In vitro and in silico Characterisation of Mdpgip 1 and MdPGIP2 Isolated from Malus domestica cv Granny Smith

Malus domestica polygalacturonase inhibiting protein 1 (Mdpgip1) and Mdpgip2 genes were isolated, inserted for expression in a tobacco genome. Crude MdPGIP extracts prepared from the Mdpgip1 and Mdpgip2 transgenics were used to determine the MdPGIPs’ inhibition spectra against polygalacturonases (PGs) prepared from Botryosphaeria obtusa (BoPGs), Botrytis cinerea (BcPGs), Diaporthe ambigua (DaPGs), Fusarium verticillioides (FvPGs) and Verticillium dahliae (VdPGs). At maximum concentrations of 200ng of protein, both MdPGIPs inhibited BoPGs and VdPGs by 100%, and DaPGs and BcPGs by 83% and 67%, respectively. Furthermore, 29% and 14% inhibition was observed against FvPGs by MdPGIP2 and MdPGIP1, respectively. Analysis of the Mdpgip1 and Mdpgip2 genes revealed that both genes comprise single open reading frames (ORFs) of 993 nucleotides that encode 330 amino acid polypeptides. In silico characterisation of the polypeptides revealed structures typical of PGIP proteins which include a 24 amino acid putative signal peptide, a potential cleavage site (Ala-Leu-Ser) for the signal peptide, potential glycosylation sites (Asn-X-Ser/Thr), leucine-rich repeat (LRR) domain, as well as N- and C-terminal domains. The secondary and tertiary structures of the MdPGIPs were modelled and revealed that a curved and elongated molecule that contains sheet B1, sheet B2 and helices on its LRR domain. The hydropathic evaluation indicated a repetitive hydrophobic motif in the LRR domain and a hydrophilic surface area consistent with a globular protein. The overall properties of the MdPGIPs are similar to those of the prototypical bean PGIP2, but detected differences in inhibition of PGs from various fungal species support its use in biotechnological applications as an inhibitor of targeted fungal PGs using a transgenic approach.