Contribution of Autophagy to Cellular Iron Homeostasis and Stress Adaptation in Alternaria alternata.

The tangerine pathotype of Alternaria alternata produces the Alternaria citri toxin (ACT), which elicits a host immune response characterized by the increase in harmful reactive oxygen species (ROS) production. ROS detoxification in A. alternata relies on the degradation of peroxisomes through autophagy and iron acquisition using siderophores. In this study, we investigated the role of autophagy in regulating siderophore and iron homeostasis in A. alternata. Our results showed that autophagy positively influences siderophore production and iron uptake. The A. alternata strains deficient in autophagy-related genes 1 and 8 (ΔAaatg1 and ΔAaatg8) could not thrive without iron, and their adaptability to high-iron environments was also reduced. Furthermore, the ability of autophagy-deficient strains to withstand ROS was compromised. Notably, autophagy deficiency significantly reduced the production of dimerumic acid (DMA), a siderophore in A. alternata, which may contribute to ROS detoxification. Compared to the wild-type strain, ΔAaatg8 was defective in cellular iron balances. We also observed iron-induced autophagy and lipid peroxidation in A. alternata. To summarize, our study indicates that autophagy and maintaining iron homeostasis are interconnected and contribute to the stress resistance and the virulence of A. alternata. These results provide new insights into the complex interplay connecting autophagy, iron metabolism, and fungal pathogenesis in A. alternata.

The Role of a Nascent Polypeptide-Associated Complex Subunit Alpha in Siderophore Biosynthesis, Oxidative Stress Response, and Virulence in Alternaria alternata.

The present study demonstrates that a nascent polypeptide-associated complex α subunit (Nac1) functions as a transcriptional regulator and plays both positive and negative roles in a vast array of functions in Alternaria alternata. Gain- and loss-of-function studies reveal that Nac1 is required for the formation and germination of conidia, likely via the regulation of Fus3 and Slt2 mitogen-activated protein kinase (MAPK)-coding genes, both implicated in conidiation. Nac1 negatively regulates hyphal branching and the production of cell wall-degrading enzymes. Importantly, Nac1 is required for the biosynthesis of siderophores, a novel phenotype that has not been reported to be associated with a Nac in fungi. The expression of Nac1 is positively regulated by iron, as well as by the Hog1 MAPK and the NADPH-dependent oxidase (Nox) complex. Nac1 confers cellular susceptibility to reactive oxygen species (ROS) likely via negatively regulating the expression of the genes encoding Yap1, Skn7, Hog1, and Nox, all involved in ROS resistance. The involvement of Nac1 in sensitivity to glucose-, mannitol-, or sorbitol-induced osmotic stress could be due to its ability to suppress the expression of Skn7. The requirement of Nac1 in resistance to salts is unlikely mediated through the transcriptional activation of Hog1. Although Nac1 plays no role in toxin production, Nac1 is required for fungal full virulence. All observed deficiencies can be restored by re-expressing a functional copy of Nac1, confirming that Nac1 contributes to the phenotypes. Thus, a dynamic regulation of gene expression via Nac1 is critical for developmental, physiological, and pathological processes of A. alternata.

The siderophore repressor SreA maintains growth, hydrogen peroxide resistance, and cell wall integrity in the phytopathogenic fungus Alternaria alternata.

