The soybean PGIP family contains members with different inhibiting properties and regulation following Sclerotinia sclerotiorum infection

Polygalacturonase-inhibiting protein (PGIP) is a plant cell wall protein that regulates the action of fungal endopolygalacturonases (PG) favouring the formation of oligogalacturonides active as elicitors of plant defense responses. In order to characterize the genomic organization of the soybean pgip family, we have screened a BAC library prepared from genomic DNA of the cv. Williams82. A total of 7 BAC clones were isolated and characterized further to identify novel pgip genes. These analysis allowed the identification of two genes, Gmpgip3 and Gmpgip4, sharing a sequence similarity higher than 60% with the previously characterized Gmpgip1 and Gmpgip2. These four genes span a region larger than 50 kbp and sequence comparison showed that they form two cluster, one containing Gmpgip1 and Gmpgip2, and the other Gmpgip3 and Gmpgip4. When compared with other legume pgips, Gmpgip3 group with the bean Pvpgip1 and Pvpgip2, suggesting that these genes are closer to the ancestral gene. To study the biochemical properties of the proteins encoded by Gmpgip1, Gmpgip2, Gmpgip3 and Gmpgip4 we have expressed them in Nicotiana bentamiana by using the Potato Virus X (PVX) as vector. All the PVX-Gmpgip constructs were able to produce in good quantity the heterologous protein, as verified by western blot and/or SDS-PAGE analyses. Only GmPGIP4 did not cross-hybridize against the bean PGIP antibody. The protein extracts from N. benthamiana containing the specific GmPGIP were used in agarose diffusion assays to test their recognition specificities against a number of PGs including Botrytis cinerea, Aspergillus niger, Fusarium moniliforme, Colletotricum acutatum and Stenocarpella maydis. GmPGIP1, GmPGIP2 and GmPGIP4 showed a very weak inhibition activity against those PGs. On the contrary, GmPGIP3 showed a strong inhibiting activity towards all PGs tested. Its inhibitory capability is similar to that of PvPGIP2 from bean, the most strong inhibitor so far characterized. Finally, in order to study whether pgip genes are differentially regulated during pathogen infection and wounding we have performed RT-PCR analyses with primers specific for each Gmpgip gene. These analyses were carried out on RNA extracted from soybean hypocotyls at different times after wounding or infection with Sclerotinia sclerotiorum. Gmpgip1, Gmpgip3 and Gmpgip4 are up regulated following wounding and infection with S. sclerotiorum, whereas Gmpgip2 is not induced by wounding and is up regulated late (at 48 h) after infection with S. sclerotiorum.