In silico analysis of the polygalacturonase inhibiting protein 1 from apple, Malus domestica.

BACKGROUND: The Malus domestica polygalacturonase inhibiting protein 1 (MdPGIP1) gene, encoding the M. domestica polygalacturonase inhibiting protein 1 (MdPGIP1), was isolated from the Granny Smith apple cultivar (GenBank accession no. DQ185063). The gene was used to transform tobacco and potato for enhanced resistance against fungal diseases. FINDINGS: Analysis of the MdPGIP1 nucleotide sequence revealed that the gene comprises 993 nucleotides that encode a 330 amino acid polypeptide. In silico characterization of the MdPGIP1 polypeptide revealed domains typical of PGIP proteins, which include a 24 amino acid putative signal peptide, a potential cleavage site [Alanine-Leucine-Serine (ALS)] for the signal peptide, a 238 amino acid leucine-rich repeat (LRR) domain, a 46 amino acid N-terminal domain and a 22 amino acid C-terminal domain. The hydropathic evaluation of MdPGIP1 indicated a repetitive hydrophobic motif in the LRR domain and a hydrophilic surface area consistent with a globular protein. The typical consensus glycosylation sequence of Asn-X-Ser/Thr was identified in MdPGIP1, indicating potential N-linked glycosylation of MdPGIP1. The molecular mass of non-glycosylated MdPGIP1 was calculated as 36.615 kDa and the theoretical isoelectric point as 6.98. Furthermore, the secondary and tertiary structure of MdPGIP1 was modelled, and revealed that MdPGIP1 is a curved and elongated molecule that contains sheet B1, sheet B2 and 310-helices on its LRR domain. CONCLUSION: The overall properties of the MdPGIP1 protein is similar to that of the prototypical Phaseolus vulgaris PGIP 2 (PvPGIP2), and the detected differences supported its use in biotechnological applications as an inhibitor of targeted fungal polygalacturonases (PGs).

Apple polygalacturonase inhibiting protein1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins.

Extracts from apple fruit (cultivar "Granny Smith") inhibited the cell-wall degrading polygalacturonase (PG) activity of Colletotrichum lupini, the causal agent of anthracnose on lupins, as well as Aspergillus niger PG. Southern blot analysis indicated that this cultivar of apple has a small gene family of polygalacturonase inhibiting proteins (pgips), and therefore heterologous expression in transgenic tobacco was used to identify the specific gene product responsible for the inhibitory activity. A previously isolated pgip gene, termed Mdpgip1, was introduced into tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. The mature MdPGIP1 protein was purified to apparent homogeneity from tobacco leaves by high salt extraction, clarification by DEAE-Sepharose and cation exchange HPLC. Purified MdPGIP1 inhibited PGs from C. lupini and PGs from two economically important pathogens of apple trees, Botryosphaeria obtusa and Diaporthe ambigua. It did not inhibit the A. niger PG, which was in contrast to the apple fruit extract used in this study. We conclude that there are at least two active PGIPs expressed in apple, which differ in their charge properties and ability to inhibit A. niger PG.