Isolation and characterization of a possible polygalacturonase: inhibiting protein from wheat

[摘要] English: The presence and possible role of polygalacturonase-inhibiting protein (PGIP) inwheat (Triticum aestivum) as part of the plant's defense reaction following leaf rust(Puccinia triticina) infection were investigated.Through its ability to inhibit fungal endopolygalacturonase (EPG) that breaks downthe plant cell wall during colonization, this protein is known to play an important rolein the natural defense arsenal of dicotyledonous plants. The presence of PGIP inmonocotyledonous cereals has never before been conclusively proved.A preliminary investigation using a polyclonal antibody raised against a purified beanPGIP (PGIP-I) revealed the induction of a possible PGIP of ±37.0 kDa followingfungal infection, while an inhibition assay of EPG from AsperglÏ/us niger showed adecrease in PGIP activity. Through ion-exchange and size exclusion chromatographythe presence of wheat PGIP was subsequently confirmed by the purification of a±36.0 kDa inhibitor, which proved specific for the EPG of Coch/iobo/us sativus andnot A. niger. Using a more specific anti-PGIP antibody (PGIP-II) the presence of thisprotein in wheat was also confirmed through immunoblotting. The expression of PGIP in wheat following salicylic acid (SA) treatment and fungalinfection in terms of C sativus EPG inhibition was recorded. While SA treatmentshowed an induction of PGIP at protein and activity levels, fungal infection repeatedthe reduction in PGIP activity as previously observed. Using PGIP-II in immunogoldlocalization the expression of PGIP in wheat leaves was confined to the plant cell walland the periphery of the haustorium in the cytosol.Attempts to clone the wheat pgip gene through the polymerase chain reaction (peR)using degenerate primers were inconclusive, as fragments amplified did not exhibitsignificant similarity to PGIP from dicotyledonous plants.These results therefore indicate that wheat expresses a ±36.0 kDa PGIP in reactionto fungal and SA treatment, but fungus-related factors originating from either theplant or the fungus apparently induce the EPG activity to higher levels, or suppressthe PGIP activity to lower levels, both recordable as a decrease in PGIP activity andhaving the potential to enhance plant disease.

[发布机构] University of the Free State