide adenine dinucleotide phosphate oxidase complexes

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) are a group of eukaryotic flavoenzymes that catalyse the reduction of dioxygen to the superoxide anion using electrons provided by NADPH. An integral membrane flavocytochrome b558 heterodimer, composed of the catalytic subunit gp91^phox and the adaptor protein p22^phox, is essential for catalytic activity of the mammalian Nox2 complex. Two homologues of the mammalian gp91^phox, NoxA and NoxB, have been identified in fungi and shown to be crucial for distinct fungal cell differentiation and developmental processes, but to date, no homologue of the p22^phox adaptor protein has been identified. Isolation of a mutant from Podospora anserina with a phenotype identical to a previously characterised PaNox1 mutant, combined with phylogenetic analysis, identified a fungal homologue of p22^phox called PaNoxD. The same adaptor protein was shown to be a component of the Botrytis cinerea NoxA complex as supported by the identical phenotypes of the bcnoxA and bcnoxD mutants and direct physical interaction between BcNoxA and BcNoxD. These results suggest that NoxA/NoxD is the fungal equivalent of the mammalian gp91^phox/p22^phox flavocytochrome complex. Tetraspanin (Pls1) mutants of P. anserina and B. cinerea have identical phenotypes to noxB mutants, suggesting that Pls1 is the corresponding integral membrane adaptor for assembly of the NoxB complex.     

Direct interaction between Tks proteins and the N-terminal proline-rich region (PRR) of NoxA1 mediates Nox1-dependent ROS generation

NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been implicated in several physiological and pathophysiological processes. To date seven members of this family have been reported, including Nox1-5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, the organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate proteins Tks4 and Tks5 as functional members of a p47^phox-related organizer superfamily. As a functional consequence of this interaction, Nox1 localizes to invadopodia, actin-rich membrane protrusions of cancer cells which facilitate pericellular proteolysis and invasive behavior.Here, we report that Tks4 and Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of NoxA1 is essential for this direct interaction with the Tks proteins. When the PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5 are unable to act as organizers for Nox2 because of their inability to interact with p67^phox, which lacks the N-terminal PRR, thus conferring a unique specificity to Tks4 and 5.Taken together, these results clarify the molecular basis for the interaction between NoxA1 and the Tks proteins and may provide new insights into the pharmacological design of a more effective anti-metastatic strategy.     

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.     

A homologue of the fungal tetraspanin Pls1 is required for Epichloë festucae expressorium formation and establishment of a mutualistic interaction with Lolium perenne

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium-like structure known as an expressorium. In plant-pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild-type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium-like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring-like structure at the point of cuticle exit as found in the wild-type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.     

Selective Recapitulation of Conserved and Nonconserved Regions of Putative NOXA1 Protein Activation Domain Confers Isoform-specific Inhibition of Nox1 Oxidase and Attenuation of Endothelial Cell Migration

Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O₂^⨪) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O₂^⨪ generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O₂^⨪ production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.     

