Molecular Characterization of a Polygalacturonase Inhibitor of Pear Fruit

Abstract

<jats:p>Higher plant inhibitors of fungal polygalacturonases are potential contributors to plant defense. To test this hypothesis we have raised antibodies against the `Bartlett' pear fruit polygalacturonase inhibitor (PGIP) and cloned a pear fruit PGIP cDNA. The pear PGIP cDNA was isolated by polymerase chain reactions based on our amino acid and nucleotide sequence information. Sequence analysis predicts a gene product of 34.5 kD with an isoelectric point of 6.02 in agreement with our biochemical data. Seven potential glycosylation sites are consistent with the glycoprotein character of these PGIPs. Southern blot analysis suggests the presence of 1 or 2 genes in the pear genome. Northern blot analysis indicates the presence of a transcript of 1.5 kb. Western blot analysis shows cross-reactivity of the anti-pear PGIP antibody to various dicot species as well as corn.</jats:p>

Structure and expression of an inhibitor of fungal polygalacturonases from tomato

“…The concentrated extract was applied to a concanavalin A (Con A)-Sepharose 4B column and separated as described previously [35]. Fractions which contained PGIP activity eluted both in the flow-through and with 0.25 M e-methyl mannoside.…”

“…These two protein preparations were purified further by cation-exchange fast-performance liquid chromatography (FPLC; Pharmacia, Uppsala, Sweden) using a Mono S HR 5/5 column as described [35]. Fractions containing PGIP activity were pooled and the buffer of the active material changed to 20 mM Tris, pH 8.0 (buffer A) using a Centricon-10 microconcentrator.…”

“…After 17 d the culture medium was collected by filtration through cheesecloth and Whatman 1 MM paper and concentrated by ultrafiltration using a PM-10 membrane. Inhibition of PG activity was determined by assaying the PG-catalyzed release of reducing sugars from sodium polypectate (Sigma, St. Louis, MO) [13] in the presence or absence of PGIP, as described previously [35]. PG and PGIP were mixed and incubated at room temperature for 5 min prior to the addition of substrate.…”

“…A genomic clone encoding bean PGIP [40] and a cDNA clone encoding pear PGIP [35] have deduced amino acid sequences which are only 50~o identical but contain conserved cysteine residues and three regions of higher homology. In pear PGIP mRNA levels were higher in fruits than in flowers and undetectable in leaves [35].…”

“…In pear PGIP mRNA levels were higher in fruits than in flowers and undetectable in leaves [35]. In addition, pear cell suspension cultures have PGIP activity and protein although mRNA levels have not been determined (unpublished).…”