Characterization and expression of an antifungal zeamatin-like protein (Zlp) gene from Zea mays.
Abstract
A cDNA clone encoding a basic thaumatin-like protein of Zea mays was recovered from a mid-development seed cDNA library. The gene, Zlp, encoded a protein that was nearly identical with maize zeamatin and alpha-amylase/trypsin inhibitor. Expression of Zlp mRNA was highest in the endosperm tissue of seed 4 weeks after pollination. Expression of zeamatin-like (ZLP) protein correlated with mRNA; also, a low basal level of ZLP expression in leaf was not appreciably induced by abiotic stresses. ZLP was expressed with its own signal peptide in insect cells and in transgenic Arabidopsis and tomato plants. ZLP was secreted in all three systems, with correct processing of the signal peptide. ZLP expressed in transgenic tomato was found to be partially subjected to a proteolytic cleavage after residue 180, by an unknown mechanism, to give a "nicked" isoform of ZLP. Purified ZLP from all three sources, as well as purified "nicked" ZLP from tomato, demonstrated fungal inhibition against Candida albicans and Trichoderma reesei, with marginal inhibition observed against Alternaria solani and Neurospora crassa.
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Abstract
A cDNA clone encoding a basic thaumatin-like protein of Zea mays was recovered from a mid-development seed cDNA library. The gene, Zlp, encoded a protein that was nearly identical with maize zeamatin and alpha-amylase/trypsin inhibitor. Expression of Zlp mRNA was highest in the endosperm tissue of seed 4 weeks after pollination. Expression of zeamatin-like (ZLP) protein correlated with mRNA; also, a low basal level of ZLP expression in leaf was not appreciably induced by abiotic stresses. ZLP was expressed with its own signal peptide in insect cells and in transgenic Arabidopsis and tomato plants. ZLP was secreted in all three systems, with correct processing of the signal peptide. ZLP expressed in transgenic tomato was found to be partially subjected to a proteolytic cleavage after residue 180, by an unknown mechanism, to give a "nicked" isoform of ZLP. Purified ZLP from all three sources, as well as purified "nicked" ZLP from tomato, demonstrated fungal inhibition against Candida albicans and Trichoderma reesei, with marginal inhibition observed against Alternaria solani and Neurospora crassa.