Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of <10 g/ml. The fungitoxic effect declined in the order zearalenone > ␣-zearalenol > -zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyil)-resorcylic acid lactone] is a mycotoxin produced by several species of Fusarium, most notably Fusarium graminearum and Fusarium culmorum. Zearalenone and its derivatives exert estrogenic and anabolic effects on mammals. Carryover of zearalenone from infected grain to feedstuff causes reproductive problems in pigs, sheep, and other farm animals, including precocious sexual development, vulva enlargement, pseudopregnancy, loss of embryos, and reduced litter size (10,14,24). When digested with grain-based food, zearalenone may cause hyperestrogenism in children (33). Apart from its estrogenicity, zearalenone is genotoxic in mice and is a suspected carcinogen (5, 15, 30). The intake of zearalenone by some human populations is close to the estimated tolerable limits (9), and the amount of zearalenone allowed in grain, feedstuff, and food is regulated in several countries (1). Current ecological and economic constraints on grain production encourage minimum tillage practices and reduced fungicide application, so infection of cereal plants with Fusarium spp. is increasing and contamination with mycotoxins is becoming a more serious threat to grain production.One promising strategy for reducing zearalenone contamination is enzymatic degradation. The only commercial product on the market claimed to enzymatically detoxify zearalenone is the feed additive Mycofix Plus (Biomin GmbH, Herzogenburg, Austria). There are no published laboratory data demonstrating the hydrolysis of zearalenone by Mycofix Plus or by its active component, and such activity was not detected by us (21). The recently discovered yeast species Trichosporon mycotoxinivorans (27) degrades zearalenone and is expected to provide fut...
Agrobacterium tumefaciens-mediated transformation of filamentous fungi consists of (i) induction of A. tumefaciens culture harbouring a binary vector, (ii) co-incubation of bacteria with fungal spores on a solid support, and (iii) selection of transformants. During the induction, vir genes on the helper component of the binary vector are activated, conditioning A. tumefaciens for transformation. During co-cultivation, T-DNA part of the binary vector system is transferred into fungal nucleus and inserted into the genome. Transformants are selected on a medium with appropriate antibiotic. In order to maximize the number of transformants, the ratio of A. tumefaciens cells to fungal spores and the duration of the co-cultivation need be optimized. The procedure takes two to three weeks for fastgrowing fungi.
The smut fungus Sporisorium reilianum occurs in two varieties (S. reilianum f. sp. reilianum and S. reilianum f. sp. zeae) that cause head smut disease on sorghum and maize, respectively. Prior to plant infection, compatible haploid sporidia of S. reilianum fuse to form infectious dikaryotic hyphae that penetrate the leaf surface, spread throughout the plant, and reach the inflorescences, in which spore formation occurs. To elucidate the basis of host specificity of the two S. reilianum varieties, we compared disease etiology of S. reilianum f. sp. reilianum and S. reilianum f. sp. zeae on sorghum and maize. Both varieties could penetrate and multiply in both hosts. However, red spots appeared on inoculated leaves after sorghum infection with S. reilianum f. sp. zeae. Using matrix-assisted laser desorption-ionization time of flight analysis of leaf extracts, we show that sorghum reacts with the production of the red and orange phytoalexins luteolinidin and apigeninidin upon colonization by S. reilianum f. sp. zeae but not by S. reilianum f. sp. reilianum. Using in vitro growth assays, we demonstrate that luteolinidin but not apigeninidin slows vegetative growth of both S. reilianum f. sp. zeae and S. reilianum f. sp. reilianum. However, the phytoalexin biosynthesis gene SbDFR3 is only induced in sorghum after infection with S. reilianum f. sp. zeae, as shown by quantitative real-time polymerase chain reaction. This suggests that regulation of luteolinidin biosynthesis determines infection success of S. reilianum on sorghum.
Root-knot nematodes are soil-borne pathogens that invade and establish feeding sites in plant roots. They have an extremely broad host range, including most vascular plants. During infection of a susceptible host, root-knot nematodes secrete molecules called effectors that help them establish an intimate interaction with the plant and, at the same time, allow them to evade or suppress plant immune responses. Despite the fact that Meloidogyne hapla is a significant pest on several food crops, no effectors have been characterized from this root-knot nematode species thus far. Using the published genome and proteome from M. hapla, we have identified and characterized two genes, MhTTL2 and Mh265. MhTTL2 encodes a predicted secreted protein containing a transthyretin-like protein domain. The expression of MhTTL2 was up-regulated during parasitic life stages of the nematode, and in situ hybridization showed that MhTTL2 was expressed in the amphids, suggesting it has a role in the nematode nervous system during parasitism. We also studied the gene Mh265. The Mh265 transcript was localized to the subventral esophageal glands. An upregulation in Mh265 expression coincided with the pre- and early-parasitic life stages of the nematode. When Mh265 was constitutively expressed in plants, it enhanced their susceptibility to nematodes. These transgenic plants were also compromised in flg22-induced callose deposition, suggesting the Mh265 is modulating plant basal immune responses.
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