Cloning and analysis of pppg1, an inducible endopolygalacturonase gene from the oomycete plant pathogen Phytophthora parasitica

Abstract

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of crops. Here, we report the isolation of a gene, named pppg1, which encodes an extracellular endopolygalacturonase in P. parasitica. Both cDNA and a genomic clone were isolated and sequenced. The pppg1 gene showed standard characteristics with respect to core promoter and intron sequences of Phytophthora. The predicted protein of pppg1 has a calculated molecular mass of 39.7 kDa and a p I value of 5.2, and contains a putative signal peptide of 20 amino acid residues on the N-terminus. The deduced amino acid sequence is highly conserved with those of other Phytophthora and fungal endopolygalacturonases. Analysis by reverse transcription followed by real-time quantitative polymerase chain reaction showed that transcription of pppg1 was repressed by glucose, but induced by pectin in the culture. Moreover, pppg1 is highly expressed during interaction of P. parasitica with the host plant, suggesting its involvement in the process of host infection. Heterologous expression of pppg1 in Pichia pastoris produced proteins with molecular mass ranging from 75 to 200 kDa, very likely due to differential glycosylation by the yeast. Deglycosylation of the recombinant protein resulted in a complete loss of the endopolygalacturonase activity.