Doctor of Philosophy / Genetics Interdepartmental Program / Christopher Toomajian / Secondary metabolites (SMs), low molecular weight molecules that are not essential for normal organism growth and development, may confer a selective advantage in some environments. Fungal SMs are structurally and functionally diverse and include mycotoxins, plant regulators and pigments, and the genes that work together in SM biosynthetic pathways are physically clustered in the genome. Fusarium, a genus of filamentous fungi, is noted for SM production, especially mycotoxins, which may contribute to plant pathogenesis. Fusarium species exhibit differences in their SM profiles, and comparative genomics studies have found corresponding differences in the SM gene clusters in some Fusarium species. The investigation of differences in the genomes and SM gene clusters between closely related species, such as F. proliferatum and F. fujikuroi, may help explain their phenotypic divergence, including differences in SM profiles. In addition, the study of intra-species SM variation may indicate how SM loci affect a pathogen’s fitness traits. My research includes three main projects that address different aspects of Fusarium SM variability. To carry out my projects, I established a feasible Genotyping-by-Sequencing (GBS) protocol for Fusarium. One project explored the genetic bases underlying phenotypic divergence related to SM profiles and pathogenicity between F. proliferatum and F. fujikuroi using a quantitative genetics approach. Specifically, I 1) constructed the first high density genetic map based on progeny from an interspecific cross between these two species; and 2) detected a novel regulatory locus for gibberellic acid production and identified a region affecting onion virulence that includes the fumonisin gene cluster. The second project characterized the F. proliferatum parent genome from the previous cross and its SM gene clusters using a comparative genomics approach. Specifically, I 1) assembled the F. proliferatum genome into 12 chromosomes with a combined length of ~43 Mb; 2) annotated this assembly and characterized its 50 SM gene clusters; and 3) detected over 100 F. proliferatum specific genes that might play roles in this species’ host specificity and plant pathogenicity. The third project used a population genomics approach to explore how different F. graminearum chemotypes, or isolates classified based on the accumulation of alternate trichothecene toxin types, may differ for fitness traits and whether trichothecene genes are directly responsible for these differences. Specifically, I 1) genotyped over 300 F. graminearum strains from New York and the upper Midwest in the U.S. and from South America using our GBS protocol; 2) detected two major subpopulations that were correlated, though imperfectly, to the predicted 3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15ADON) chemotypes in the U.S.; 3) identified a rapid linkage disequilibrium decay over a few tens of kb followed by a slower decay to background levels over a distance of 200 kb to 400 kb in selected subpopulations in the U.S.; and 4) found that neither chemotype has a clear fitness advantage in a small set of isolates from New York, but that isolates belonging to one genetic subpopulation may on average have a fitness advantage over isolates from the other subpopulation.

Doctor of Philosophy / Genetics Interdepartmental Program / Christopher Toomajian / Secondary metabolites (SMs), low molecular weight molecules that are not essential for normal organism growth and development, may confer a selective advantage in some environments. Fungal SMs are structurally and functionally diverse and include mycotoxins, plant regulators and pigments, and the genes that work together in SM biosynthetic pathways are physically clustered in the genome. Fusarium, a genus of filamentous fungi, is noted for SM production, especially mycotoxins, which may contribute to plant pathogenesis. Fusarium species exhibit differences in their SM profiles, and comparative genomics studies have found corresponding differences in the SM gene clusters in some Fusarium species. The investigation of differences in the genomes and SM gene clusters between closely related species, such as F. proliferatum and F. fujikuroi, may help explain their phenotypic divergence, including differences in SM profiles. In addition, the study of intra-species SM variation may indicate how SM loci affect a pathogen’s fitness traits. My research includes three main projects that address different aspects of Fusarium SM variability. To carry out my projects, I established a feasible Genotyping-by-Sequencing (GBS) protocol for Fusarium. One project explored the genetic bases underlying phenotypic divergence related to SM profiles and pathogenicity between F. proliferatum and F. fujikuroi using a quantitative genetics approach. Specifically, I 1) constructed the first high density genetic map based on progeny from an interspecific cross between these two species; and 2) detected a novel regulatory locus for gibberellic acid production and identified a region affecting onion virulence that includes the fumonisin gene cluster. The second project characterized the F. proliferatum parent genome from the previous cross and its SM gene clusters using a comparative genomics approach. Specifically, I 1) assembled the F. proliferatum genome into 12 chromosomes with a combined length of ~43 Mb; 2) annotated this assembly and characterized its 50 SM gene clusters; and 3) detected over 100 F. proliferatum specific genes that might play roles in this species’ host specificity and plant pathogenicity. The third project used a population genomics approach to explore how different F. graminearum chemotypes, or isolates classified based on the accumulation of alternate trichothecene toxin types, may differ for fitness traits and whether trichothecene genes are directly responsible for these differences. Specifically, I 1) genotyped over 300 F. graminearum strains from New York and the upper Midwest in the U.S. and from South America using our GBS protocol; 2) detected two major subpopulations that were correlated, though imperfectly, to the predicted 3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15ADON) chemotypes in the U.S.; 3) identified a rapid linkage disequilibrium decay over a few tens of kb followed by a slower decay to background levels over a distance of 200 kb to 400 kb in selected subpopulations in the U.S.; and 4) found that neither chemotype has a clear fitness advantage in a small set of isolates from New York, but that isolates belonging to one genetic subpopulation may on average have a fitness advantage over isolates from the other subpopulation.

