A cutinase-encoding gene from Phytophthora capsici isolated by differential-display RT-PCR

To detect and ultimately isolate genes of Phytophthora capsici the expression of which is induced during its interaction with pepper, a comparative analysis of gene expression in the wild-type pathogenic fungus with expression in a non-pathogenic (Nop) mutant reduced in cutinase and esterase activities was performed by the differential display of mRNAs. Discrimination of fungal genes induced in planta, from plant genes induced in response to the pathogen, was accomplished by exposure of the mycelium to bare-rooted seedlings of pepper (Capsicum annuum) in sterile water, to allow the initiation of infection, and then physical removal of the induced mycelium. With six sets of primer combinations, eight cDNA fragments (representing fungal genes) were present in planta only for the pathogenic strain. RNA-blot analysis showed that the transcripts detected accumulated to detectable levels only at early stages of the interaction. Sequence analysis and database searches revealed homology of one of the cDNA clones to fungal cutinases. The 218 amino-acid sequence predicted from sequencing a genomic clone of P. capsici suggested a protein of molecular weight of 23 980 Da with similarity to fungal cutinases previously characterized. These results indicated that differential-display analysis is sufficiently sensitive to be applied for the detection and isolation of fungal genes induced during a plant-pathogen interaction.