Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.10

In vitro anti-fungal activity of the wheat puroindoline proteins

C. Balconi*^,**, N. Berardo*, J.E. Sherwood**, M.Motto*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo

**) Dept. Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA

puroindolines, anti-fungal activity, in vitro, wheat

The puroindolines (PIN) are wheat endosperm proteins believed to be involved in wheat grain texture, determining grain softness. These proteins have also been shown to have in vitro anti-microbial properties and in vivo activity in transgenic rice plants that constitutively express the puroindoline genes. Because the PIN proteins normally are in wheat endosperm and thus are routinely ingested by humans and animals alike, their use in transgenic crops may be less objectionable than gene products normally not present in plants or in the edible portion of plants.

The goal of our research has been to determine mode and range of action for the anti-fungal activity of the puroindolines. The first step was to set up bioassays to test the activity of seed purified PIN proteins against different fungi. Bioassays were developed comparing the anti-fungal activities from a non-PIN-containing control (Langdon Durum wheat, -PIN) and a PIN-containing variety (Langdon 5D(5B), created by substituting chromosome 5B of Langdon with chromosome 5D from Chinese Spring, which contains pin genes, +PIN). We used different approaches to test the sensitivity of different fungi to PIN proteins. The bioassay for yeast-like fungi like Ustilago hordei, Saccharomyces cerevisiae,Candida albicans and Crytococcus neoformans was based on growth in broth culture and quantified by microscopic cell count. All fungi tested were sensitive to PIN, although U. hordei was the most sensitive. High concentrations of PIN extract were fungicidal against U. hordei and fungistatic against the other fungi. Cell morphology was distorted, indicating the PIN was affecting cell membranes or walls. The effect of PIN extract on filamentous fungi was determined using Magnaporthe grisea and Fusarium culmorum. Known amounts of the +PIN and –PIN extracts were spread on agar medium and the plates were inoculated with a small plug of the test organism. Radial growth was measured over time. Growth of both fungi was inhibited by the +PIN extract. Research is in progress to amplify the knowledge about the range of fungi sensitive to puroindolines and to determine the mechanism for anti-fungal activity of these proteins.