This thesis is divided into two parts and covers biosynthetic studies on two secondary metabolites, tropic acid in Part I and piliformic acid, in Part II.(S)-Tropic acid is the acid moiety of the alkaloids hyoscyamine and scopolamine, which are produced by a number of plants of the Solanacae family. An intriguing rearrangement of the L-phenylalanine side chain gives rise to the isopropanoid (S)-tropic acid skeleton. The detailed nature of the rearrangement has however remained elusive despite continued interest over the years. In chapter two the identification of intermediates between L-phenylalanine and (S)-tropic acid is discussed, which has placed (R)-D-phenyllactic acid as an immediate precursor. The stereochemical features of the rearrangement are described in chapter 3 and finally in chapter 4 a mechanism for the rearrangement is proposed. This is based on information obtained from the incorporation of various isotopically labelled precursors to tropic acid into two of the minor alkaloids, 3a-2'-hydroxyacetoxytropane and 3a- phenylacetoxytropane. This work was carried out in collaboration with Dr Richard Robins at the AFRC Institute of Food Research in Norwich. Piliformic acid is elaborated by the slow growing fungus Poronia piliformis. The incorporation of a number of isotopically labelled substrates into piliformic acid has revealed a mixed biosynthetic origin, comprising C(_8) and C(_3) fragments. These have been shown to be of acetogenic and citric acid cycle origins respectively. The C(_8) fragment has been further demonstrated to be a degradation product of a longer chain fatty acid. The mode of coupling of the two fragments has been investigated and suggests the intermediacy of a novel a-carboxyoctanoate. A pathway for the assembly of piliformic acid, involving a 1,3-hydrogen shift, is proposed, consistent with the above findings. These results are the subject of chapter 6.

Fusarium graminearum is responsible for causing Fusarium head blight in cereals and maize imposing a significant impact in Canadian agriculture. While a handful of secondary metabolites produced by F. graminearum are recognized as contributors to disease virulence, the functions of numerous molecular products arising from biosynthetic gene clusters expressed during infection remain undiscovered. Presented here are the results of CRISPR-Cas9 mediated gene-deletion experiments disrupting core biosynthetic genes from four biosynthetic gene clusters with reported in-planta transcription: C08, C16, C13 and C70. Both wheat head infection assays and coleoptile infection assays were used to evaluate the pathology phenotypes of transformant strains illustrating potential links between C16 and pathogenicity. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products and attempt to identify unknown secondary metabolites of the biosynthetic gene clusters. While C08 secondary metabolite remained elusive, confirmation of C16 secondary metabolites led to hypotheses regarding their potential connections to the inhibition of plant immune response and untargeted secondary metabolite profiling of the C13/C70 transformant strains suggests that this BGC may have significant implications for global secondary metabolite production.