The siderophore-mediated iron uptake machinery is required by the tangerine pathotype of Alternaria alternata to colonize host plants. The present study reports the functions of the GATA-type transcription regulator SreA by analyzing loss- and gain-of-function mutants. The expression of sreA is transiently upregulated by excess iron. The sreA deficiency mutant (ΔsreA) shows severe growth defect but produces ACT toxin and incites necrotic lesions on citrus leaves as efficiently as wild type. SreA suppresses the expression of genes encoding polypeptides required for siderophore biosynthesis and transport under iron-replete conditions. Under iron-replete conditions, SreA impacts the expression of the genes encoding the NADPH oxidase complex involved in H2O2 production. SreA negatively impacts H2O2 resistance as ΔsreA increases resistance to H2O2. However, sreA deficiency has no effects on the expression of genes encoding several key factors (Yap1, Hog1, and Skn7) involved in oxidative stress resistance. ΔsreA increases resistance to calcofluor white and Congo red, which may suggest a role of SreA in the maintenance of cell wall integrity. Those are novel phenotypes associated with fungal sreA. Overall, our results indicate that SreA is required to protect fungal cells from cytotoxicity caused by excess iron. The results also highlight the regulatory functions of SreA and provide insights into the critical role of siderophore-mediated iron homeostasis in resistance to oxidative stress in A. alternata.

Carbon catabolite repression gene creA regulates morphology, aflatoxin biosynthesis and virulence in Aspergillus flavus.

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.

Thioredoxin and Glutaredoxin Systems Required for Oxidative Stress Resistance, Fungicide Sensitivity, and Virulence of Alternaria alternata.

This study determined the function of thioredoxin and glutaredoxin systems in the phytopathogenic fungus Alternaria alternata via analyzing mutants obtained from the targeted deletion of genes encoding thioredoxin peroxidase (Tsa1), thioredoxin reductase (Trr1), and glutathione reductase (Glr1). Trr1 and Glr1, but not Tsa1, are required for growth and conidiation. The reduced growth and conidiation seen in the Trr1 or Glr1 deletion mutant can be restored by glutathione. Deletion mutants showing growth inhibition by oxidants are defective for H2O2 detoxification and induce smaller lesions on citrus leaves. Trr1 and Glr1, but not Tsa1, also contribute to NaCl resistance. Glr1 is required for sorbitol resistance and is responsible for resistance to mancozeb and boscalid but not chlorothalonil fungicides, a novel phenotype that has not been reported in fungi. Trr1 is required for resistance to boscalid and chlorothalonil fungicides but confers susceptibility to mancozeb. The Tsa1 deletion mutant displays wild-type sensitivity to the tested fungicides. The expression of Tsa1 and Trr1 is regulated by the oxidative stress responsive regulators Yap1, Hog1, and Skn7. The expression of Tsa1, but not Trr1, is also regulated indirectly by the NADPH oxidase. The results indicate that the capability to resist oxidative stress is required for virulence of A. alternataIMPORTANCE The thioredoxin and glutaredoxin systems are important thiol antioxidant systems in cells, and knowledge of these two systems in the plant-pathogenic fungus A. alternata is useful for finding new strategies to reduce the virulence of this pathogen. In this study, we demonstrated that thiol antioxidant system-related genes (Tsa1, Trr1, and Glr1) are required for H2O2 detoxification and virulence in A. alternata Moreover, deletion of Trr1 results in hypersensitivity to the fungicides chlorothalonil and boscalid, and Glr1 deletion mutants are highly sensitive to mancozeb, which is the fungicide mostly used in citrus fields. Therefore, our findings demonstrate that the ability to detoxify reactive oxygen species (ROS) plays a critical role in pathogenesis on citrus and provide novel insights into the physiological functions of thiol-containing systems in fungicide sensitivity for A. alternata.

CoII(Chromomycin)2 Complex Induces a Conformational Change of CCG Repeats from i-Motif to Base-Extruded DNA Duplex.

We have reported the propensity of a DNA sequence containing CCG repeats to form a stable i-motif tetraplex structure in the absence of ligands. Here we show that an i-motif DNA sequence may transition to a base-extruded duplex structure with a GGCC tetranucleotide tract when bound to the (CoII)-mediated dimer of chromomycin A3, CoII(Chro)2. Biophysical experiments reveal that CCG trinucleotide repeats provide favorable binding sites for CoII(Chro)2. In addition, water hydration and divalent metal ion (CoII) interactions also play a crucial role in the stabilization of CCG trinucleotide repeats (TNRs). Our data furnish useful structural information for the design of novel therapeutic strategies to treat neurological diseases caused by repeat expansions.

