Browsing by Subject "trichothecene"

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    A barley UDP-glucosyltransferase provides high levels of resistance to trichothecenes and Fusarium Head Blight in cereals
    (2017-08) Li, Xin
    Fusarium Head Blight (FHB) is a devastating disease that leads to severe economic losses to worldwide wheat (Triticum aestivum) and barley (Hordeum vulgare L.) production. During FHB disease development, the main causal fungal pathogen, Fusarium graminearum can infect a single spikelet and cause disease symptoms throughout the spike in susceptible wheat. Moreover, F. graminearum produces trichothecene mycotoxins (eg., deoxynivalenol (DON), nivalenol (NIV) and their derivatives) that facilitate FHB disease development, and toxin contamination of the grain poses a significant threat to human and animal health. Two major types of resistance have been identified and deployed in breeding programs including: type I (resistance to initial infection) and type II (resistance to spread of the symptoms). Although worldwide efforts have focused on germplasm screening and QTL mapping of FHB resistance, unfortunately, resistance to FHB is quantitatively controlled and only partial. Thus, research efforts have expanded to identifying genes and deploying them in transgenic wheat. Previous gene expression analysis in the barley cultivar ‘Morex’ led to discovery of a UDP-glucosyltransferase gene, HvUGT13248, that was shown to provide DON resistance in transgenic yeast and Arabidopsis thaliana via conjugation of DON to DON-3-O-glucoside (D3G), a much less toxic derivative. To test this promising gene in wheat, we developed transgenic wheat expressing HvUGT13248 and showed that these transgenic plants exhibited significant reduction of disease spread (type II resistance) in the spike compared with nontransformed controls. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G. Under field conditions, FHB severity was variable between different transgenic lines; however, in some years the transgenic wheat showed significantly less severe disease phenotypes compared with the nontransformed controls. Moreover, HvUGT13248 is also capable of converting NIV to the detoxified derivative, NIV-3-O-β-D-glucoside (NIV3G). An enzymatic assay using HvUGT13248 purified from Escherichia coli showed that HvUGT13248 efficiently catalyzed NIV to NIV3G. Overexpression in yeast, Arabidopsis thaliana and wheat showed enhanced NIV resistance when grown on media containing different levels of NIV. Increased ability to convert nivalenol to NIV3G was observed in transgenic wheat, which also exhibits type II resistance to a NIV-producing F. graminearum strain. Transgenic wheat expressing HvUGT13248 also provides type II resistance to F. graminearum strains producing 3-ADON and NX-2, and resistance to root growth inhibition in 3,15-diANIV-containing media. Moreover, the HvUGT13248 transgene was introgressed into the elite wheat cultivars ‘Rollag’ and ‘Linkert’, and greenhouse point inoculation results showed that HvUGT13248 improved FHB resistance in elite backgrounds. HvUGT13248 overexpression in transgenic barley also enhanced DON resistance observed in a root assay. In conclusion, HvUGT13248 is a highly effective FHB resistant gene that can detoxify a wide spectrum of trichothecene chemotypes. Thus, the mechanism of resistance is by rapidly detoxifying trichothecenes to their glucoside forms.
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    Identification and characterization of fungal sesquiterpenoid biosynthetic pathways
    (2016-05) Flynn, Christopher
    Fungi produce terpenoids for a wide variety of functions, primarily as signaling and defense compounds. Several highly bioactive sesquiterpenoids are produced only in fungi, particularly those derived from the protoilludene and hirsutene scaffolds. Many sesquiterpenoids have pharmaceutical applications; however, chemical synthesis of sesquiterpenoids is expensive, and biosynthesis in their native fungal hosts is hindered by low concentration and cultivation that is difficult or impossible. Incredible improvements in engineering terpenoid biosynthetic pathways in heterologous hosts have been made in recent years, rendering terpenoid biosynthesis economically viable. However, developing recombinant engineered systems requires the identification of the enzymes and the respective biosynthetic pathways involved in the production of terpenoids from the native fungal host. The primary goal of this work was to identify and characterize the enzymes producing two classes of sesquiterpenoids, derived from protoilludene and hirsutene scaffolds, from Stereum hirsutum. The first step in understanding terpenoid biosynthesis is the identification and characterization of the key scaffold producing enzymes, sesquiterpene synthases (STS). Previously, we developed a predictive framework based on STS protein sequence to accurately guide identification of fungal sesquiterpene synthases based on their initial cyclization mechanism. I successfully implemented this framework to identify three novel protoilludene synthases in the