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 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.

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.

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.

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.

Biotin biosynthesis affected by the NADPH oxidase and lipid metabolism is required for growth, sporulation and infectivity in the citrus fungal pathogen Alternaria alternata.

The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and cell-wall-degrading enzymes and to mitigate toxic reactive oxygen species is crucial for A. alternata pathogenesis to citrus. Little is known about nutrient availability within citrus tissues to the fungal pathogen. In the present study, we assess the infectivity of a biotin deficiency mutant (ΔbioB) and a complementation strain (CP36) on citrus leaves to determine how biotin impacts A. alternata pathogenesis. Growth and sporulation of ΔbioB are highly dependent on biotin. ΔbioB retains its ability to acquire and transport biotin from the surrounding environment. Growth deficiency of ΔbioB can also be partially restored by the presence of oleic acid or Tween 20, suggesting the requirement of biotin in lipid metabolism. Experimental evidence indicates that de novo biotin biosynthesis is regulated by the NADPH oxidase, implicating in the production of H2O2, and is affected by the function of peroxisomes. Three genes involved in the biosynthesis of biotin are clustered and co-regulated by biotin indicating a transcriptional feedback loop activation. Infectivity assays using fungal mycelium reveal that ΔbioB cultured on medium without biotin fails to infect citrus leaves; co-inoculation with biotin fully restores infectivity. The CP36 strain re-expressing a functional copy of bioB displays wild-type growth, sporulation and virulence. Taken together, we conclude that the attainability or accessibility of biotin is extremely restricted in citrus cells. A. alternata must be able to synthesize biotin in order to utilize nutrients for growth, colonization and development within the host.

Proper Functions of Peroxisomes Are Vital for Pathogenesis of Citrus Brown Spot Disease Caused by Alternaria alternata.

In addition to the production of a host-selective toxin, the tangerine pathotype of Alternaria alternata must conquer toxic reactive oxygen species (ROS) in order to colonize host plants. The roles of a peroxin 6-coding gene (pex6) implicated in protein import into peroxisomes was functionally characterized to gain a better understanding of molecular mechanisms in ROS resistance and fungal pathogenicity. The peroxisome is a vital organelle involved in metabolisms of fatty acids and hydrogen peroxide in eukaryotes. Targeted deletion of pex6 had no impacts on the biogenesis of peroxisomes and cellular resistance to ROS. The pex6 deficient mutant (Δpex6) reduced toxin production by 40% compared to wild type and barely induce necrotic lesions on citrus leaves. Co-inoculation of purified toxin with Δpex6 conidia on citrus leaves, however, failed to fully restore lesion formation, indicating that toxin only partially contributed to the loss of Δpex6 pathogenicity. Δpex6 conidia germinated poorly and formed fewer appressorium-like structures (nonmelanized enlargement of hyphal tips) than wild type. Δpex6 hyphae grew slowly and failed to penetrate beyond the epidermal layers. Moreover, Δpex6 had thinner cell walls and lower viability. All of these defects resulting from deletion of pex6 could also account for the loss of Δpex6 pathogenicity. Overall, our results have demonstrated that proper peroxisome functions are of vital importance to pathogenesis of the tangerine pathotype of A. alternata.

The basal transcription factor II H subunit Tfb5 is required for stress response and pathogenicity in the tangerine pathotype of Alternaria alternata.

The basal transcription factor II H (TFIIH) is a multicomponent complex. In the present study, we characterized a TFIIH subunit Tfb5 by analysing loss- and gain-of-function mutants to gain a better understanding of the molecular mechanisms underlying stress resistance and pathogenicity in the citrus fungal pathogen Alternaria alternata. Tfb5 deficiency mutants (ΔAatfb5) decreased sporulation and pigmentation, and were impaired in the maintenance of colony surface hydrophobicity and cell wall integrity. ΔAatfb5 increased sensitivity to ultraviolet light, DNA-damaging agents, and oxidants. The expression of Aatfb5 was up-regulated in the wild type upon infection in citrus leaves, implicating the requirement of Aatfb5 in fungal pathogenesis. Biochemical and virulence assays revealed that ΔAatfb5 was defective in toxin production and cellwall-degrading enzymes, and failed to induce necrotic lesions on detached citrus leaves. Aatfb5 fused with green fluorescent protein (GFP) was localized in the cytoplasm and nucleus and physically interacted with another subunit, Tfb2, based on yeast two-hybrid and co-immunoprecipitation analyses. Transcriptome and Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) analyses revealed the positive and negative roles of Aatfb5 in the production of various secondary metabolites and in the regulation of many metabolic and biosynthetic processes in A. alternata. Aatfb5 may play a negative role in oxidative phosphorylation and a positive role in peroxisome biosynthesis. Two cutinase-coding genes (AaCut2 and AaCut15) required for full virulence were down-regulated in ΔAatfb5. Overall, this study expands our understanding of how A. alternata uses the basal transcription factor to deal with stress and achieve successful infection in the plant host.

Aaprb1, a subtilsin-like protease, required for autophagy and virulence of the tangerine pathotype of Alternaria alternata.

