The role of proline in osmoregulation in Phytophthora nicotianae

A cDNA encoding Δ¹-pyrroline-5-carboxylate reductase (P5CR), the enzyme that catalyzes the final step in proline biosynthesis in plants and bacteria, has been cloned from the oomycete plant pathogen Phytophthora nicotianae. Genomic DNA blots indicated that P. nicotianae and P. cinnamomi each contain a single P5CR gene, whereas P. infestans contains one or two genes. Complementation of a strain of Escherichia coli defective in the P5CR protein by the P. nicotianae P5CR cDNA confirmed that the gene encoded a functional P5CR. RNA blots revealed that P5CR was expressed at a much higher level in P. nicotianae zoospores than in vegetative hyphae, sporulating hyphae, or germlings. Furthermore, P5CR mRNA levels increased with time in zoospores, demonstrating that transcription occurs in zoospores. mRNA encoding histidine and tryptophan biosynthetic enzymes was not highly and specifically expressed in zoospores, indicating that the developmental pattern of P5CR expression was not simply a reflection of overall amino acid biosynthesis as might be required for protein synthesis. Measurement of free proline in P. nicotianae at different stages of the life cycle revealed that proline concentration was highest in sporulating hyphae. Vegetative hyphae and germ-lings contained about 50% of this concentration of proline, and zoospores contained only about 1% of this level. Substantial amounts of proline were measured in the medium into which the zoospore had been released. Hypoosmotic shock of P. nicotianae hyphae led to an approximately 50% decrease in free proline concentration within 30 min of transfer to low-osmolarity medium and was accompanied by an increase in the level of P5CR mRNA. These data are discussed in terms of the possible role of proline in osmoregulation in Phytophthora.