Pretorius, Z. A.Van der Westhuizen, A. J.Bergmann, C. W.Kemp, Gabré2017-10-192017-10-192001http://hdl.handle.net/11660/7309English: The presence and possible role of polygalacturonase-inhibiting protein (PGIP) in wheat (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 down the plant cell wall during colonization, this protein is known to play an important role in the natural defense arsenal of dicotyledonous plants. The presence of PGIP in monocotyledonous cereals has never before been conclusively proved. A preliminary investigation using a polyclonal antibody raised against a purified bean PGIP (PGIP-I) revealed the induction of a possible PGIP of ±37.0 kDa following fungal infection, while an inhibition assay of EPG from AsperglÏ/us niger showed a decrease in PGIP activity. Through ion-exchange and size exclusion chromatography the 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 and not A. niger. Using a more specific anti-PGIP antibody (PGIP-II) the presence of this protein in wheat was also confirmed through immunoblotting. The expression of PGIP in wheat following salicylic acid (SA) treatment and fungal infection in terms of C sativus EPG inhibition was recorded. While SA treatment showed an induction of PGIP at protein and activity levels, fungal infection repeated the reduction in PGIP activity as previously observed. Using PGIP-II in immunogold localization the expression of PGIP in wheat leaves was confined to the plant cell wall and 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 exhibit significant similarity to PGIP from dicotyledonous plants. These results therefore indicate that wheat expresses a ±36.0 kDa PGIP in reaction to fungal and SA treatment, but fungus-related factors originating from either the plant or the fungus apparently induce the EPG activity to higher levels, or suppress the PGIP activity to lower levels, both recordable as a decrease in PGIP activity and having the potential to enhance plant disease.Afrikaans: Hierdie studie het die moontlike rol van poligalakturonase-inhiberings proteïen (PGIP) teenwoordig in koring (Triticum aestivum), wat aktief optree as deel van die plant se verdedigingsreaksie tydens blaarroes (Puccinia triticina) -infeksie, ondersoek. Hierdie proteïen en sy rol in die verdedigingsarsenaal in diktotiel plante is goed bekend. PGIP beskerm die plant deur die hidrolitiese ensiem, endopoligalakturonase (EPG), wat deur die patogeen vrygestel word om die selwand te vernietig, te inhibeer. Die rol van hierdie proteïen in monokotiele en veral die graangewasse is nog nooit bestudeer nie. 'n Voorlopige immunologiese ondersoek met 'n poliklonale teenliggaampie wat opgewek is teen gesuiwerde boontjie PGIP (PGIP-I) het aangetoon dat 'n moontlike PGIP van ±37.0 kDa geïnduseer word met blaarroesinfeksie. Die gepaardgaande aktiwiteit soos gemeet aan die inhibisie van Aspergillus niger EPG het 'n afname in PGIP aangetoon. Met behulp van ioon-uitruilings en molekulêre siftingschromatografie is die teenwoordigheid van PGIP in koring finaal bevestig. 'n Proteïen van ±36.0 kDa is gesuiwer wat spesifiek is vir Cochliobolus sativus EPG, maar nie vir A. niger nie. Hierdie proteïen is ook herken met 'n meer spesifieke anti-PGIP poliklonale teenliggaampie (PGIP-II) tydens 'n immunologiese studie. Hierdie EPG is vervolgens gebruik om die uitdrukking van koring PGIP te bestudeer na onderskeidelik salisielsuurbehandeling en swaminfeksie. Salisielsuurbehandeling het 'n induksie in beide PGIP aktiwiteit en op proteïenvlak aangetoon, terwyl swaminfeksie 'n afname in PGIP aktiwiteit te weeg gebring het met 'n toename op proteïenvlak soos waargeneem met PGIP-II. Deur middel van immunogoudmerking is vasgestel dat PGIP uitdrukking in die koringselle beperk is tot die selwand en die haustorium van die patogeen in die plant sitosol. Pogings om die koring pgip geen te kloneer deur middel van die polimerase kettingreaksie en nie-spesifieke DNA inleiers was onsuksesvol. Fragmente wat sodoende geamplifiseer is het geen betekenisvolle ooreenkomste met PGIP van dikotiel plante getoon nie. Hierdie studie het aangetoon dat koring oor 'n 36.0 kDa PGIP beskik wat tydens salisielsuur en swaminfeksie geïnduseer word, maar 'n swamverwante faktor induseer EPG aktiwiteit of onderdruk PGIP aktiwiteit wat 'n afname in laasgenoemde veroorsaak. So 'n afname in PGIP aktiwiteit kan lei tot die swam se ontwikkeling en die gevolglike verlaging in plantproduktiwiteit.enWheat -- BreedingWheat -- Disease and pest resistancePhytopathogenic fungi -- Host plantsFungal diseases of plantsThesis (Ph.D. (Botany and Genetics))--University of the Free State, 2001ThesisUniversity of the Free State