The glutathione peroxidase-mediated reactive oxygen species resistance, fungicide sensitivity and cell wall construction in the citrus fungal pathogen Alternaria alternata.

The ability to detoxify reactive oxygen species (ROS) is critical for pathogenicity in the necrotrophic fungus Alternaria alternata. We report a glutathione peroxidase 3 (AaGPx3) involved in the complex signalling network that is essential for the detoxification of cellular stresses induced by ROS and for A. alternata pathogenesis in citrus. AaGPx3 deletion mutants displayed increased sensitivity to H2 O2 and many ROS-generating compounds. AaGPx3 is required for correct fungal development as the AaGPx3 mutant strains showed a severe reduction in conidiation. AaGPx3 mutants accumulated higher chitin content than the wild-type and were less sensitive to the cell wall-targeting compounds calcofluor white and Congo red, as well as the fungicides fludioxonil and vinclozolin, suggesting a role of the glutathione systems in fungal cell wall construction. Virulence assays revealed that AaGPx3 is required for full virulence. The expression of AaGPx3 was downregulated in fungal strains carrying defective NADPH oxidase (Nox) or the oxidative stress responsive regulators YAP1 and HOG1, all implicated in ROS resistance. These results further support the important role of ROS detoxification during A. alternata pathogenesis in citrus. Overall, our study provides genetic evidence to define the central role of AaGPx3 in the biological and pathological functions of A. alternata.

Calcineurin phosphatase and phospholipase C are required for developmental and pathological functions in the citrus fungal pathogen Alternaria alternata.

Excessive Ca(2+) or compounds interfering with phosphoinositide cycling have been found to inhibit the growth of the tangerine pathotype of Alternaria alternata, suggesting a crucial role of Ca(2+) homeostasis in this pathotype. The roles of PLC1, a phospholipase C-coding gene and CAL1, a calcineurin phosphatase-coding gene were investigated. Targeted gene disruption showed that both PLC1 and CAL1 were required for vegetative growth, conidial formation and pathogenesis in citrus. Fungal strains lacking PLC1 or CAL1 exhibited extremely slow growth and induced small lesions on calamondin leaves. Δplc1 mutants produced fewer conidia, which germinated at slower rates than wild-type. Δcal1 mutants produced abnormal hyphae and failed to produce any mature conidia, but instead produced highly melanized bulbous hyphae with distinct septae. Fluorescence microscopy using Fluo-3 dye as a Ca(2+) indicator revealed that the Δplc1 mutant hyphae emitted stronger cytosolic fluorescence, and the Δcal1 mutant hyphae emitted less cytosolic fluorescence, than those of wild-type. Infection assessed on detached calamondin leaves revealed that application of CaCl2 or neomycin 24 h prior to inoculation provided protection against Alt. alternata. These data indicate that a dynamic equilibrium of cellular Ca(2+) is critical for developmental and pathological processes of Alt. alternata.

Resistance to oxidative stress via regulating siderophore-mediated iron acquisition by the citrus fungal pathogen Alternaria alternata.

The ability of the necrotrophic fungus Alternaria alternata to detoxify reactive oxygen species (ROS) is crucial for pathogenesis to citrus. We report regulation of siderophore-mediated iron acquisition and ROS resistance by the NADPH oxidase (NOX), the redox activating yes-associated protein 1 (YAP1) regulator, and the high-osmolarity glycerol 1 (HOG1) mitogen-activated protein kinase (MAPK). The A. alternata nonribosomal peptide synthetase (NPS6) is essential for the biosynthesis of siderophores, contributing to iron uptake under low-iron conditions. Fungal strains impaired for NOX, YAP1, HOG1 or NPS6 all display increased sensitivity to ROS. Exogenous addition of iron at least partially rescues ROS sensitivity seen for NPS6, YAP1, HOG1, and NOX mutants. Importantly, expression of the NPS6 gene and biosynthesis of siderophores are regulated by NOX, YAP1 and HOG1, supporting a functional link among these regulatory pathways. Although iron fully rescues H2O2 sensitivity seen in mutants impaired for the response regulator SKN7, neither expression of NPS6 nor biosynthesis of siderophores is controlled by SKN7. Our results indicate that the acquisition of environmental iron has profound effects on ROS detoxification.

