RESUMO
Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.
Assuntos
Ascomicetos/enzimologia , Ascomicetos/patogenicidade , Micotoxinas/biossíntese , Perileno/análogos & derivados , Perileno/metabolismo , Policetídeo Sintases/metabolismo , Sequência de Aminoácidos , Ascomicetos/genética , Expressão Gênica , Dados de Sequência Molecular , Folhas de Planta/microbiologia , Policetídeo Sintases/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nicotiana/microbiologia , Virulência/genéticaRESUMO
Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Deltactb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Deltactb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Deltactb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Deltactb3 disruptants drastically reduced lesion formation.
Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Metiltransferases/genética , Oxigenases de Função Mista/genética , Perileno/análogos & derivados , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Solanum lycopersicum/microbiologia , Metiltransferases/metabolismo , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Perileno/química , Perileno/metabolismo , Análise de Sequência de DNA , Nicotiana/microbiologia , Virulência/genéticaRESUMO
SUMMARY Colletotrichum acutatum causes Key lime anthracnose (KLA) and postbloom fruit drop (PFD) of citrus. We utilized restriction enzyme-mediated integration (REMI) mutagenesis to produce six non-pathogenic mutants from a KLA isolate after screening 1064 transformants on detached Key lime leaves. Subsequently, a gene designated KLAP1 (Key Lime Anthracnose Pathogenicity) was identified from one of the mutants and was demonstrated genetically to be required for pathogenicity to Key lime leaves. The predicted polypeptide encoded by KLAP1 contains a cAMP and cGMP-dependent protein kinase phosphorylation site, and two RGD (Arg-Gly-Asp) cell attachment sequences, a bipartite nuclear targeting sequence, a fungal G-protein alpha subunit signature, a putative metal-binding zinc finger (Cys(2)His(2)) and a putative HMG-I/Y ('high mobility group' non-histone chromatin protein encoding genes) DNA-binding domain (A+T hook), suggesting that KLAP1 may function as a transcription activator in C. acutatum. Sequences homologous to KLAP1 were detected in most C. acutatum isolates examined, and similarity was found in several classes of fungi, animals, plants and bacteria, indicating that KLAP1 is a putative, uncharacterized, conserved transcription activator in fungi. Targeted gene disruption of KLAP1 yielded mutants that were blocked in the penetration stage and were completely defective in pathogenicity on Key lime leaves, but remained pathogenic to flower petals. Complementation of a klap1-null mutant with a full-length KLAP1 gene clone restored complete ability to incite lesions on Key lime. The results indicate that KLAP1 is an important pathogenicity factor in C. acutatum.