5.4.11
APPLE POLYGALACTURONASE-INHIBITING PROTEIN cDNA CLONING AND TRANSCRIPTION

WS CONWAY1, C YAO1, GS ROSS2 and CE SAMS3

1Horticultural Crops Quality Lab, USDA -ARS, BARC-West, Beltsville, MD 20705, USA; 2The Horticulture and Food Research Institute of New Zealand Ltd., Albert Research Center, Auckland, New Zealand; 3Department of Plant and Soil Science, University of Tennessee, Knoxville, TN 37901, USA

Background and objectives
Fungal polygalacturonase (PG) has been proposed as a pathogenicity factor, particularly for soft-rotting pathogens. Polygalacturonase-inhibiting proteins (PGIPs) in the plant cell wall have been shown to effectively inhibit the activity of this enzyme. However, overexpression of pear and bean PGIPs did not enhance resistance of tomato to fungal pathogens [1]. We have purified and characterized a PGIP in mature apple fruit [2]. The objective of this study was to clone the cDNA encoding apple PGIP and determine its gene expression patterns during fruit development and when placed under different stress conditions.

Results and conclusions
A PGIP cDNA from mature apple fruit has been cloned and characterized. The open reading frame encodes a polypeptde of 330 amino acids, in which 24 amino acids at the N-terminus comprise the signal peptide. The mature polypeptide has a predicted molecular mass of 34 kDa, a calculated isoelectric point of 7.0, and seven potential N-glycosylation sites. Apple PGIP contains 10 imperfect leucine-rich repeat sequence motifs averaging 24 amino acids in length. In addition to the 1.35-kb PGIP transcript, the cloned cDNA also hybridized to RNA molecules with sizes of 3.16 and 4.96 kb. Genomic DNA analysis revealed that the apple PGIP probably belongs to a small family of genes. Amounts of PGIP gene transcripts varied in fruit collected at different maturities, suggesting that R is developmentally regulated. A very high level of PGIP gene expression in the decayed area and the adjacent area on fruit inoculated with Penicillium expansum and Botrytis cinerea was detected. However, there was no apparent increase in the amount of PGIP transcript in tissue distant from the decayed region. Wounding of fruit also induced PGIP gene expression but to a lesser extent than in the decayed area. Following storage at OC for 1 month, the amount of PGIP transcript in ripe fruit was significantly increased. The PGIP gene in both immature and ripe fruit was readily activated by fungal infections, while in stored fruit the induction was very limited and concurred with an increase of fruit susceptibility to fungal colonization of host tissue. Since PGIP gene activation is regulated by fruit development and responds to wounding, fungal infection and cold storage, these observations suggest that apple PGIP may have multiple roles during fruit development and stress response.

References
1. Desiderio A, Aracri B, Leckie F et al., 1997. Molecular Plant-Microbe Interactions 10, 852-860.
2. Yao C, Conway WS, Sams CE, 1995. Phytopathology 85, 1373-1377.