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Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

The electron rich and aromatic character of the indole group allows for a wide range of oxidative and substitution reactions, creating a versatile platform for generating structurally diverse molecules. This thesis explores enzyme and synthetic chemistries that act upon indoles and related molecules. Chapter 1 describes the results of in vivo studies of RebC, an enzyme that plays a pivotal role in the biosynthesis of the indolocarbazole alkaloid rebeccamycin. A homologous enzyme, StaC, exists in the biosynthetic pathway for staurosporine, a related indolocarbazole. Structural differences between the RebC and StaC active sites were hypothesized to play a pivotal role in determining the oxidation state in the corresponding natural products. Sequence alignment of RebC and StaC with homologous enzymes from related indolocarbazole biosynthetic pathways revealed six non-conserved residues in the active site. Three RebC variants were generated by replacement of all six, four, or two specific residues with their StaC counterparts. It was demonstrated that only two substitutions, F216V and R239N, are required to convert the specificity of RebC to that of StaC. Analysis of the structure of the RebC bound to a putative reaction intermediate supports the importance of F216 and R239 in catalysis. Based on these results, contrasting mechanisms for RebC and StaC are proposed to account for their differing specificities. Chapter 2 describes a synthetic approach to primarily heterocyclic analogues of lycogarubin C. Suzuki coupling of appropriately functionalized 3,4-dibromopyrrole or 3,4-bis(trifluoromethanesulfonyl)pyrrole was effective for numerous π-excessive five-membered heterocyclic-3-boronic acids. The optimized conditions were less effective for cross-couplings involving heteroaromatic-2-boronic acids, π-deficient heteroaromatic boronic acids, and heteroaromatic boropinacolate esters. Oxidative cyclization of the 3,4-bis(thiophen-3-yl)pyrrole and 3,4-bis(benzothiophen-3-yl)pyrrole to give analogues of the corresponding indolocarbazoles was demonstrated. Chapter 3 describes preliminary results on the development of regioselective C-5 and C-7 indole metalation tactics of indole-6-carboxamides, in order to provide new functionalized indoles. The use of an indole C-2 silicon protection strategy in combination with a sterically bulky C-6 N,N-di-isopropyl carboxamide directed metalation group overcame undesired side reactions observed with the analogous N,N-diethyl indole-6-carboxamide, affording the C-5 and C-7 substituted products in 40% and 13% yields, respectively. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-02-13 11:14:49.599

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the family Pooideae in which they can synthesise an array of bioprotective alkaloids. Some strains produce the ergot alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyteinfected grasses. Cloning and analysis of a plant-induced non-ribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii and analysis of the E. festucae E2368 genome sequence revealed a complex gene cluster for ergot alkaloid biosynthesis. The EAS cluster contained a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Functional analysis of lpsB confirmed its role in ergovaline synthesis and bioassays with the lpsB mutant unexpectedly suggested that ergovaline was not required for black beetle (Heteronychus arator) feeding deterrence from epichloë-infected grasses. Southern analysis showed the cluster was linked with previously identified ergot alkaloid biosynthetic genes, dmaW and lpsA, at a subtelomeric location. The ergovaline genes are closely associated with transposon relics, including retrotransposons, autonomous DNA transposons and miniature inverted-repeat transposable elements (MITEs), which are very rare in other fungi. All genes in the cluster were highly expressed in planta but expression was very low or undetectable in mycelia from axenic culture, including under nitrogen-, carbonor phosphate-limited conditions. Comparative analysis of the EAS gene cluster in four different epichloë strains showed marked differences in gene expression and ergot alkaloid synthesis. Gene order is conserved in each strain although evidence for recombination between two MITEs and expansion or reduction of a simple sequence repeat (SSR) at a single intergenic region was observed. Heterologous expression of a candidate regulatory gene, laeA, from Aspergillus nidulans, which is a global regulator of secondary metabolism in aspergilli, did not affect eas gene expression. This, along with phylogeny and microsynteny analysis, suggests there is not an orthologue of this gene in epichloë. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.