Subtilisin-like serine protease secreted by pathogenic fungi can facilitate the infection and acquisition of nutrients. Functions of subtilisin-like serine proteases in the phytopathogenic fungus Alternaria alternata remains unknown. In the current study, 15 subtilisin-like serine proteases were individually deleted in the citrus fungal pathogen A. alternata. Only one, designated AaPrb1, was found to be required for A. alternata pathogenesis. The AaPrb1 deficiency strain (ΔAaprb1) reduced growth, conidiation, the formation of aerial hyphae, protease production, and virulence on citrus leaves. However, biochemical analyses and bioassays revealed that ΔAaprb1 plays no role in the production of ACT toxin. Through Y2H assays, Aaprb1 was found to interact with Aapep4, a vacuole-localized proteinase A in A. alternata. Furthermore, silencing AaPep4 in A. alternata resulted in phenotypes similar with those of ΔAaprb1. Expression of AaPrb1 was found to be regulated by AaPep4. TEM showed that AaPrb1and AaPep4 were involved in the suppression of the degradation of autophagosomes. Deletion of the autophagy gene AaAtg8 in A. alternata decreased conidiation, the formation of aerial hyphae and pathogenicity similar to ΔAaprb1, implying that some phenotypes of ΔAaprb1 were due to the impairment of autophagy. Overall, this study expands our understanding of how A. alternata utilizes the subtilisin-like serine protease to achieve successful infection in the plant host.

A Synergistic Effect of Chitosan and Lactic Acid Bacteria on the Control of Cruciferous Vegetable Diseases.

Two lactic acid bacteria (LAB) designated J02 and J13 were recovered from fermented vegetables based on their ability to suppress soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) on radish. J02 and J13 were identified as Lactobacillus pentosus and Leuconostoc fallax, respectively. The ability of J02 and J13 to suppress plant diseases is highly dependent on chitosan. LAB alone has no effect and chitosan alone has only a moderate effect on disease reduction. However, J02 or J13 broth cultures plus chitosan display a strong inhibitory effect against plant pathogens and significantly reduces disease severity. LAB strains after being cultured in fish surimi (agricultural waste) and glycerol or sucrose-containing medium and mixed with chitosan, reduce three cruciferous vegetable diseases, including cabbage black spot caused by Alternaria brassicicola, black rot caused by Xanthomonas campestris pv. campestris, and soft rot caused by Pcc. Experimental trials reveal that multiple applications are more effective than a single application. In-vitro assays also reveal the J02/chitosan mixture is antagonistic against Colletotrichum higginsianum, Sclerotium rolfsii, and Fusarium oxysporum f. sp. rapae, indicating a broad-spectrum activity of LAB/chitosan. Overall, our results indicate that a synergistic combination of LAB and chitosan offers a promising approach to biocontrol.

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.

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.

Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ΔSte12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ΔSte12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.

Fungichromin Production by Streptomyces padanus PMS-702 for Controlling Cucumber Downy Mildew.

Streptomyces padanus PMS-702 strain produces a polyene macrolide antibiotic fungichromin and displays antagonistic activities against many phytopathogenic fungi. In the present study, experimental formulations were assessed to improve the production of fungichromin, the efficacy of PMS-702 on the suppression of sporangial germination, and the reduction of cucumber downy mildew caused by Pseudoperonospora cubensis. PMS-702 strain cultured in a soybean meal-glucose (SMG) medium led to low levels of fungichromin accumulation and sporangial germination suppression. Increasing medium compositions and adding plant oils (noticeably coconut oil) in SMG significantly increased fungichromin production from 68 to 1,999.6 µg/ml. Microscopic examination reveals that the resultant suspensions significantly reduced sporangial germination and caused cytoplasmic aggregation. Greenhouse trials reveal that the application of PMS-702 cultural suspensions reduced downy mildew severity considerably. The addition of Tween 80 into the synthetic medium while culturing PMS-702 further increased the suppressive efficacy of downy mildew severity, particularly when applied at 24 h before inoculation or co-applied with inoculum. Fungichromin at 50 µg/ml induced phytotoxicity showing minor necrosis surrounded with light yellowish halos on cucumber leaves. The concentration that leads to 90% inhibition (IC90) of sporangial germination was estimated to be around 10 µg/ml. The results provide a strong possibility of using the S. padanus PMS-702 strain as a biocontrol agent to control other plant pathogens.

A Major Facilitator Superfamily Transporter Regulated by the Stress-Responsive Transcription Factor Yap1 Is Required for Resistance to Fungicides, Xenobiotics, and Oxidants and Full Virulence in Alternaria alternata.

Alternaria alternata relies on the ability to produce a host-selective toxin and to detoxify reactive oxygen species to successfully colonize the host. An A. alternata major facilitator superfamily transporter designated AaMFS54 was functionally characterized by analysis of loss- and gain-of-function mutations to better understand the factors required for fungal pathogenesis. AaMFS54 was originally identified from a wild-type expression library after being subtracted with that of a mutant impaired for the oxidative stress-responsive transcription regulator Yap1. AaMFS54 contains 14 transmembrane helixes. Fungal mutant lacking AaMFS54 produced fewer conidia and increased sensitivity to many potent oxidants (potassium superoxide and singlet-oxygen generating compounds), xenobiotics (2,3,5-triiodobenzoic acid and 2-chloro-5-hydroxypyridine), and fungicides (clotrimazole, fludioxonil, vinclozolin, and iprodione). AaMFS54 mutant induced necrotic lesion sizes similar to those induced by wild-type on leaves of susceptible citrus cultivars after point inoculation with spore suspensions. However, the mutant produced smaller colonies and less fluffy hyphae on the affected leaves. Virulence assays on citrus leaves inoculated by spraying with spores revealed that AaMFS54 mutant induced less severe lesions than wild-type, indicating the requirement of AaMFS54 in pathogenesis. All defective phenotypes were restored in a strain re-acquiring a functional copy of AaMFS54. Northern blotting analysis revealed that the expression of AaMFS54 was suppressed by xenobiotics. The current studies indicate that the Yap1-mediated transporter plays a role in resistance to toxic oxidants and fungicides in A. alternata. The relationships of MFS transporters with other regulatory components conferring stress resistance and A. alternata pathogenesis are also discussed.

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.

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.

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.