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22^phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H₂O₂ production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22^phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O₂⁻ generation. In contrast, Nox4 released H₂O₂ and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.The family of NADPH oxidases consists of seven members termed Nox/Duox that differ in their tissue expression profiles, modes of activation, reactive oxygen species (ROS) outputs, and physiological functions. Understanding their distinguishing features is a prerequisite for rational inhibitor design and thus targeted intervention in ROS-mediated pathophysiologies (4). The coexpression of different Nox isoforms, each with potentially distinct functional profiles, in the same cell type necessitates a more discriminating approach than application of pan-Nox inhibitors. Detailed structure-function studies are necessary to identify unique regions and their impact with respect to catalytic function or localization of the enzyme. All Nox/Duox enzymes share a Nox backbone with six predicted transmembrane domains and an intracellular carboxyl-terminal domain which harbors FAD and NADPH binding sites. Nox5 and Duox1/2 enzymes contain additional structural elements such as amino terminal EF-hand motifs, a hallmark of their regulation by the intracellular calcium concentration (13, 30).The founding member of the NADPH oxidase family, the phagocyte oxidase, consists of membrane-bound Nox2 in a complex with the smaller subunit p22^phox (3). Heterodimerization of these two proteins is required for maturation and translocation of the enzyme complex to the plasma membrane or to intracellular vesicles. The Nox family members Nox1, Nox3, and Nox4 follow this paradigm (1, 14, 21, 25, 31). Heterodimer formation and association of the Nox/p22^phox complex at particular cellular membranes is essential for catalytic activity, i.e., for ROS generation. Nox2, and to a lesser degree Nox1 and Nox3, remain dormant under resting conditions and rely on stimulus-dependent translocation and assembly of oxidase components such as p47^phox and p67^phox, or NoxO1 and NoxA1 in the case of Nox1 and Nox3 (16). These steps, together with activation and translocation of the GTPase Rac, ultimately lead to the assembled, catalytically active oxidase and to ROS generation.Nox4 differs from the usual theme of multimeric assembly of active NADPH oxidases found in Nox1 to Nox3 (21, 22, 28, 32). Constitutive H₂O₂ production by Nox4 localized at perinuclear vesicles has been reported (1, 21, 28). Since NADPH oxidases catalyze the one-electron reduction of molecular oxygen to superoxide anion, the current dogma suggests that Nox4 generates intracellular superoxide. The superoxide produced will then dismutate rapidly to H₂O₂, diffusing from the cell into the extracellular milieu. Cytosolic proteins, which regulate the activity of Nox1 to Nox3 by binding to the carboxyl-terminal domains of Nox1 to Nox3, seem to be irrelevant for Nox4 function. The membrane-bound subunit p22^phox is to date the only known protein associated with Nox1 to Nox4. Heterodimerization, translocation, and enzymatic function of these oxidases require p22^phox. Recent structure-function analyses of complexes between Nox2 or Nox4 and the subunit p22^phox documented specific regions and amino acid residues in p22^phox necessary for complex formation and oxidase activity (35, 37). Interestingly, a p22^phox mutant (p22^phox Y121H) is capable of distinguishing between Nox1 to Nox3 and Nox4 by forming a functional complex only with Nox4, further suggesting unique structural features in Nox4 (35).In this study, we expand structure-function analysis of the oxidase complex by comparing Nox4/Nox2 chimeric enzymes with respect to NADPH oxidase activity, type of reactive oxygen species produced, requirement for additional oxidase components, and detailed subcellular localization.     

A p67Phox-like regulator is recruited to control hyphal branching in a fungal-grass mutualistic symbiosis

Key requirements for microbes to initiate and establish mutualistic symbiotic interactions with plants are evasion of potential host defense responses and strict control of microbial growth. Reactive oxygen species (ROS) produced by a specific NADPH oxidase isoform, NoxA, regulate hyphal growth in the mutualistic interaction between the fungal endophyte Epichlo? festucae and its grass host Lolium perenne. Unlike mammalian systems, little is known about the fungal NADPH oxidase complex and its response to differentiation signals. We identify an E. festucae p67(phox)-like regulator, NoxR, dispensable in culture but essential in planta for the symbiotic interaction. Plants infected with a noxR deletion mutant show severe stunting and premature senescence, whereas hyphae in the meristematic tissues show increased branching leading to increased fungal colonization of pseudostem and leaf blade tissue. Inhibition of ROS production or overexpression of noxR recapitulates the hyperbranching phenotype in culture. NoxR interacts in vitro with the small GTP binding protein RacA and requires a functional RacA binding site to complement the noxR mutant and restore the wild-type plant interaction phenotype. These results show that NoxR is a key regulator of NoxA in symbiosis, where it acts together with RacA to spatially regulate ROS production and control hyphal branching and patterning.     

HIF-1α Activation by Intermittent Hypoxia Requires NADPH Oxidase Stimulation by Xanthine Oxidase

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O₂ saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O₂ for 30 sec) and normoxia (21% O₂ for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47^phox and p67^phox to the plasma membrane and their interaction with gp91^phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.     

Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding

Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3zeta protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.     

