Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma

Abstract

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.Trichothecenes are a group of over 200 sesquiterpenoidderived secondary metabolites that vary by the pattern of oxygenations and esterifications of a core tricyclic structure with an epoxide function. These metabolites are produced by species in at least six genera of the fungal order Hypocreales (class Sordariomycetes): Fusarium, Myrothecium, Spicellum, Stachybotrys, Trichoderma, and Trichothecium. Trichothecenes are considered mycotoxins because they are often found as contaminants in food and animal feed and they can induce vomiting, alimentary hemorrhaging, and dermatitis in humans and livestock. These symptoms most likely result from the ability of trichothecenes to inhibit protein synthesis (40) and/or to induce apoptosis in eukaryotic cells (45). Trichothecenes also can act as immunosuppressors (51) and neurotoxins (36). Trichothecenes are phytotoxic (17). Previously (56), we observed that overproduction of trichodermin in Trichoderma brevicompactum reduced tomato seed germination and plant growth while increasing the size of the lesions produced when the pathogen was artificially inoculated on tomato plants previously colonized by Trichoderma spp.

Involvement of Trichoderma Trichothecenes in the Biocontrol Activity and Induction of Plant Defense-Related Genes

“…The explanation for this gene location still remains unclear; however, a plausible explanation would be that the ancestral tri cluster included this gene and that during evolution, tri5 was located outside the cluster in the Trichoderma trichothecene producers. Once trichodiene is produced in Trichoderma, it is then oxygenated by the tri4-encoded cytochrome P450 monooxygenase, utilizing three consecutive oxygenation steps at the C-2, C-12, and C-11 positions to produce isotrichodiol (11). The 3-step oxygenation by Tri4 is also found for Myrothecium (37).…”

“…In Fusarium, the known genes for trichothecene biosynthesis are located in three different loci: one locus composed of a main cluster containing 12 genes, including Tri5, and the other two loci composed of 1 or 2 genes (43). In Trichoderma, only the tri5 gene is located outside the main cluster (11). In both genera, Tri5 encodes trichodiene synthase, a terpene cyclase that is the first committed enzyme of the trichothecene biosynthetic pathway, which converts farnesyl pyrophosphate (FPP) to trichodiene (24,25).…”

“…Trichothecenes are produced by the genera Fusarium, Stachybotris, Myrothecium, and Trichoderma (14) as well as others (12). The trichothecene biosynthetic pathways in Trichothecium (33), Fusarium (5,8,23,26,29,59), Myrothecium (53), and, recently, Trichoderma (11) have been studied. In Fusarium and Trichoderma, most of the genes involved in trichothecene biosynthesis are clustered, but the two genera display important genetic organizational differences.…”

Trichoderma Research in the Genome Era

“…Trichoderma spp. also produce trichothecenes, such as trichodermin and harzianum A, via a TRI cluster that is significantly different from that of Fusarium TRI clusters (12,79). Deletion of Tri4, a Cyt P450 monooxygenase resulted in reduced antifungal activity of T. arundinaceum against B. cinerea and R. solani and also reduced induced resistance in tomato (79).…”

“…The transcription factor Thctf1 regulates the biosynthesis of the volatile antifungal compound 6-pentyl-2H-pyran-2-one (6-PP) (106). A trichothecene-like (TRI) cluster has been identified in Trichoderma brevicompactum, producing the phytotoxin trichodermin (12). Trichoderma arundinaceum, in contrast, is beneficial to plants and produces harzianum A, a growth-promoting trichothecene (79).…”