Discovery, purification and characterization of antifungal proteins and other defense-related compounds from tropical plants.

Abstract

To date, we have screened 164 extracts from over 100 different plant species for antifungal activity against Fusarium chlamydosporum, a saprophytic/pathogenic fungus. Out of these 164 crude extracts, 111 had antifungal activity. Thirty-five aqueous crude extracts exhibited very strong to complete inhibition of growth of F. chlamydosporum. Forty of the 164 extracts retained activity after dialysis (3,500 molecular weight cut off) suggesting that proteins may be responsible for the activity. Five of the dialyzed extracts showed strong to complete inhibition of fungal growth. Antifungal activities present in several of these plant extracts were purified further.

Novel antifungal activities were discovered in extracts from seeds of three different legumes, Swartzia simplex, S. cubensis and Pentaclethra macroloba. These activities included chitinases and other defense-related proteins and peptides. Chitinases were purified from all three sources using a combination of chitin-affinity chromatography followed by anion-exchange chromatography. Alternatively, chitinases were purified by a combination of affinity-chromatography followed by size-exclusion chromatography. Acidic chitinases isolated from all three species inhibited growth of A. flavus in a liquid bioassay.

The physical and biochemical properties of selected chitinases were determined. Purified chitinases were stable in 0.1 M HCl, 0.1 M acetic acid and 0.1 M NaOH. The molecular weight, pI, glycosylation, amino acid composition and partial amino acid sequence of several forms of the purified chitinases were determined. The partial amino acid sequence revealed a high degree (over 60%) of sequence homology to class three lysozyme/chitinase and endochitinase precursor from several plant species.

In addition to chitinases, other antifungal proteins/peptides were detected in all three species mentioned above. Agglutination and beta-1,3-glucanase activities present in these extracts were associated with several fractions that inhibited the growth of A. flavus. Other novel activities were detected and purified. These activities await further characterization.

A protein-containing fraction purified from Coccoloba sp. exhibited a broad range of antifungal activity. This material strongly inhibited conidial germination and hyphal growth of A. flavus, Fusarium chlamydosporum, F. moniliforme, Sclerotinia minor and Sclerotium rolfsii. The antifungal activity was stable in 0.1 M NaOH, 0.1 M HCl, 0.1 M acetic acid, survived boiling for 5 min and was retained after dialysis using 3,500 to 14,000 molecular weight cut off dialysis membranes. Purified materials caused changes in fungal morphology including hyperbranching and extremely reduced hyphal elongation. These observations are consistent with the effects of many morphogenic plant defensins suggesting that the active component may be a defensin-like protein.

Every year we loose billions of dollars in agriculture because of fungal pathogens. Chemical controls of these pathogens provide some protection but the continued use of chemical pesticides is under attack because of economic, environmental and health issues. The focus of our research is to find alternative ways to protect crop plants from fungal pathogens and pests. Our specific goal is to discover, purify and characterize antifungal proteins and to isolate genes encoding these proteins. Once isolated these genes can be engineered into crop plants to increase their resistance to fungal diseases. Our research has focused on plant species from the tropical rainforest because of the diversity of species present in these areas and because of the virtually untapped wealth of novel defenses used by these plants.