Isolation and sequencing of an endopolygalacturonase gene in Diaporthe helianthi

Diaporthe helianthi, causal agent of stem canker, is an important pathogen of sunflower in Europe. Its pathogenetic mechanisms are still poorly understood, although the production of phytotoxic compounds has been demonstrated. Degradation of cell walls by fungal specific enzymes, primarily pectinases, has been shown to be involved in plant pathogenesis. By now many fungal endopolygalacturonase (endoPG) sequences have been isolated from phytopathogenic microorganisms. In D. helianthi there is no information about endoPG enzymes produced or about their coding regions. In order to analyse the endoPG genomic region in a French D. helianthi isolate (8/96), heterologous primers, designed on fungal conserved endoPG genes, have been exploited. The endoPG genic region has been isolated by screening four differently digested D. helianthi genomic libraries, constructed according to the Genome Walker system (Clontech). Several amplicons obtained by walking both in 5’ and in 3’ direction have been cloned and sequenced: the partial sequences obtained have been overlapped and specific external primers have been designed on the D. helianthi region. By amplification and cloning of the complete genomic region, the full gene sequence has been determined. Sequence analysis by means of BLAST programs has confirmed its endoPG nature. Further studies about the relatedness of D. helianthi sequence to the known fungal endoPG genes and about functional and structural domains in its deduced protein are in progress.