Impaired induction of allergic lung inflammation by Alternaria alternata mutant MAPK homologue Fus3.

The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.

Cyclic AMP-dependent protein kinase A negatively regulates conidia formation by the tangerine pathotype of Alternaria alternata.

The necrotrophic fungal pathogen Alternaria alternata causes brown spot diseases in many citrus cultivars. The FUS3 and SLT2 mitogen-activated protein kinases (MAPK)-mediated signaling pathways have been shown to be required for conidiation. Exogenous application of cAMP to this fungal pathogen decreased conidia formation considerably. This study determined whether a cAMP-activated protein kinase A (PKA) is required for conidiation. Using loss-of-function mutations in PKA catalytic and regulatory subunit-coding genes, we demonstrated that PKA negatively regulates conidiation. Fungal mutants lacking PKA catalytic subunit gene (PKA ( cat )) reduced growth, lacked detectable PKA activity, and produced higher amounts of conidia compared to wild-type. Introduction of a functional copy of PKA ( cat ) into a null mutant partially restored PKA activity and produced wild-type level of conidia. In contrast, fungi lacking PKA regulatory subunit gene (PKA ( reg )) produced detectable PKA activity, exhibited severe growth reduction, formed swelling hyphal segments, and produced no mature conidia. Introduction of the PKA ( reg ) gene to a regulatory subunit mutant restored all phenotypes to wild type. PKA ( reg )-null mutants induced fewer necrotic lesions on citrus compared to wild-type, whereas PKA ( cat ) mutant displayed wild-type virulence. Overall, our studies indicate that PKA and FUS3-mediated signaling pathways apparently have very different roles in the regulation of conidia production and A. alternata pathogenesis in citrus.

The Regulatory Hub of Siderophore Biosynthesis in the Phytopathogenic Fungus Alternaria alternata.

A GATA zinc finger-containing repressor (AaSreA) suppresses siderophore biosynthesis in the phytopathogenic fungus Alternaria alternata under iron-replete conditions. In this study, targeted gene deletion revealed two bZIP-containing transcription factors (AaHapX and AaAtf1) and three CCAAT-binding proteins (AaHapB, AaHapC, and AaHapE) that positively regulate gene expression in siderophore production. This is a novel phenotype regarding Atf1 and siderophore biosynthesis. Quantitative RT-PCR analyses revealed that only AaHapX and AaSreA were regulated by iron. AaSreA and AaHapX form a transcriptional feedback negative loop to regulate iron acquisition in response to the availability of environmental iron. Under iron-limited conditions, AaAtf1 enhanced the expression of AaNps6, thus playing a positive role in siderophore production. However, under nutrient-rich conditions, AaAtf1 plays a negative role in resistance to sugar-induced osmotic stress, and AaHapX plays a negative role in resistance to salt-induced osmotic stress. Virulence assays performed on detached citrus leaves revealed that AaHapX and AaAtf1 play no role in fungal pathogenicity. However, fungal strains carrying the AaHapB, AaHapC, or AaHapE deletion failed to incite necrotic lesions, likely due to severe growth deficiency. Our results revealed that siderophore biosynthesis and iron homeostasis are regulated by a well-organized network in A. alternata.

The Pex3-mediated peroxisome biogenesis plays a critical role in metabolic biosynthesis, stress response, and pathogenicity in Alternaria alternata.

