CLONING OF A NOVEL CONSTITUTIVELY EXPRESSED PECTATE LYASE GENE PELB FROM FUSARIUM-SOLANI F-SP PISI (NECTRIA-HAEMATOCOCCA, MATING-TYPE-VI) AND CHARACTERIZATION OF THE GENE-PRODUCT EXPRESSED IN PICHIA-PASTORIS

Since plant-pathogenic fungi must penetrate through pectinaceous layer s of the host cell wall, pectin-degrading enzymes are thought to be im portant for pathogenesis, Antibodies prepared against a pectin-inducib le pectate lyase (pectate lyase A [PLA]) produced by a phytopathogenic fungus, Fusarium solani f, sp, pisi (Nectria haematococca, mating typ e VI), was previously found to protect the host from infection. The ge ne (peLA) and its cDNA were cloned and sequenced, Here we report the i solation of a new pectate lyase gene, pelB, from a genomic library of F. solani f. sp. pisi with the pelA cDNA as the probe, A 2,6-kb DNA fr agment containing pelB and its flanking regions was sequenced, The cod ing region of pelB was amplified by reverse transcription-mediated PCR , using total RNA isolated from F. solani pisi culture grown in the pr esence of glucose as the sole carbon source, The predicted open readin g frame of pelB would encode a 25,6-kDa protein of 244 amino acids whi ch has 65% amino acid sequence identity with PLA from F. solani f, sp. pisi but no significant homology with other pectinolytic enzymes, The first 16 amino acid residues at the N terminus appeared to be a signa l peptide, The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa. PLB was purified to homogeneity by using a two-step procedure involvin g ammonium sulfate precipitation followed by Superdex G75 gel filtrati on chromatography. Purified PLB showed optimal lyase activity at pH 10 .0, A rapid drop in the viscosity of the substrate and Mono Q anion-ex change chromatography of the products generated by the lyase showed th at PLB cleaved polygalacturonate chains in an endo fashion, Western bl otting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other, The 5' flankin g regions of both pelA and pelB were translationally fused to the beta -glucuronidase gene and introduced into F. solani f. sp. pisi, and bet a-glucuronidase activities of the transformants were measured, Express ion of the marker gene by the transformants showed that pelA expressio n is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression, Revers e transcription-mediated PCR showed that both pelA and pelB are expres sed when F. solani f. sp. pisi infects pea epicotyl.