Identification of a novel pelD gene expressed uniquely in planta by Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of its protein product as an endo-pectate lyase

Abstract

Antibodies prepared against a pectin-inducible pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) were previously found to protect the host from infection. The gene (pelA) and two of its homologs were cloned and sequenced. Here we report the isolation of a new pectate lyase gene, pelD, from a genomic library of F. solani pisi. A 1.5-kb DNA fragment containing pelD and its flanking regions was sequenced. The nucleotide sequence of pelD would encode a protein of 24.5 kDa which shares 49, 44, and 65% amino acid sequence identity with PLA, PLB, and PLC, respectively, from the same fungus. Because the first 19 amino acid residues appeared to be a signal peptide, the mature enzyme could be a 22.7-kDa protein. pelD transcripts and PLD protein could not be detected in fungus cultured in glucose, pectin, pea epicotyl extract, or a pea cell wall preparation. However, pelD transcripts were readily found by RT-PCR with RNA isolated from infected pea tissues. The cDNA of pelD, thus obtained, was expressed in Pichia pastoris with the putative pelD signal sequence. The secreted PLD was purified and characterized to be an endopectate lyase, and its lyase activity could be inhibited by anti-PLA IgG. Thus, protection of the host observed with the anti-PLA antibodies could reflect inhibition of immunologically related pectate lyases including PLD which is expressed uniquely in planta.