Peroxisomes are microbodies involved in the metabolism of fatty acids and hydrogen peroxide (H2O2) in eukaryotes. In the current study, an AaPex3 gene encoding a peroxisome membrane protein was demonstrated to be required for peroxisome biogenesis and resistance to peroxides and superoxide-generating compounds. Deleting AaPex3 affected the expression of the genes encoding the NADPH oxidase (NoxA) and the Yap1 stress-responsive transcription regulator, both of which have been implicated in ROS resistance. The AaPex3-mediated peroxisome biogenesis negatively affected resistance to singlet oxygen-generating compounds, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA), novel phenotypes associated with peroxisomes. Nile red staining revealed that ΔAaPex3 accumulated more lipid bodies than the wild type. ΔAaPex3 conidia had thinner cell walls than the wild type, suggesting the involvement of AaPex3 in maintaining cell wall integrity. Genetic evidence has also demonstrated that the AaPex3-mediated peroxisome biogenesis is required for conidiogenesis, conidia germination, siderophore biosynthesis, toxin production, and virulence. Biotin or lipids could restore ΔAaPex3 growth in axenic culture and on the surface of citrus leaves. In contrast, co-application of ΔAaPex3 with biotin and oleic acid on citrus leaves failed to induce necrotic lesions. Our results revealed the multifaceted functions of peroxisomes in the phytopathogenic fungus.

Roles for SKN7 response regulator in stress resistance, conidiation and virulence in the citrus pathogen Alternaria alternata.

"Two-component" histidine kinase (HSK1) is the primary regulator of resistance to sugar osmotic stress and sensitivity to dicarboximide or phenylpyrrole fungicides in the citrus fungal pathogen Alternaria alternata. On the other hand, the mitogen-activated protein kinase HOG1 confers resistance solely to salts and oxidative stress. We report here independent and shared functions of the SKN7-mediated signaling pathway with HSK1 and HOG1. SKN7, a putative transcription downstream regulator of HSK1, is primarily required for cellular resistance to oxidative and sugar-induced osmotic stress. SKN7, perhaps acting in parallel with HOG1, is required for resistance to H(2)O(2), tert-butyl hydroperoxide, and cumyl peroxide, but not to the superoxide-generating compounds - menadione, potassium superoxide, and diamide. Because of phenotypic commonalities, SKN7 is likely involved in resistance to sugar-induced osmotic stress via the HSK1 signaling pathway. However, mutants lacking SKN7 displayed wild-type sensitivity to NaCl and KCl salts. SKN7 is constitutively localized in the nucleus regardless of H(2)O(2) treatment. When compared to the wild type, skn7 mutants exhibited lower catalase, peroxidase, and superoxide dismutase activities and induced significantly fewer necrotic lesions on the susceptible citrus cultivar. The skn7 mutant exhibited fungicide resistance at levels between the hsk1 and the hog1 mutant strains. Skn7/hog1 double mutants exhibited fungicide resistance, similar to the strain with a single AaHSK1 gene mutation. Moreover, the A. alternata SKN7 plays a role in conidia formation. Conidia produced by the skn7 mutant are smaller and have fewer transverse septae than those produced by wild type. All altered phenotypes in the mutant were restored by introducing and expressing a wild-type copy of SKN7 under control of the endogenous promoter.

Enhanced production of ganoderic acids and cytotoxicity of Ganoderma lucidum using solid-medium culture.

