An Extracellular Protein from Phytophthora parasitica var nicotianae Is Associated with Stress Metabolite Accumulation in Tobacco Callus

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Abstract
The most abundant extracellular protein produced by Phytophthora parasitica var nicotianae at early stages of rapid growth in culture has a molecular weight of 46 kilodaltons and has been designated Ppn 46e. Culture conditions for the production of this protein have been optimized and the protein has been purified by gel filtration and ion-exchange chromatography. Ppn 46e is a soluble, acidic protein (pI 4.67). The amino acids Asx (aspartic acid or asparagine), alanine, glycine, Glx (glutamic acid or glutamine), and serine are the most abundant at 13.4%, 12.3%, 12.1%, 9.3%, and 9.3% of the residues, respectively. The purified protein is, by weight, 1.8% glucose, 1.6% mannose, and 0.5% galactose. A bioassay for Ppn 46e based on tobacco callus has been developed. In this assay as little as 20 nanograms (4.3 × 10−13 mole) Ppn 46e causes the accumulation of the sesquiterpenoid phytoalexin, capsidiol, as estimated by gas chromatography. Levels of capsidiol of 25 micrograms per gram fresh weight were elicited by 80 nanograms Ppn 46e per callus piece. Pretreatment of the protein with either pronase or by boiling resulted in a loss of elicitor activity. Periodate treatment, which inactivates glucan elicitors, did not affect the ability of Ppn 46e to cause capsidiol accumulation. Monospecific antibodies to Ppn 46e were raised in mice. Western blotting experiments employing these antibodies showed that Ppn 46e was present in infected tobacco plants. Dot blotting experiments revealed the presence of the Ppn 46e epitope(s) in Phytophthora megasperma, P. cactorum, P. cinnamomi, and P. infestans but not in Fusarium.