Submitted to: Journal of Toxicology Toxins Reviews
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/19/2008
Publication Date: 12/2/2009
Citation: Petthambaran, B., Hawkins, L.K., Windham, G.L., Williams, W.P., Luthe, D.S. 2009. Anti-fungal Activity of Maize Silk Proteins and Role of Chitinases in Aspergillus flavus Resistance. Journal of Toxicology Toxins Reviews. 29:27-39. Interpretive Summary: Aflatoxin contamination caused by the fungus Aspergillus flavus is a chronic problem in corn in the southeastern United States. Corn inbreds have been identified by USDA-ARS researchers that are resistant to A. flavus kernel infection and to aflatoxin contamination. Very little is known about biochemical factors that might be associated with resistance. Since corn silks are speculated to be the site of initial infection by A. flavus, we conducted lab studies to determine if anti-fungal proteins were present in silks. Resistant and susceptible corn inbreds were grown in the field and silks were collected from inside the husks. Two-dimensional gel electrophoresis was used to identify proteins that were specific to the resistant inbreds. Three chitinases, enzymes that are often associated with resistance, were found in the resistant inbreds. Lab studies were conducted to determine if the proteins found in the corn silks would have an effect on fungal growth. Assays were conducted in Petri dishes inoculated with A. flavus and containing small paper discs coated with proteins from the resistant and susceptible inbreds. Fungal growth was determined by measuring fluorescence (our test fungus glowed under ultraviolet light) and ergosterol content (a chemical found in fungi but not plants). Fungal growth was highest on paper discs containing proteins from the silks of susceptible plants. Proteins from silks of resistant plants suppressed or did not stimulate fungal growth. Our studies indicate that proteins are present in silks that have anti-fungal properties and these proteins may be associated with A. flavus/aflatoixn resistance.
Technical Abstract: Proteins were extracted from silks of two Aspergillus flavus resistant maize (Zea mays L.) inbreds, two susceptible inbreds, and one intermediately-resistant inbred grown in the field. Two-dimensional gel electrophoresis was used to identify and compare expression patterns of the proteins in the maize inbreds. Proteomic analyses indicated the presence of three chitinases (PRm chitinase, chitinase I, and chitinase A). Chitinase assays on silk protein from crude extracts showed significantly higher activity in the resistant maize inbreds compared to the susceptible inbreds (P < 0.01). Antifungal activity of the extracted silk proteins was quantified using an agar plate assay. The assay involved placing paper discs containing silk proteins from each inbred in Petri dishes containing V-8 agar inoculated with an A. flavus isolate tagged with a green fluorescent protein (GFP). Fungal growth on the paper discs was quantified by measuring GFP-fluorescence intensity and ergosterol content. Fungal growth, as indicated by fluorescence intensity levels, was significantly greater on discs treated with protein extracts from the two susceptible inbreds compared to fungal growth on untreated discs. Fluorescence intensity levels for discs treated with resistant protein extracts were significantly less compared to fluorescence intensity levels on untreated discs. Discs treated with proteins from the intermediately resistant inbred had fluorescence intensity levels comparable to the untreated discs. Ergosterol concentrations were significantly higher on discs containing proteins extracted from silks of susceptible inbreds compared to the untreated discs or discs containing proteins from silks of resistant inbreds. Fungal growth (based on ergosterol concentrations) on disc treated protein extracts from the resistant inbreds was comparable to growth on the untreated paper discs. The differential expression of antifungal proteins in maize silks, especially PR-3 proteins such as chitinases, suggest that these proteins may be contributing in the resistance to A. flavus.