Submerged cultures of Ganoderma lucidum are used to produce fungal mycelium, which is used as a functional food and in the production of various triterpenoids, including ganoderic acids (GAs). Specific culture approaches that produce fungal mycelium with high levels of GAs and good biological activity are critical in the functional food industry. In this study, a solid-medium culture approach to producing mycelium was compared to the submerged culture system. Production of GAs, biomass, intracellular polysaccharides, and cytotoxicity of the cultured mycelium were compared as between solid and submerged culture. Growing G. lucidum strains on solid potato dextrose agar medium increased biomass, the production of ganoderic acid 24 (lanosta-7,9(11), 24-trien-3α-o1-26-oic acid), GAs, and total intracellular polysaccharides as compared to fungi grown in submerged culture. Triterpenoid-enriched methanol extracts of mycelium from solid-medium culture showed higher cytotoxicity than those from submerged culture. The IC(50) values of methanol extracts from solid-medium culture were 11.5, 8.6, and 9.9 times less than submerged culture on human lung cancer cells CH27, melanoma cells M21, and oral cancer cells HSC-3 respectively. The squalene synthase and lanosterol synthase coding genes had higher expression on the culture of solid potato dextrose medium. This is the first report that solid-medium culture is able to increase GA production significantly as compared to submerged culture and, in the process, produces much higher biological activity. This indicates that it may be possible to enhance the production of GAs by implementing mycelium culture on solid medium.

A zinc finger suppressor involved in stress resistance, cell wall integrity, conidiogenesis, and autophagy in the necrotrophic fungal pathogen Alternaria alternata.

The tangerine pathotype of Alternaria alternata can withstand high-level reactive oxygen species (ROS). By analyzing loss- and gain-of-function mutants, this study demonstrated that a Cys2His2 zinc finger-containing transcription regulator, A. alternata Stress Response Regulator 1 (AaSRR1), plays a negative role in resistance to peroxides and singlet-oxygen-generating compounds. AaSRR1 plays no role in cellular susceptibility or resistance to superoxide-producing compounds. AaSRR1 also negatively regulates conidiogenesis, maintenance of cell wall and membrane integrities, and chitin biosynthesis. Some wild-type hyphae displayed necrosis after exposure to 30 mM H2O2, whereas AaSRR1 deficient mutant (ΔAaSRR1) hyphae had visible granules and vacuoles. sGFP-AaATG8 proteolysis assays revealed that H2O2 and starvation could trigger autophagy formation in both wild type and ΔAaSRR1. Autophagy occurred at higher rates in ΔAaSRR1 than wild type under both conditions, particularly after H2O2 treatments, indicating that autophagy might contribute to ROS resistance. Upon exposure to H2O2 or under starvation, AaSRR1 was translocated into the nucleus, even though the expression of AaSRR1 was decreased. AaSRR1 is required for vegetative growth but is dispensable for fungal virulence as assayed on detached calamondin leaves. AaSRR1 suppressed the expression of the gene encoding a HOG1 mitogen-activated protein (MAP) kinase implicated in ROS resistance. Mutation of AaSRR1 increased catalase activity but decreased superoxide dismutase activity, leading to fewer ROS accumulation in the cytosol. Nevertheless, our results indicated that AaSRR1 is a transcription suppressor for ROS resistance. This study also revealed tradeoffs between stress responses and hyphal growth in A. alternata.

Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata.

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy-mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP-AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H2 O2 ) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H2 O2 triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down-regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium-like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild-type toxin levels in axenic culture, the mutant induced a lower level of H2 O2 and smaller necrotic lesions on citrus leaves. In addition to H2 O2 , nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy-mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.

Cellular responses required for oxidative stress tolerance, colonization, and lesion formation by the necrotrophic fungus Alternaria alternata in citrus.

The pathogenic capability of the tangerine pathotype of Alternaria alternata relies on the production of host-selective ACT toxin. Inoculation of A. alternata in leaves of the citrus quickly induced rapid lipid peroxidation, accumulation of hydrogen peroxide (H(2)O(2)), and cell death, indicative of host defensive response. We previously demonstrated an essential role of the A. alternata AaAP1 gene, encoding a redox-responsive YAP1-like transcription factor, to contribute to fungal pathogenicity. The AaAP1 null mutant fails to incite necrotic lesions. In this study, we show further that the fungal mutant defective at the AaAP1 locus displayed reduced activities for glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and ligninolytic peroxidase, yet retained normal production of ACT toxin. In contrast to the wild-type progenitor and the genetically reverted strain, the mutant strain was unable to detoxify H(2)O(2) effectively and was killed upon exposure to H(2)O(2). The mutant strain induced lower levels of H(2)O(2) accumulation in citrus leaves, compared to those induced by the wild-type or by the genetically reverted strain. Upon exposure to H(2)O(2), A. alternata apparently changed expression of a wide array of the genes regulated by AaAP1. Thus, the impairment of the AaAP1 null mutants to incite necrotic lesions is apparently a consequence of their inability to alleviate the toxicity of ROS, and circumvention of plant defenses is important for the disease process.

