A Corn Trypsin Inhibitor with Antifungal Activity Inhibits Aspergillus flavus α-Amylase

Chen, Z.-Y., Brown, R. L., Russin, J. S., Lax, A. R., and Cleveland, T. E. 1999. A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus α-amylase. Phytopathology 89:902-907. In this study, we found that the inhibition of fungal growth in potato dextrose broth (PDB) medium by the 14-kDa corn trypsin inhibitor (TI) protein, previously found to be associated with host resistance to aflatoxin production and active against various fungi, was relieved when exogenous α-amylase was added along with TI. No inhibitory effect of TI on fungal growth was observed when Aspergillus flavus was grown on a medium containing either 5% glucose or 1% gelatin as a carbon source. Further investigation found that TI not only inhibited fungal production of extracellular α-amylase when A. flavus was grown in PDB medium containing TI at 100 μg ml–1 but also reduced the enzymatic activity of A. flavus αamylase by 27%. At a higher concentration, however, TI stimulated the production of α-amylase. The effect of TI on the production of amyloglucosidase, another enzyme involved in starch metabolism by the fungus, was quite different. It stimulated the production of this enzyme during the first 10 h at all concentrations studied. These studies suggest that the resistance of certain corn genotypes to A. flavus infection may be partially due to the ability of TI to reduce the production of extracellular fungal α-amylase and its activity, thereby limiting the availability of simple sugars for fungal growth. However, further investigation of the relationship between TI levels and fungal α-amylase expression in vivo is needed. Additional keywords: antifungal protein, maize. Plants do not contain an immune system and must rely on other mechanisms to protect themselves from fungal infection. These mechanisms include synthesis of inhibitory compounds such as phenols, melanins, tannins, or phytoalexins as well as accumulation of proteins that can directly inhibit fungal growth (20). A number of such antifungal proteins have been isolated from a variety of plant tissues (11,22,24,25,30–32). These include protease inhibitors, lectins, ribosomal inactivating proteins, zeamatin, chitinases, β-1,3-glucanases, and thionins. The most extensively studied protease inhibitors are the trypsin inhibitors (TI), proteins of different size and amino acid sequence that can inhibit the activity of trypsin. They have been isolated from various grains including corn kernels (29,35). Some TI have been reported to be inhibitors of both trypsin and α-amylase (9,28,29,33). Gatehouse et al. (16) indicated that elevated levels of TI observed in cowpea (Vigna unguiculata) cv. Tvu2027 played a role in resistance to damage caused by the bruchid beetle Callosobruchus maculatus. Feeding studies using artificial diets found that ingestion of TI caused a significant elevation of the levels of trypsin in insect guts. Therefore, it was proposed that the true mode of action of the inhibitor was to cause pernicious hyperproduction of trypsin, which in turn leads to depletion of the sulfur-containing amino acids, this being detrimental to the insects (5). Direct evidence of involvement of TI in plant defense was demonstrated by expression of the cowpea TI gene in tobacco, which increased its resistance against herbivorous insects (18). Peng and Black (26) showed that TI and chymotrypsin inhibitor levels in tomato plants increased following infection by the pathogenic fungus Phytophthora infestans, but this host response only occurred in those varieties of tomato that were resistant to the fungus and not in susceptible varieties. The possible involvement of TI in plant defense against fungal pathogens has also been implicated by their antifungal activities (20,36). However, the mode of action of many of these proteins against fungi has not been clearly demonstrated. A 14-kDa protein from corn kernels shown to be a specific inhibitor of bovine trypsin (35) also inhibited activated Hageman factor (factor XIIa) of the intrinsic blood-clotting process (19) as well as α-amylase from the red flour beetle (Tribolium castaneum) and the yellow mealworm (Tenebrio molitor) (9). However, this protein did not inhibit α-amylases from the fungi Aspergillus niger, A. fumigatus, or A. oryzae, the bacterium Bacillus subtilis, or human saliva (2). We recently demonstrated that this 14-kDa TI was associated with resistance to A. flavus infection and aflatoxin production in 11 corn genotypes (11). The purified TI inhibited both in vitro conidia germination and hyphal growth of A. flavus, the major aflatoxin-producing fungus in crops such as corn, cotton, peanut, and tree nuts. The reduced fungal growth of A. flavus has been shown to correlate with reduced aflatoxin production in laboratory studies (8,17). The ability of TI to inhibit the growth of A. flavus and some other fungal pathogens (10) suggests its potential importance in the control of aflatoxin contamination of corn and other crops as well as of some other fungal diseases. Characterization of the mode of action of the 14-kDa TI could constitute a first step leading to the development of selectable marker traits for corn breeders as well as isolation of genes for use in developing resistant transgenic plants. Since certain TI have been shown to inhibit α-amylase activity, the current study sought to determine if the 14-kDa TI from corn resistant to A. flavus infection and aflatoxin production inhibited A. flavus α-amylase, thereby inhibiting fungal growth. Preliminary reports have been published (7,12). Corresponding author: R. L. Brown; E-mail address: [email protected] Publication no. P-1999-0819-01R This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological