VIEW ARTICLE

Biochemistry and Cell Biology

Purification and Characterization of a Polygalacturonase Produced by Penicillium expansum in Apple Fruit. Chenglin Yao, Horticultural Crops Quality Laboratory, USDA-ARS, BARC-West, Beltsville, MD 20705; William S. Conway(2), and Carl E. Sams(3). (2)Horticultural Crops Quality Laboratory, USDA-ARS, BARC-West, Beltsville, MD 20705; (3)Department of Plant and Soil Science, University of Tennessee, Knoxville 37901. Phytopathology 86:1160-1166. Accepted for publication 14 August 1996. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1996. DOI: 10.1094/Phyto-86-1160.

A polygalacturonase (PG) was purified from rotted cortical tissue of cv. Golden Delicious apple fruit inoculated with Penicillium expansum. The purified protein had a pI of 8.10 and a molecular mass of 34 kDa. The PG was heat labile and most active at pH 5.5. There were no detectable O- or N-linked glycans associated with the PG polypeptide. The purified enzyme macerated apple tissue in situ and in vitro. PG activity was not affected by purified apple polygalacturonase-inhibiting protein. Based on the amino acid sequences determined, three degenerate oligonucleotides were synthesized and used as primers to amplify the flanking regions on the fungal genome by polymerase chain reaction (PCR). Amino acid sequences predicted from the cloned PCR products matched perfectly with the determined amino acid sequences, indicating the fungal origin of the purified PG. When a cloned 212-bp PCR product was used as a probe, it hybridized with 1.5-kb RNA molecules extracted from P. expansum in rotted apple tissue. However, no hybridized signals were readily detected for RNA isolated either from nonrotted apple tissue or fungal mycelia grown in cultures with apple pectin as the sole carbon source. This demonstrated that the fungal PG was mainly, if not specifically, expressed in the invasion and colonization of fruit.