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The production and usage of biomaterials is no new feat for humanity. We have constantly used the natural resources around us to progress as a species but now that we enter a more advanced age the reliance on primitive biomaterials and their extraction methods has started to become a threat to the way of life to our species. The unsustainable production of materials for use has led us to a resource and climate crisis and the need for more sustainable, renewable, and smarter biomaterials has become one of the priorities for our research and development sectors. Utilising Synthetic Biology and its tools to engineer novel biomaterials has recently started to progress. Engineering microorganisms to synthesis materials that can be used for applications such as sensing-and-responding to their environment to having unique filtration properties able to purify contaminated water sources has recently been achieved. Bacteria from the Acetobacter genus know to produce a biomaterial called bacterial cellulose (BC) have been of increasing interest. One specific Acetobacter species named Komagateaibacter rhaeticus iGEM is a model bacterial cellulose producing organism and is able to synthesis copious amounts of this biomaterial. As it is a genetically amenable strain it allows us to engineer in functionalities and increase the amounts of BC that the organism can produce making it a smarter, engineerable, living biomaterial. In this work we have set out to apply Synthetic Biology principles to K. rhaeticus iGEM strain. By metabolically engineering this organism to overexpress a number of genes in bacterial cellulose synthesis pathway and the fructose utilisation pathway we were able to increase the yield of BC produced by K. rhaeticus. Additionally, we investigated the secretome of the organism in order to utilise its native Sec and Tat secretion pathways in order to secrete reporter proteins to achieve proof-of-concept of the ability to functionalise BC using K. rhaeticus as a secretion platform. This would provide the basis for future work in which one could grow a functional biomaterial capable of bioremediation, biosensing and wound repair with a reduced burden on current energy demanding processes and at both a low and high scale depending on the needs of the situation.

Goss’s wilt is a bacterial disease of maize caused by the Gram-positive bacterium Clavibacter michiganensis subsp. nebraskensis (Cmn). Goss’s wilt was discovered for the first time in South Central Nebraska in 1969. Following its discovery, the disease spread to the neighboring states over the next decade. Maize germplasm was screened for resistance to Goss’s wilt, and possibly due to the deployment of partially resistant hybrids, Goss’s wilt did not cause any significant damage during the 1980s and 1990s. However, Goss’s wilt re-emerged around 2006 and has been spreading to major maize growing areas in the United States and Canada. It is important to understand the genetic basis of resistance to Goss’s wilt to devise strategies for breeding resistance into maize hybrids. The main objectives of this dissertation were to (i) map quantitative trait loci (QTL) for resistance to Goss’s wilt using linkage mapping, joint linkage mapping, and genome-wide association mapping; (ii) identify differentially expressed genes in resistant and susceptible inbred lines in response to Cmn using RNA-seq; and (iii) to explore the prospects of genomic prediction of resistance to Goss’s wilt. Three bi-parental linkage mapping families including B73 x Oh43, B73 x HP301, and B73 x P39 that were evaluated for Goss’s wilt were used for joint linkage and linkage mapping. Eleven QTL were detected for resistance to Goss’s wilt on chromosomes 1, 2, 3, 4, 5, and 10 through joint linkage mapping. Linkage mapping in each of the three families identified nine, six, and four QTL in the families B73 × Oh43, B73 × HP301, and B73 × P39, respectively. Genome-wide association analysis conducted using a diversity panel of 555 maize inbred lines and 450 recombinant inbred lines (RILs) from three bi-parental mapping populations found three SNPs in the diversity panel and 10 SNPs in the combined dataset of diversity panel and RILs that were associated with Goss’s wilt resistance. Two modules of correlated genes were discovered that showed differential regulation in response to Cmn between resistant (N551) and susceptible (B14A) inbred lines using a weighted gene co-expression network analysis. Gene ontology analysis revealed that the genes inside one of the modules were enriched in defense related functions. Genomic prediction of Goss’s wilt resistance was conducted on the data obtained from bi-parental families and the diversity panel. Highest predictive ability of 0.56 and 0.64 was achieved in the diversity panel and B73 x Oh43 population respectively. Effect of training population size, composition, and adding diverse lines to training population on predictive ability was also assessed. Results indicated that predictive ability is not highly benefited when training population is designed by adding equal number of lines from each of the three families. Adding diverse lines to the training population lead to minor changes in predictive ability. Overall, the results improved our understanding of the genetic architecture of Goss’s wilt resistance and showed that the resistance to Goss’s wilt is a complex trait, controlled by small effect QTL.

Leptographium Lagerb. & Melin and Grosmannia Goid. root-inhabiting fungi are the important contributing biotic factors associated with declining loblolly (Pinus taeda L.) and slash pine (P. elliottii Englem.) species in the southeastern United States. The research in this thesis is focused on exploring the variation among the commonly out planted genotypes of loblolly and slash pine by the timber industries in the southeastern United States. Artificial inoculation experiments were conducted on the seedlings and the mature tree roots. Following inoculations, lesion parameters were recorded to access the relative virulence of fungal species and to determine the variation in lesion parameters among the families. Grosmannia and Leptographium species produced dark brown resin filled lesions on the seedling stems and the mature tree roots also exhibited darkened pitch-filled lesions and occlusions around the point of inoculation. In an effort to study the role of nitrogen and carbon allocation pattern of the genotypes on Grosmannia huntii infection, a greenhouse experiment was conducted. Although, the lesion response indicated successful fungal infection, no significant differences in lesion parameters were noticed between normal and high nitrogen treatment levels. Family differences were noticed in stem total phenolic concentration. Also, the positive correlations between stem total phenolic concentration and family morphological traits (total seedling dry weight, fraction of total dry weight allocated to foliage) for several families show that morphological variables may provide a basis for predicting preformed defense potential. Leptographium terebrantis followed by G. huntii caused larger lesions on seedlings stems and tree roots in the present research. Significant differences in lesion parameters were observed among the seedling families in year 2011 and 2012 experiments. Seedlings inoculation experiments identified some families (L-5, L-20, L-8, and, L-13) that developed consistently smaller lesions, while other families (L-1, L-2, L-3 and L-4) were found to have consistently larger lesions. Variability in lesions was observed between the two fungal species from inoculations on the mature tree roots. However, lesion parameters did not vary significantly among the families. Overall results indicated that ophiostomatoid fungi produced local symptomology and are capable of causing damage to the southern pines. Deployment of these families in the field should take into account the relative risk of other abiotic factors and root-feeding beetles associated with the ophiostomatoid fungi.