Isolation and analysis of a novel inducible pectate lyase gene from the phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca mating population VI).

摘要

A pectate lyase produced by Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI) was previously shown to be essential for host infection (M. S. Crawford and P. E. Kolattukudy, Arch. Biochem. Biophys. 258:196-205, 1987). Pectate lyase genes have not been cloned from any phytopathogenic fungi. A gene, designated pelA, encoding an inducible pectate lyase was isolated from F. solani f. sp. pisi. A probe was synthesized by polymerase chain reaction with oligonucleotide primers based on the known amino acid sequences of two regions of the mature protein and first-strand cDNA as template. Both cDNA and the gene were isolated and sequenced. That the cloned cDNA represents the previously purified pectate lyase is shown by the complete match of the sequences of the N-terminal 38 amino acid residues and the 20 amino acid residues of an internal peptide with the sequence deduced from the cDNA sequence. This lyase sequence shows little homology to those of other pectolytic enzymes. The pelA gene shows standard characteristics with respect to promoter, intron, and polyadenylation sequences. As determined by primer extension and nuclease S1 analysis of the origin of the transcription, there are multiple initiation sites clustered in a region of 12 nucleotides located about 55 bp upstream of the start codon. Northern (RNA) blot analysis showed a single band of mRNA at about 1 kb. The pelA gene mRNA was detected only when F. solani f. sp. pisi was grown with pectin, and there was no detectable transcript accumulation when the fungus was grown with glucose as the sole carbon source. When both carbon sources were present, the pelA gene was transcribed only after glucose was completely depleted, indicating carbon catabolite repression. Moreover, the levels of transcription decreased rapidly prior to maximal enzyme accumulation, suggesting a mechanism of self catabolite repression.