Cloning and characterization of the gene encoding the endo polygalacturonase‐inhibiting protein (PGIP) of Phaseolus vulgaris L.

Abstract

Summary: Polygalacturonase‐inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N‐terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758‐bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb Sa/I–Sa/I DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)⁺ RNAs, isolated from various tissues of bean seedlings or from suspension‐cultured bean cells, were also performed using the cloned PCR‐generated DNA as a probe. A 1.2 kb transcript was detected in suspension‐cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers. A cDNA clone containing part of the coding region for the PGIP (from nt 345 to the poly(A) tail) was isolated from a λgt11 cDNA library prepared from poly(A)⁺ RNA isolated from suspension‐cultured cells of P. vulgaris cv. Pinto. The nucleotide and deduced amino acid sequences of the PGIP genes from the two different cultivars shared 96.4% and 97.4% identity, respectively.