Molecular and genetic determination of the role of elsinochrome toxins produced by Elsinoe fawcettii causing citrus scab.

摘要

Citrus scab disease, caused by the fungus Elsinoe fawcettii (Bitancourt & Jenkins), affects all varieties of citrus, resulting in serious fruit blemishes and economic losses in Florida. My study focused on genetic determination of the role of elsinochrome phytotoxins produced by E. fawcettii.;Results indicated that elsinochromes function as photosensitizing compounds that exert toxicity to plant cells due to production of reactive oxygen species such as singlet oxygen and superoxides. Upon irradiation to light, elsinochromes alone rapidly killed suspension-cultured citrus and tobacco cells, induced necrotic lesions on rough lemon leaves, and provoked a steady increase of electrolyte leakage. The toxicity was drastically decreased or alleviated by the singlet oxygen quenchers, such as bixin, DABCO, or reduced glutathione. Accumulation of singlet oxygen and superoxides induced by elsinochromes after irradiation was also detected.;An EfPKS1 gene encoding a fungal polyketide synthase (PKS) was cloned and characterized from E. fawcettii to confirm the roles of elsinochromes in fungal pathogenesis and lesion formation by genetic disruption and complementation strategies. In addition, accumulation of the EfPKS1 transcript and elsinochromes by the wild-type strain were coordinately regulated by light, carbon, nitrogen and pH. The results clearly indicate that elsinochromes play a consequential role in fungal pathogenesis.;The genes involved in the biosynthesis of fungal secondary metabolites are often clustered. I sequenced a 30-kb region beyond EfPKS1 and identified nine putative genes; some of them encoding polypeptides are likely required for elsinochrome biosynthesis and regulation. In addition to EfPKS1, five genes, RDT1 encoding a reductase, OXR1 encoding an oxidoreductase, TSF1encoding a transcriptional factor, ECT1encoding a membrane transporter, and PRF1 encoding a prefoldin protein subunit were identified. Other four genes (designated as EfHP1-4) encode hypothetical proteins that are likely not associated with biosynthetic functions. The involvement of these genes in elsinochrome production was evident by analyzing the TSF1 gene encoding a putative pathway-specific regulator. Targeted disruption specifically occurred at the TSF1 gene created fungal mutants that are defective in elsinochrome production. Expression of the adjacent genes in the TSF1-disrupted mutants was markedly down-regulated.;In addition, elsinochromes were extracted, for the first time, from affected lesions. A survey of 52 field-collected E. fawcettii isolates in Florida revealed that most of them were able to produce elsinochromes in cultures and/or in planta. A single isolate (Ef41) with distinct genetic traits from all others failed to infect rough lemon, grapefruit, and sour orange and produced no elsinochromes. Overall, my studies employing biochemical, molecular, and pathological approaches clearly demonstrated that elsinochromes play critical roles for fungal pathogenesis and lesion development.