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Biochemistry and Cell Biology

Endopolygalacturonase from Fusarium oxysporum f. sp. lycopersici. Purification, Characterization, and Production During Infection of Tomato Plants. Antonio Di Pietro , Departamento de Genetica, Facultad de Ciencias, Universidad de C6rdoba, 14071 C6rdoba, Spain. M. Isabel G. Roncero. Departamento de Genetica, Facultad de Ciencias, Universidad de C6rdoba, 14071 C6rdoba, Spain. Phytopathology 86:1324-1330. Accepted for publication 3 September 1996. Copyright 1996 The American Phytopathological Society. DOI: 10.1094/Phyto-86-1324.

Production of extracellular pectinolytic enzymes by Fusarium oxysporum f. sp. lycopersici on different carbon sources was analyzed by zymograms of isoelectric focusing (IEF) gels. Pectinolytic isoforms with pIs ranging from 4.0 to 9.0 were detected. A polygalacturonase (PG; EC 3.2.1.15) was purified by preparative IEF and denominated PG1. The enzyme consisted of one major unglycosylated isoform with pI 7.0 and a molecular weight (MW) of 35,000 and three major N-glycosylated isoforms with pIs 6.0, 6.5, and 6.75 and a MW of 37,500. Absence of PG1 from the filtrate after tunicamycin treatment indicated that N-glycosylation was essential for its secretion. PG1 was active as a monomer and apparently hydrolyzed polygalacturonic acid simultaneously in an endo-and exomanner; hydrolysis of substrate bonds was 3% at 50% viscosity reduction indicating random cleavage, whereas analysis of reaction products showed early appearance of galacturonic acid. pH and temperature optima were 4.0 and 37?C, respectively. Sequencing of 11 N-terminal amino acids showed 91% identity to a Fusarium moniliforme endoPG. PG1 was approximately twice as resistant to inhibition by purified tomato PG-inhibitor protein as endoPGs from F. moniliforme and Aspergillus niger. Zymograms revealed the presence of the main PG1 isoform (pI 7.0) in stems of tomato plants infected with F. oxysporum f. sp. lycopersici, while a pectate lyase (pI 8.7) was detected in roots during early stages of infection.