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 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 are continuing this study by proteolytically cleaving the protein backbone and analyzing the resulting peptide/glycopeptide mixtures by MALDI-TOF analysis. 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.
Showing the most recent 10 out of 245 publications
