Purification and molecular characterization of polygalacturonase from Colletotrichum acutatum

Colletotrichum acutatum, bitter rot of apple, is responsible for severe losses in the apple crop in the southeastern US. In spite of increasing losses in apple orchards, fundamental studies to develop control methods have not been extensively conducted. To understand the pathology and physiology of C. acutatum, polygalacturonase (PG), thought to be one of the major virulence factors involved in fruit decay, was studied using molecular and biochemical methods. A total of 6 isozymes were found by chromatofocusing, among which the two acidic isozymes were further purified by preparative isoelectricfocusing and anion exchange chromatography. The molecular weights of the two acidic isozymes were 35 kDa and the optimal pH for enzyme activity was 4.5. Both PGs showed exo- and endo-PG activity.; Two PG genes and their promoters were cloned by degenerate PCR and genome walking. These two PG genes were designated as capg1 and capg2. The deduced protein of capg1 was composed of 358 amino acids and the calculated pI was 8.78. The predicted protein of capg2 consisted of 363 amino acids and the pI was 9.11. The two cloned capgs showed high nucleotide and amino acid identities (83--89%) with three other PG genes of Colletotrichum. Reverse transcription-PCR showed that the expression of the two capgs was influenced by the carbon source and pH.; Apple fruit were treated with calcium (Ca) by pressure infiltration to determine if calcium, which confers decay resistance in apple, also affects the activity and expression of PGs produced by C. acutatum. The decayed area was reduced as the calcium concentration of the solution with which the fruit were treated increased. PG activity was lower in calcium treated apples than in the non-treated fruit. Apple cell walls were extracted and used as the sole carbon source in minimal medium in which C. acutatum was grown. PG activity was lower in the medium amended with calcium treated apple cell walls than in the medium amended with cell walls from non-treated fruit. The expression of capg1 was also reduced in the calcium treated cell wall medium.