Maize anthracnose, caused by the fungus Colletotrichum graminicola, is an economically important species contributing to major yield losses. C. graminicola is a hemibiotroph; initially it invades its host while it is alive, and then it switches to destructive necrotrophic growth and the host is killed. Establishment of compatible interactions by biotrophic pathogens is usually associated with suppression of host defenses and cell death, while necrotrophic pathogens typically secrete phytotoxic compounds and induce cell death. To understand the relationship of hemibiotrophy in C. graminicola to biotrophy and necrotrophy, I compared a compatible and an incompatible interaction, utilizing a non-pathogenic mutant strain that is very similar to the wild type in vitro. I developed an assay to visualize in detail living fungal and host cells during pathogenic and nonpathogenic interactions. My results provided evidence that C. graminicola produces diffusible substances during colonization that predispose nearby living host cells for fungal invasion. My observations further suggested that the mutant is nonpathogenic because it fails to produce these substances. To explore the possibility that the C. graminicola mutant is impaired in the production and/or secretion of one or more secondary metabolites (SM), I characterized the range of SM-associated genes in C. graminicola. C. graminicola has a large and diverse repetoire of these genes, indicating significant capacity for the production of SM. I then characterized the global expression of fungal genes during different developmental phases in both compatible and incompatible interactions. I found that SM-associated genes are expressed during early and late stages of maize infection. Secreted proteins and putative effectors were overrepresented among differentially regulated predicted gene products. There were relatively few differences in expression between the mutant and wild type, suggesting that differences between them may relate to post-transcriptional events. The transcriptional analysis indicated that the mutant was defective very early in biotrophy. This study indicates that biotrophy and necrotrophy coexist in this pathosystem in different cells, and that arrays of differentially regulated and locally expressed genes are involved in maintaining this balance. Understanding the nature of induced susceptibility may lead to new therapeutic targets for management of this damaging disease.

Marine organisms have attracted much attention as potential source for drugs over recent years. Soft corals have yielded many bioactive metabolites. Some of them have been examined for their pharmacological properties. For the process of drug discovery, we have examined bioactive metabolites from the organic extracts of two soft corals Sinularia gibberosa and Sarcophyton sp. collected off Formosan coast. This study had led to the isolation of forty-two natural products (1¡V42), including one new £]-caryophyllene-type sesquiterpenoid (1), four new xeniaphyllane-type norditerpenoids (2, 14, 16, and 17), fourteen new xeniaphyllane-type diterpenoids (3¡V13 and 18¡V20), one novel nor-humulene (15), seven new xeniaphyllane-type diterpenoids (21¡V26) with cyclic peroxyhemiketal (3,6-dihydro-1,2-dioxin-3-ol) moiety, and one new steroid (27), along with five known compounds (28¡V32) from Sinularia gibberosa. Three new cembrane-type diterpenoids (33¡V35), along with seven known cembranolides (36¡V42) were isolated from Sarcophyton sp. The structures of metabolites 1¡V42 including their stereochemistry have been established by detailed spectroscopic analyses, particularly mass, 2D NMR (1H¡V1H COSY, HMQC, HMBC, and NOESY) spectroscopy and by comparison with the related physical and spectral data from other known compounds. In above metabolites, two compounds (8, 9) exhibited cytotoxicity against the growth of MCF-7, Hep 3B, Ca9-22 cancer cell lines. Furthermore, nine compounds (4, 8, 9, 11, 12, 13, 21, 31, 39) exhibited cytotoxicity against the growth of MDA-MB-231, Hep G2 and A-549 cancer cell lines.

Jadomycins are secondary metabolites produced by Streptomyces venezuelae ISP5230 VS1099 in response to conditions of stress such as heat or ethanol shock. They have been shown to exhibit antibiotic and anticancer activity. Unique structural features of the jadomycins include a rare 2,6-dideoxysugar, L-digitoxose, and an oxazolone ring with an amino acid component. Previous studies have revealed that jadomycin derivatives can be produced by altering the amino acid in S. venezuelae ISP5230 VS1099 culture media which becomes incorporated into the oxazolone ring. One jadomycin from a proteogenic amino acid and three new jadomycins from non-proteogenic amino acids have been successfully produced on a large scale (4 mg/L to 12 mg/L, 2 L) and characterized using mass spectrometry, infrared spectroscopy, UV-visible spectroscopy, and nuclear magnetic resonance spectroscopy. One of these contains a terminal alkyne functionality and has been used in cycloaddition reactions with various azides to produce a library of triazole-containing jadomycins.