Peroxisomes Implicated in the Biosynthesis of Siderophores and Biotin, Cell Wall Integrity, Autophagy, and Response to Hydrogen Peroxide in the Citrus Pathogenic Fungus Alternaria alternata.

Little is known about the roles of peroxisomes in the necrotrophic fungal plant pathogens. In the present study, a Pex6 gene encoding an ATPase-associated protein was characterized by analysis of functional mutations in the tangerine pathotype of Alternaria alternata, which produces a host-selective toxin. Peroxisomes were observed in fungal cells by expressing a mCherry fluorescent protein tagging with conserved tripeptides serine-lysing-leucine and transmission electron microscopy. The results indicated that Pex6 plays no roles in peroxisomal biogenesis but impacts protein import into peroxisomes. The number of peroxisomes was affected by nutritional conditions and H2O2, and their degradation was mediated by an autophagy-related machinery termed pexophagy. Pex6 was shown to be required for the formation of Woronin bodies, the biosynthesis of biotin, siderophores, and toxin, the uptake and accumulation of H2O2, growth, and virulence, as well as the Slt2 MAP kinase-mediated maintenance of cell wall integrity. Adding biotin, oleate, and iron in combination fully restored the growth of the pex6-deficient mutant (Δpex6), but failed to restore Δpex6 virulence to citrus. Adding purified toxin could only partially restore Δpex6 virulence even in the presence of biotin, oleate, and iron. Sensitivity assays revealed that Pex6 plays no roles in resistance to H2O2 and superoxide, but plays a negative role in resistance to 2-chloro-5-hydroxypyridine (a hydroxyl radical-generating compound), eosin Y and rose Bengal (singlet oxygen-generating compounds), and 2,3,5-triiodobenzoic acid (an auxin transport inhibitor). The diverse functions of Pex6 underscore the importance of peroxisomes in physiology, pathogenesis, and development in A. alternata.

Genome-Wide Identification and Functional Characterization of GATA Transcription Factor Gene Family in Alternaria alternata.

In the present study, we identified six GATA transcription factors (AaAreA, AaAreB, AaLreA, AaLreB, AaNsdD, and AaSreA) and characterized their functions in response to environmental stress and virulence in the tangerine pathotype of Alternaria alternata. The targeted gene knockout of each of the GATA-coding genes decreased the growth to varying degrees. The mutation of AaAreA, AaAreB, AaLreB, or AaNsdD decreased the conidiation. All the GATA transcription factors were found to be required for tolerance to cumyl hydroperoxide and tert-butyl-hydroperoxide (oxidants) and Congo red (a cell-wall-destructing agent). Pathogenicity assays assessed on detached citrus leaves revealed that mutations of AaAreA, AaLreA, AaLreB, or AaNsdD significantly decreased the fungal virulence. A comparative transcriptome analysis between the ∆AreA mutant and the wild-type strain revealed that the inactivation of AaAreA led to alterations in the expression of genes involved in a number of biological processes, including oxidoreductase activity, amino acid metabolism, and secondary metabolite biogenesis. Taken together, our findings revealed that GATA-coding genes play diverse roles in response to environmental stress and are important regulators involved in fungal development, conidiation, ROS detoxification, as well as pathogenesis. This study, for the first time, systemically underlines the critical role of GATA transcription factors in response to environmental stress and virulence in A. alternata.