genome of Stereum hirsutum. Furthermore, data shows that both gene structure as well as protein sequence is highly conserved between fungal STS, reinforcing the hypothesis that fungal STS have evolved based upon initial cyclization mechanisms. In addition, application of this same predictive framework led to the identification and cloning of the first hirsutene synthase from Stereum hirsutum. Unexpectedly, I discovered that the STS was not a typical, single domain enzyme, but instead an unprecedented two-domain STS, 3-hydroxy-3-methylglutaryl-(HMG)-CoA synthase, HS-HMGS. HMG-CoA synthase is a key enzyme that catalyzes the second step in the isoprenoid-precursor mevalonate (MVA) pathway; duplications of this gene in isoprenoid pathway gene clusters may represent a mechanism for increasing MVA pathway flux in fungi, potentially increasing isoprenoid/sesquiterpenoid yield. Following my discovery of this novel bifunctional HS-HMGS, I conducted a large-scale search of all sequenced fungal genomes for duplications of MVA and isoprenoid pathway genes. I found that duplication of early MVA pathway genes were both common and widespread throughout the fungal kingdom, with many HMG-CoA synthases being found in isoprenoid biosynthetic gene clusters as well. This suggests duplication of early MVA pathway genes may be a common mechanism utilized by fungi to increase production of specific isoprenoids. Following identification of the the protoilludene and hirsutene synthases, I then cloned the P450 and oxidase enzymes found in their gene clusters. These types of enzymes are predicted to be required for the modification of the sesquiterpene scaffold, yielding the final bioactive sesquiterpenoids. Extensive testing of these potential sesquiterpene modifying enzymes identified two, Omp7a and Omp7b, which modify the protoilludene scaffold. Work to purify sufficient quantities of these compounds to determine the exact chemical modifications by NMR is ongoing, and would be the first demonstration of protoilludene scaffold modification to date. Finally, while isolating STS and refactoring their biosynthetic pathways is the major goal of this work, understanding the biological function of sesquiterpenoids in the native host is also key, because it provides insights into the bioactivities of sesquiterpenoids and helps us understand the factors that govern expression of the biosynthetic pathways. Therefore, I initiated a collaboration to identify the sesquiterpenes produced by Fusarium graminearum, the cause of the crop disease Fusarium Head Blight (FHB). F. graminearum is known to produce deoxynivalenol (DON), a toxic sesquiterpenoid derived via trichodiene synthase (Tri5). However, genomic analysis identified eight sesquiterpene synthases, while only two STS had been characterized to date, suggesting genomic potential to produce non-trichothecene sesquiterpenoids (NTS) that may affect pathogenesis, a topic that has not been comprehensively studied. I therefore set out to determine what NTS are produced by F. graminearum cultures, and if their production is increased under pathogenic (inducing) conditions. GC/MS analysis identified several NTS produced only in induced cultures. Surprisingly, induced Δtri5 deletion strains not only ceased production of the anticipated Tri5-derived trichothecenes, but also stopped producing sesquiterpenes not produced by directly by Tri5. Thus, while Tri5 expression is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 is not sufficient to explain the observed diversity of sesquiterpenoids. These findings suggest that tri5 expression, through an as-of-yet unidentified mechanism, is required for production of NTS. To determine the mechanism through which Tri5 influences NTS biosynthesis, either via protein:protein interactions, or via signaling by trichodiene, a catalytically inactive Tri5 was created that retains its secondary structure, and is currently being prepared for testing. While the role of trichothecenes in phytotoxicity is known, the biological function of non-trichothecene sesquiterpenes and their recently discovered co-regulation has not yet been determined. Understanding the role of NTS in pathogenesis may aid in breeding resistant crops, and prove valuable in controlling FHB. In summary, this work has identified novel fungal sesquiterpene synthases, and demonstrated success in refactoring sesquiterpenoid biosynthetic pathways. My work has provided insights into evolutionary conservation and adaption of gene and protein structures of STS, which will facilitate future discovery and characterization of novel types of STS. In addition, my work with F. graminearum has highlighted that sesquiterpene(oids) may play a potential role in gene regulation of biosynthetic pathways, an important consideration when rebuilding pathways in a heterologous host. Finally, the conservation of early MVA pathway genes in isoprenoid biosynthetic gene clusters suggests a previously neglected mechanism of isoprenoid pathway regulation in fungi, which has wide implications for fungal isoprenoid biosynthesis, genome architecture, and mechanisms pathway regulation.