<p>The world is facing a public health threat due to increasing emergence of antibiotic resistance in pathogens. <em>Streptomyces </em>the soil-dwelling, Gram-positive, filamentous bacteria belonging to the family actinomycetes, are proven to be rich sources of natural antibiotics. Genome sequencing of <em>Streptomyces coelicolor, </em>a model organism of this genus, has revealed that in addition to the five antibiotics characterized so far, it possesses abundant genetic architecture of unexpressed biosynthetic or cryptic clusters for secondary metabolite production. The reason for their silence appears to be the poor understanding of their specific activation stimuli. In <em>Streptomyces coelicolor,</em> a pleiotropic regulator belonging to the two-component system family, <em>afsQ1</em>, has shown to activate the production of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA). The aim of this research was to employ the genetically engineered <em>afsQ1</em> allele (named <em>afsQ1*</em>), which mimics the phosphorylated active form and obviates the need for specific external stimulus, and screen for novel antibiotic production. In this study, <em>afsQ1* </em>was introduced in various wild actinomycete isolates from the Wright Actinomycetes Collection (WAC) by conjugation and the resulting mutants were screened for antibiotic production. Two out of six WAC strains showed <em>afsQ1*- </em>induced antimicrobial activity. Interestingly, we were able to purify two antibiotic compounds, namely 1082 [M+2H]²⁺<strong> </strong>and 782 [M+H]⁺<strong> </strong>from the strain WAC00263. 1082 [M+2H]²⁺,<strong> </strong>a potentially novel antimicrobial peptide, exhibited activity against a wide range of Gram-positive bacteria including resistant pathogens such as vancomycin-resistant <em>Enterococcus</em> ATCC# 51299, a clinical isolate of methicillin resistant <em>Staphylococcus aureus</em>, and a clinical isolate of <em>S. aureus</em> BM3002. Moreover, it also showed activity against an opportunistic Gram-negative multi-drug resistant pathogen <em>Acinetobacter baumannii</em> B0098426R and a virulent strain of the fungus <em>Cryptococcus neoformans </em>H99<em>. </em>The second newly expressed molecule, 782 [M+H]⁺<strong> </strong>was not as potent as 1082 [M+2H]²⁺,<strong> </strong>so<strong> </strong>far only exhibited antimicrobial activity against the Gram-positive laboratory strains <em>Bacillus subtilis</em> #168 and <em>Micrococcus luteus</em>. These results reiterate that the technique of heterologous expression of the pleiotropic regulator, <em>afsQ1*</em>, in diverse actinomycetes is an excellent tool to induce novel antimicrobial production.</p> / Master of Science (MSc)

