Citrus brown spot disease is caused by the necrotrophic fungus Alternaria alternata. Its pathogenic capability has been thought to depend exclusively on the production of host-selective ACT toxin. However, circumvention of plant defenses is also likely to be important for the disease process. To investigate the fungal response to host-generated reactive oxygen species (ROS), we cloned and characterized the AaAP1 gene of A. alternata, which encodes a polypeptide resembling yeast YAP1-like transcriptional activators implicated in cellular responses to stress. Expression of the AaAP1 gene in a wild-type strain was primarily induced by H(2)O(2) or ROS-generating oxidants. Using a loss-of-function mutation in the AaAP1 gene, we demonstrated an essential requirement for oxidative tolerance during the host invasion step. Mutants lacking AaAP1 showed increased sensitivity to H(2)O(2) and loss of fungal pathogenicity. The DeltaAaAP1 null mutant did not cause any visible necrotic lesions on wounded or unwounded leaves of citrus cv. Minneola. Compared with the wild type, the null mutant displayed lower catalase, peroxidase, and superoxide dismutase activities. All mutant phenotypes were restored to the wild type in fungal strains expressing a functional copy of AaAP1. Upon exposure to H(2)O(2), the AaAP1::sGFP (synthetic green fluorescent protein) fusion protein became localized in the nucleus. Inoculation of the mutant with NADPH oxidase inhibitors partially restored fungal pathogenicity. Our results highlight the global regulatory role of a YAP1 homolog in response to oxidative stress in A. alternata and provide insights into the critical role of ROS detoxification in the pathogenicity of A. alternata.

No abstract

Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.

Alternaria species produce and excrete dimethyl coprogen siderophores to acquire iron. The Alternaria alternata gene AaNPS6, encoding a polypeptide analogous to fungal nonribosomal peptide synthetases, was found to be required for the production of siderophores and virulence on citrus. Siderophores purified from culture filtrates of the wild-type strain did not induce any phytotoxicity on the leaves of citrus. Fungal strains lacking AaNPS6 produced little or no detectable extracellular siderophores and displayed an increased sensitivity to H₂O₂, superoxide-generating compounds (KO₂ and menadione) and iron depletion. Δnps6 mutants were also defective for the production of melanin and conidia. The introduction of a wild-type AaNPS6 under the control of its endogenous promoter to a Δnps6 null mutant at least partially restored siderophore production and virulence to citrus, demonstrating a functional link between iron uptake and fungal pathogenesis. Elevated sensitivity to H₂O₂, seen for the Δnps6 null strain could be relieved by exogenous application of ferric iron. The expression of the AaNPS6 gene was highly up-regulated under low-iron conditions and apparently controlled by the redox-responsive yeast transcriptional regulator YAP1. Hence, the maintenance of iron homeostasis via siderophore-mediated iron uptake also plays an important role in resistance to toxic reactive oxygen species (ROS). Our results demonstrate further the critical role of ROS detoxification for the pathogenicity of A. alternata in citrus.

Elsinochrome pigments produced by many phytopathogenic Elsinoë spp. are nonhost-selective toxins which react with oxygen molecules after light activation to produce highly toxic reactive oxygen species. The structures and chemical properties of four derivatives are well known. However, the biological roles of elsinochromes in fungal pathogenesis are poorly understood. Many isolates of Elsinoë fawcettii causing citrus scab are able to produce elsinochromes under axenic conditions. In this article, we report the cloning, expression, and functional characterization of the polyketide synthase-encoding gene, EfPKS1, which we show is required for the production of elsinochromes and fungal pathogenesis. Targeted disruption of EfPKS1 in E.fawcettii completely abrogated elsinochrome production, drastically reduced conidiation, and significantly decreased lesion formation on rough lemon leaves. All mutant phenotypes were restored to the wild type in fungal strains expressing a functional copy of EfPKS1. Accumulation of the EfPKS1 transcript and elsinochromes by a wild-type strain appears to be coordinately regulated by light, nutrients, and pH. The results indicate that the product of EfPKS1 is involved in the biosynthesis of elsinochromes via a fungal polyketide pathway, and that elsinochromes play an important role in fungal pathogenesis.

No abstract

No abstract

Fungi respond and adapt to different environmental stimuli via signal transduction systems. We determined the function of a yeast SLT2 mitogen-activated protein (MAP) kinase homologue (AaSLT2) in Alternaria alternata, the fungal pathogen of citrus. Analysis of the loss-of-function mutant indicated that AaSLT2 is required for the production of a host-selective toxin, and is crucial for fungal pathogenicity. Moreover, the A. alternata slt2 mutants displayed hypersensitivity to cell wall-degrading enzymes and chemicals such as Calcofluor white and Congo red. This implicates an important role of AaSLT2 in the maintenance of cell wall integrity in A. alternata. The A. alternata slt2 mutants were also hypersensitive to a heteroaromatic compound, 2-chloro-5-hydroxypyridine, and a plant growth regulator, 2,3,5-triiodobenzoic acid. Developmentally, the AaSLT2 gene product was shown to be critical for conidial formation and hyphal elongation. Compared with the wild-type, the mutants produced fewer but slightly larger conidia with less transverse septae. The mutants also accumulated lower levels of melanin and chitin. Unlike the wild-type progenitor, the A. alternata slt2 mutants produced globose, swollen hyphae that did not elongate in a straight radial direction. All defective phenotypes in the mutant were restored by transformation and expression of a wild-type copy of AaSLT2 under the control of its endogenous promoter. This study highlights an important role of the AaSLT2 MAP kinase-mediated signalling pathway, regulating diverse physiological, developmental and pathological functions, in the tangerine pathotype of A. alternata.