Discovery and characterization of a class of fungal endoglucanase inhibitor proteins from higher plants

Abstract

Microbial pathogens secrete an array of enzymes that cleave polysaccharides in the cell walls of their plant hosts. These enzymes include endoglucanases, polygalacturonase, pectin lyases, xylanases, and various glycosidases. Many of these cellwall degrading enzymes are inhibited by previously identified and characterized plant proteins, such as polygalacturonase inhibiting protein (PGIP), xylanase inhibiting protein (XIP), and pectin lyase inhibiting protein (PNLIP). This dissertation describes the identification and characterization of a plantderived xyloglucan-specific endoglucanase inhibitor protein (XEGIP) from suspensioncultured tomato (Lycopersicon esculentum) cells. Previously, no endoglucanase inhibiting proteins have been identified from any plant source, although endoglucanase substrates (cellulose and hemicelluloses) are major components of the plant cell wall. XEGIP inhibits a xyloglucan-specific endoglucanase (XEG) from Aspergillus aculeatus by forming a 1:1 protein:protein complex with a Ki of approximately 0.5 nM. XEGIP also inhibits a previously unknown xyloglucan-specific endoglucanase (CfXEG) secreted by a fungal pathogen of tomato (Cladosporium fulvum). CfXEG was purified from the culture medium of C. fulvum grown on xyloglucan-rich tamarind seed powder as the sole carbon source. The cDNA encoding XEGIP was cloned and sequenced. These results were used to perform a database analysis, which revealed that XEGIP-like proteins are widely distributed in the plant kingdom. XEGIP homologs include EDGP, a carrot protein that had previously been implicated in disease resistance, although its precise function was unknown. Based on its homology to XEGIP, EDGP was isolated and found to inhibit XEG. Sequence alignment of XEGIP with TAXI (Triticum aestivum xylanase inhibitor), a recently characterized wheat protein, suggests that both proteins belong to a protein super family, hemicellulase inhibitor proteins (HIPs). Interestingly, Family 12 endoglucanases and Family 11 xylanases, which are the known HIP ligands, have very similar 3-D structures even though their sequence similarities are low. Based on their in vitro activity, in vivo localization, and enhanced expression in response to wounding and microbial infection, HIPs are proposed to function as disease resistance factors. In order to facilitate the evaluation of this hypothesis, an immunoaffinity chromatography based method was developed to screen for the binding of HIPs to their ligands. Further examination of the roles of HIPs in vivo may shed light on the mechanisms by which plants limit colonization by pathogenic microbes.