Molecular evolution of <Emphasis Type="Italic">Phox</Emphasis>-related regulatory subunits for NADPH oxidase enzymes

Background  

The reactive oxygen-generating N ADPH ox idases (Noxes) function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22 phox, and this heterodimer binds to the regulatory subunits p47 phox, p67 phox, p40 phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 (NOXA1), respective homologs of p47 phox and p67 phox, together with p22 phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22 phox also regulate Nox3, whereas Nox4 requires only p22 phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits.     

c-Src-mediated phosphorylation of NoxA1 and Tks4 induces the reactive oxygen species (ROS)-dependent formation of functional invadopodia in human colon cancer cells

The NADPH oxidase family, consisting of Nox1-5 and Duox1-2, catalyzes the regulated formation of reactive oxygen species (ROS). Highly expressed in the colon, Nox1 needs the organizer subunit NoxO1 and the activator subunit NoxA1 for its activity. The tyrosine kinase c-Src is necessary for the formation of invadopodia, phosphotyrosine-rich structures which degrade the extracellular matrix (ECM). Many Src substrates are invadopodia components, including the novel Nox1 organizer Tks4 and Tks5 proteins. Nox1-dependent ROS generation is necessary for the maintenance of functional invadopodia in human colon cancer cells. However, the signals and the molecular machinery involved in the redox-dependent regulation of invadopodia formation remain unclear. Here, we show that the interaction of NoxA1 and Tks proteins is dependent on Src activity. Interestingly, the abolishment of Src-mediated phosphorylation of Tyr110 on NoxA1 and of Tyr508 on Tks4 blocks their binding and decreases Nox1-dependent ROS generation. The contemporary presence of Tks4 and NoxA1 unphosphorylable mutants blocks SrcYF-induced invadopodia formation and ECM degradation, while the overexpression of Tks4 and NoxA1 phosphomimetic mutants rescues this phenotype. Taken together, these results elucidate the role of c-Src activity on the formation of invadopodia and may provide insight into the mechanisms of tumor formation in colon cancers.     

NADPH oxidases in fungi: diverse roles of reactive oxygen species in fungal cellular differentiation

Synthesis of reactive oxygen species (ROS) by specific NADPH oxidases (Nox) can serve both defense and differentiation signaling roles in animals and plants. Fungi have three subfamilies of NADPH oxidase. NoxA and NoxB have a structure very similar to the human gp91(phox). NoxC has in addition a Ca(2+) binding motif as found in the human Nox5 and plant Rboh families of NADPH oxidases. A survey of fungal genomes identified up to four Nox genes in some fungal species, but Nox genes are absent from available genomes of the hemiascomycete yeasts, unicellular Basidiomycetes and Zygomycetes, reflecting the diversity of fungal life forms. Specific isoforms of Nox have been shown by genetic analysis to be required for various physiological processes and cellular differentiations, including development of sexual fruiting bodies, ascospore germination, hyphal defense, hyphal growth in both mutualistic and antagonistic plant-fungal interactions. This review provides an overview of our current knowledge of fungal NADPH oxidases, including Nox distribution in the fungal kingdom, Nox structure and regulation, and known biological functions of this important group of enzymes.     

A cAMP‐Dependent Protein Kinase Gene,aapk1, Is Required for Mycelia Growth,Toxicity and Pathogenicity of Alternaria alternata on Tobacco

The fungus Alternaria alternata is a common spot‐producing plant pathogen. During the past decade, tobacco brown spot disease caused by this fungus has became prevalent in China and lead to significant losses. To better understand the molecular pathogenesis of this fungus, the aapk1 gene encoding a cAMP‐dependent protein kinase catalytic subunit was cloned, sequenced and characterized. The aapk1 deletion mutants were identified from hygromycin‐resistant transformants by PCR strategy and confirmed by Southern blot analysis and RT‐PCR. The aapk1 deletion mutant exhibited reduced vegetative growth and was less toxic than the wild‐type strain sd1. Deletion of aapk1 also delayed disease development on detached tobacco leaves. Thus, we propose that the cAMP signalling pathway is involved in mycelia growth and pathogenic phenotype of Alternaria alternata.     

Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk desensitizes EGF-induced ROS generation

Accumulating evidence indicates that protein phosphorylation regulates Nox activity. In this report, we show that serine282 residue of Nox activator 1 (NoxA1) is phosphorylated by Erk in response to EGF resulting in desensitization of Nox1 activity. Specifically, murine NoxA1 is detected as two independent protein bands in SDS PAGE, and the form of protein with higher mobility shifted to and merged with the one with lower mobility in response to EGF treatment. Pretreatment with PD98059 resulted in inhibition of NoxA1 migration in response to EGF indicating that Erk was involved in the process. Site-directed mutagenesis showed that S282A mutant but not S239A mutant failed to respond to EGF, demonstrating that serine282 is the target amino acid of Erk. Expression of S282A mutant of NoxA1 in these cells led to increased superoxide anion production in response to EGF compared to expression of the wild type, whereas the expression of S282E, a phosphomimetic mutant, resulted in significantly decreased superoxide anion generation. We also tested whether the phosphorylation of serine282 of NoxA1 affects Rac activation. Expression of S282A mutant NoxA1 up-regulated the Rac activity, whereas expression of S282E mutant led to the abrogation of Rac activation. Taken together, these results demonstrate that phosphorylation of NoxA1 is a part of the feedback mechanism that functions through activation of Rac with a net outcome of negative modulation of Nox1 activity.     

NoxA activation by the small GTPase RacA is required to maintain a mutualistic symbiotic association between Epichloë festucae and perennial ryegrass

Small GTPases of the Rac group play a key regulatory role in NADPH oxidase catalysed production of reactive oxygen species (ROS) in mammals and plants, but very little evidence is available for a corresponding role in fungi. We recently showed that ROS produced by a specific fungal NADPH oxidase isoform, NoxA, are crucial in regulating hyphal morphogenesis and growth in the mutualistic symbiotic interaction between Epichloë festucae and perennial ryegrass. We demonstrate here that E. festucae RacA is required for NoxA activation and regulated production of ROS to maintain a symbiotic interaction. Deletion of racA resulted in decreased ROS production, reduction of radial growth and hyper‐branching of the hyphae in culture. In contrast, in planta the racA mutant showed extensive colonization of the host plant, resulting in stunting and precocious senescence of the host plants. Strains expressing a dominant active (DA) allele of RacA had increased ROS production, increased aerial hyphae and reduced radial growth. These results demonstrate that RacA plays a crucial role in regulating ROS production by NoxA, in order to control hyphal morphogenesis and growth of the endophyte in planta.     

How the Pathogenic Fungus Alternaria alternata Copes with Stress via the Response Regulators SSK1 and SHO1

The tangerine pathotype of Alternaria alternata is a necrotrophic fungal pathogen causing brown spot disease on a number of citrus cultivars. To better understand the dynamics of signal regulation leading to oxidative and osmotic stress response and fungal infection on citrus, phenotypic characterization of the yeast SSK1 response regulator homolog was performed. It was determined that SSK1 responds to diverse environmental stimuli and plays a critical role in fungal pathogenesis. Experiments to determine the phenotypes resulting from the loss of SSK1 reveal that the SSK1 gene product may be fulfilling similar regulatory roles in signaling pathways involving a HOG1 MAP kinase during ROS resistance, osmotic resistance, fungicide sensitivity and fungal virulence. The SSK1 mutants display elevated sensitivity to oxidants, fail to detoxify H₂O₂ effectively, induce minor necrosis on susceptible citrus leaves, and displays resistance to dicarboximide and phenylpyrrole fungicides. Unlike the SKN7 response regulator, SSK1 and HOG1 confer resistance to salt-induced osmotic stress via an unknown kinase sensor rather than the “two component” histidine kinase HSK1. SSK1 and HOG1 play a moderate role in sugar-induced osmotic stress. We also show that SSK1 mutants are impaired in their ability to produce germ tubes from conidia, indicating a role for the gene product in cell differentiation. SSK1 also is involved in multi-drug resistance. However, deletion of the yeast SHO1 (synthetic high osmolarity) homolog resulted in no noticeable phenotypes. Nonetheless, our results show that A. alternata can sense and react to different types of stress via SSK1, HOG1 and SKN7 in a cooperative manner leading to proper physiological and pathological functions.