Plants produce a vast number of low-molecular-weight chemicals (so called secondary or specialized metabolites) that confer a selective advantage to the plant, such as defense against herbivory or protection from changing environmental conditions. Many of these specialized metabolites are used for their medicinal properties, as lead compounds in drug discovery, or to impart our food with different tastes and scents. These chemicals are produced by various pathways of enzyme-mediated reactions in plant cells. It is suspected that enzymes in plant specialized metabolism evolved from those in primary metabolism. Understanding how plants evolved to produce these diverse metabolites is of primary interest, as it can lead to the engineering of plants to be more resistant to both biotic and abiotic stress, or to produce more complex small molecule compounds that are difficult to derive. To that end, the first objective was to develop a schema for rational protein engineering using meta-analyses of a well-characterized sesquiterpene synthase family encoding two closely-related but different types of enzymes, using quantitative measures of natural selection on amino-acid positions previously demonstrated as important for neofunctionalization between two terpene synthase gene families. The change in the nonsynonymous to synonymous mutation rate ratio (dN/dS) between these two gene families was large at the sites known to be responsible for interconversion. This led to a metric (delta dN/dS) that might have some predictive power. This natural selection-oriented approach was tested on two related enzyme families involved in either nicotine/tropane alkaloid biosynthesis (putrescine N-methyltransferase) or primary metabolism (spermidine synthase) by attempting to interconvert a spermidine synthase to encode putrescine N-methyltransferase activity based upon past patterns of natural selection. In contrast to the HPS/TEAS system, using delta dN/dS metrics between SPDS and PMT and site directed mutagenesis of SPDS did not result in the desired neofunctionalization to PMT activity. Phylogenetic analyses were performed to investigate the molecular evolution of plant N-methyltransferases involved in three alkaloid biosynthetic pathways. The results from these studies indicated that unlike O-MTs that show monophyletic origins, plant N-MTs showed patterns indicating polyphyletic origins. To provide the foundation for future molecular-oriented studies of urushiol production in poison ivy, the complete poison ivy root and leaf transcriptomes were sequenced, assembled, and analyzed. / Ph. D.

Plant alkaloids are secondary metabolites that may be synthesized in an inducible defense response to herbivory (Baldwin 1999). Genetic engineering of secondary metabolic pathways in plants to enhance or reduce metabolite production is limited by the current understanding of these pathways and their regulation in response to environmental conditions. This study was intended to provide new insights into the mechanism and regulation of alkaloid biosynthesis in N. tabacum by identifying genes that are coordinately regulated during conditions that induce alkaloid biosynthesis and by comparing their expression in regulatory mutant backgrounds that differ at two quantitative alkaloid loci, A and B. In order to identify novel genes that are differentially expressed during alkaloid biosynthesis, the transcriptional profiling procedure, fluorescent differential display (FDD), was used to screen total RNA isolated from Burley 21 (WT, AABB) and LA21 (low alkaloid regulatory mutant, aabb) tobacco root cultures that were induced for alkaloid synthesis. Four of thirteen cloned FDD fragments showed sequence homology to genes with defense-related functions. The differential expression of genes represented by selected FDD gene fragments was confirmed by comparing Northern blots of transcripts of those genes to known alkaloid biosynthetic genes, putrescine methyl transferase (PMT3), ornithine decarboxylase (ODC3), arginine decarboxylase (ADC1), and quinolinate phosphoribosyltransferase (QPRT). The role of the A and B loci in differential expression of genes represented by FDD clones and of known nicotine biosynthetic genes was examined using quantitative real time polymerase chain reaction (QRT-PCR) to measure transcript levels of these genes in four tobacco genotypes differing in alkaloid content, Burley 21(AABB), HI21 (AAbb), LI21(aaBB), and LA21 (aabb). Results of this study suggest that the A/B regulon is not limited to alkaloid biosynthetic genes, but includes multiple genes with defense-related functions. QRT-PCR analysis of nicotine biosynthetic genes and genes represented by confirmed differentially expressed FDD clones showed increased mRNA accumulation in response to alkaloid induction in all the tested genotypes, which suggests that the A and B mutations affect overall mRNA accumulation levels, rather than gene inducibility, per se. Baldwin, I.T. 1999. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. Journal of Chem. Ecol. 25: 3-30. / Master of Science

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis. Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. For functional characterization, fusion constructs of G10H promoter fragments with the GUS reporter gene were generated and expression was analyzed in a tobacco protoplast transient expression assay. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between -191 and -147, -266 and -188, and -318 and -266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings, GUS expression was tissue-specific, restricted to the leaf and actively growing cells around the root tip. GUS expression was not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitors and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade. The majority of TIA pathway gene promoters contain typical W-box elements, which are frequently found to be the binding sites of WRKY transcription factors. CrWRKY1 and CrWRKY2 transcription factors were isolated using a degenerate PCR method. The C. roseus WRKY transcription factor, CrWRKY1 is preferentially expressed in roots and induced by phytohormones, jasmonate, gibberellic acid and ethylene. Overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially tryptophan decarboxylase (TDC), as well as transcriptional repressors ZCT1, ZCT2 and ZCT3. In contrast, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3 and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX-repressor domain to CrWRKY1, resulted in down-regulation of TDC and ZCTs but up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W-box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid and C. roseus protoplast assays. In CrWRKY1 hairy roots, up-regulation of TDC increased TDC activity, tryptamine concentration and resistance to 4-methyl tryptophan inhibition. Compared to control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors, including ORCA3, CrMYC2 and ZCTs, may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. CrWRKY2 is induced by methyljasmonate induction. In plant, CrWRKY2 expression is mainly found in young leaves and the stem. The stable transformation of CrWRKY2 in C. roseus hairy roots up-regulated many pathway genes, especially the genes in vindoline biosynthesis. The accumulation of vindoline was observed in CrWRKY2 hairy roots.