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) are considered to be among the first CWDEs released by the attacking fungi. EPGs 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 have been shown to 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 have been shown to exist in a variety of non-glycosylated and/or glycosylated states, depending upon the origin of these glycoproteins. It is generally believed that 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. Unfortunately, the availability of only limited quantities of these glycoproteins has prevented the use of classic carbohydrate analytical techniques. To overcome this problem, we have developed and utilized a strategy based upon matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to 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.
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