This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An essential process in the attack of plants by phytopathogenic fungi is the degradation of plant cell walls by cell wall-degrading enzymes (CWDE). Endopolygalaturonases (EPGs), among the first CWDEs released by attacking fungi, break down homogalacturonan in the cell wall, a process that provides access for other CWDEs while supplying nutrients to the fungi. Polygalacturonase-inhibiting protein (PGIP) is a glycoprotein present in plant cell walls that inhibits greater than 99% of fungal EPG activity, and the PGIP-EPG interaction results in the formation of mid-sized oligomers of homogalacturonan that elicit other plant defense responses. Thus, the interactions of fungal EPGs and plant PGIPs are prime candidates for involvement in the resistance to fungal attack. Both EPGs and PGIPs exist in a variety of non-glycosylated and/or glycosylated states, depending upon their origin. Knowledge of the structures and site-specificity of the carbohydrates from both EPGs and PGIPs will lead to a better understanding of the role of these glycoproteins in the resistance of plants to fungal attack. Classical carbohydrate analytical techniques require larger amounts of sample of these glycoproteins than can be obtained. Therefore, we have developed a strategy based upon matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry that can characterize the site-specific glycoforms present in both EPG and PGIP. We have shown by this process that the four glycoforms exhibited by Fusarium moniliforme EPG and at least three of the four glycoforms of Phaseolus vulgaris PGIP differ primarily by the number of N-linked carbohydrate chains attached to these two proteins. We expect this study to provide us with information on the glycosylation site-specificity needed for the inhibition of EPG by PGIP, which ultimately is expected to lead us toward a better understanding of how plants resist fungal attacks.
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