RESUMEN
Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5' noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.
Asunto(s)
Diterpenos de Tipo Kaurano/biosíntesis , Fusarium/enzimología , Fusarium/genética , Prueba de Complementación Genética , Giberelinas/biosíntesis , Fosfatos de Poliisoprenilo/biosíntesis , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Secuencia de Aminoácidos , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Diterpenos , Farnesiltransferasa/química , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análisis de Secuencia de ADNRESUMEN
Gibberella fujikuroi is a species complex with at least nine different biological species, termed mating populations (MPs) A to I (MP-A to MP-I), known to produce many different secondary metabolites. So far, gibberellin (GA) production is restricted to Fusarium fujikuroi (G. fujikuroi MP-C), although at least five other MPs contain all biosynthetic genes. Here, we analyze the GA gene cluster and GA pathway in the closest related species, Fusarium proliferatum (MP-D), and demonstrate that the GA genes share a high degree of sequence homology with the corresponding genes of MP-C. The GA production capacity was restored after integration of the entire GA gene cluster from MP-C, indicating the existence of an active regulation system in F. proliferatum. The results further indicate that one reason for the loss of GA production is the accumulation of several mutations in the coding and 5' noncoding regions of the ent-kaurene oxidase gene, P450-4.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Fusarium/enzimología , Fusarium/genética , Genes Fúngicos , Gibberella/enzimología , Gibberella/genética , Familia de Multigenes , Oxigenasas/genética , Secuencia de Bases , Sistema Enzimático del Citocromo P-450/metabolismo , ADN de Hongos/genética , Genes Reguladores , Genes Reporteros , Giberelinas/biosíntesis , Datos de Secuencia Molecular , Oxigenasas/metabolismo , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Conidia of the phytopathogenic fungus Calonectria morganii were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. Agrobacterium tumefaciens-mediated transformation yielded stable hygromycin B-resistant clones (average number (106 per 10(7)) [corrected] conidia). Putative transformants appeared to be mitotically and meiotically stable. The presence of the hph gene was checked by PCR. In four randomly chosen transformants, single-copy integrations of the marker gene at different chromosomal sites were proven by Southern analysis.