PURIFICATION, STRUCTURE, AND DETECTION OF THE HELMINTHOSPORIUM CARBONUM TOXIN AND ITS INACTIVE CONVERSION PRODUCT
Abstract
The host-specific toxin (HC-toxin) produced by Helminthosporium carbonum race 1 was purified by preparative thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Purified toxin selectively inhibited root growth of susceptible maize genotypes by 50% at 180 ng/ml. Structural analyses were determined in cooperation with Dr. J. M. Daly, Dr. H. W. Knoche and Mr. M. R. Pope at the University of Nebraska. Mass spectroscopic and ('1)H and ('13)C-nmr studies established the structure as a cyclic tetrapeptide with an empirical formula of C(,21)H(,32)N(,4)O(,6), a molecular weight of 436, and a sequence of cyclo{proline-alanine-alanine-aeo}, were aeo is an unusual (alpha)-amino acid, 2-amino-9,10-epoxy-8-oxodecanoic acid. Selective biological activity of the toxin was substantially reduced when toxin preparations were incubated at low pH, exposed dry to air, or stored for long periods of time at 4(DEGREES)C. The decrease in toxicity was associated with the appearance of a second peak in HPLC. This contaminant, called conversion product, was biologically inactive at concentrations greater than 100 times the EC(,50) of purified HC-toxin and did not competitively inhibit or stimulate the activity of HC-toxin in bioassays. Large quantities of the conversion product, formed by treatment of toxin with trifluoroacetic acid, were purified for structural analyses. Mass spectra, although nearly identical to those of HC-toxin, indicated that the epoxide moiety of aeo was hydrolyzed to form a 9,10-dihydroxy derivative with molecular weight of 454 and an empirical formula of C(,21)H(,34)N(,4)O(,7). The structure of conversion product was proposed to be cyclo{proline-alanine-alanine-2-amino-9,10-dihydroxy-8-oxodecanoic acid}. Thus, the epoxide moiety is required for selective biological activity of HC-toxin. HPLC was used to detect HC-toxin in extracts of leaves inoculated with H. carbonum. HC-toxin was detected in and isolated from leaf extracts from susceptible maize 72, 96, and 120 h after inoculation. The presence of alanine and proline in HPLC eluates was confirmed by TLC of dansylated hydrolyzates.
Degree
Ph.D.
Subject Area
Plant pathology
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