Os fungos endofíticos constituem uma fonte promissora para descoberta de novos compostos ativos de interesse biotecnológico. Nas últimas décadas, os fungos endofíticos têm recebido atenção no meio científico devido aos compostos que produzem, com estruturas únicas e atividades biológicas de relevância em diversas áreas, inclusive agronômica. Um dos maiores problemas nesta área são as doenças causadas por fungos fitopatogênicos que acometem as culturas. Dentre elas, a antracnose é uma das principais doenças que atinge diversas culturas no Brasil, gerando grandes perdas agrícolas, principalmente as culturas do morango, guaraná e citrus, causadas por fungos do gênero Colletotrichum. Esse trabalho teve como objetivo investigar extratos de linhagens dos fungos endofíticos Fusarium e Penicillium, isolados de folhas do guaranazeiro, com atividade à fitopatógenos do gênero Colletotrichum isolados das culturas de guaraná, morango e citrus. Para tanto foi realizado uma triagem inicial de duas linhagens de fungos, dentre nove, por ensaios biológicos in vitro de cultivo pareado na atividade antifúngica aos três fitopatógenos. As duas linhagens selecionadas, apresentaram os maiores índices de antagonismo contra os três fitopatógenos e foram identificadas como Fusarium sp e Penicillum pinophilum. Os extratos e as frações semipurificadas se mostraram com grande potencial na inibição do crescimento do fitopatógeno do gênero Colletotrichum, causador da antracnose, que provoca grandes perdas nas culturas de importância econômica do Brasil. / Endophitic fungi are a promising source for discovery of new active compounds of biotechnological interest. In recent decades, endophytic fungi have received attention in the scientific community because of compounds they produce, with unique structures and biological activities of relevance in several areas, including agronomy. One of the greatest problems in this is diseases caused by phytopathogenic fungi that affect the crops. Among them, anthracnose is a major disease that affects several crops in Brazil, generating large agricultural losses, especially strawberry crops, guarana and citrus caused by fungi of the genus Colletotrichum. This study investigated endophytic fungi strains of Fusarium and Penicillium extracts isolated from guarana leaves, with activity to phytopathogens of the genus Colletotrichum isolated from guarana crops, strawberry and citrus. For that, it was conducted an initial screening of two strains of fungi, among nine, using in vitro biological assays of cultivation paired in antifungal activity to the three pathogens. The two strains selected had the highest rates of antagonism against the three phytopathogens and were identified as Fusarium sp and Penicillium pinophilum. Extracts and semi-purified fractions were shown with great potential to inhibit pathogen growth of the genus Colletotrichum, which causes anthracnose and, thus, great losses to crops of economic importance in Brazil.

Os fungos endofíticos constituem uma fonte promissora para descoberta de novos compostos ativos de interesse biotecnológico. Nas últimas décadas, os fungos endofíticos têm recebido atenção no meio científico devido aos compostos que produzem, com estruturas únicas e atividades biológicas de relevância em diversas áreas, inclusive agronômica. Um dos maiores problemas nesta área são as doenças causadas por fungos fitopatogênicos que acometem as culturas. Dentre elas, a antracnose é uma das principais doenças que atinge diversas culturas no Brasil, gerando grandes perdas agrícolas, principalmente as culturas do morango, guaraná e citrus, causadas por fungos do gênero Colletotrichum. Esse trabalho teve como objetivo investigar extratos de linhagens dos fungos endofíticos Fusarium e Penicillium, isolados de folhas do guaranazeiro, com atividade à fitopatógenos do gênero Colletotrichum isolados das culturas de guaraná, morango e citrus. Para tanto foi realizado uma triagem inicial de duas linhagens de fungos, dentre nove, por ensaios biológicos in vitro de cultivo pareado na atividade antifúngica aos três fitopatógenos. As duas linhagens selecionadas, apresentaram os maiores índices de antagonismo contra os três fitopatógenos e foram identificadas como Fusarium sp e Penicillum pinophilum. Os extratos e as frações semipurificadas se mostraram com grande potencial na inibição do crescimento do fitopatógeno do gênero Colletotrichum, causador da antracnose, que provoca grandes perdas nas culturas de importância econômica do Brasil. / Endophitic fungi are a promising source for discovery of new active compounds of biotechnological interest. In recent decades, endophytic fungi have received attention in the scientific community because of compounds they produce, with unique structures and biological activities of relevance in several areas, including agronomy. One of the greatest problems in this is diseases caused by phytopathogenic fungi that affect the crops. Among them, anthracnose is a major disease that affects several crops in Brazil, generating large agricultural losses, especially strawberry crops, guarana and citrus caused by fungi of the genus Colletotrichum. This study investigated endophytic fungi strains of Fusarium and Penicillium extracts isolated from guarana leaves, with activity to phytopathogens of the genus Colletotrichum isolated from guarana crops, strawberry and citrus. For that, it was conducted an initial screening of two strains of fungi, among nine, using in vitro biological assays of cultivation paired in antifungal activity to the three pathogens. The two strains selected had the highest rates of antagonism against the three phytopathogens and were identified as Fusarium sp and Penicillium pinophilum. Extracts and semi-purified fractions were shown with great potential to inhibit pathogen growth of the genus Colletotrichum, which causes anthracnose and, thus, great losses to crops of economic importance in Brazil.