Individual isolates of the fungus Nectria haematococca exist in a wide range of habitats and part of this diversity is attributed to the presence of conditionally dispensable (CD) chromosomes that carry habitat-defining genes. In the current study a new factor located on one of these CD chromosomes was found. This trait allows pea pathogenic isolates of N. haematococca to grow in homoserine, a compound present in large amounts on pea root exudates. The gene(s) for homoserine utilization (HUT) are located on the same CD chromosome that carries the cluster of genes for pea pathogenicity, the PEP cluster. The PDA1 gene, a member of the PEP cluster, is routinely used as a marker for the presence of this CD chromosome, therefore it has been called the PDA1-CD chromosome. For the purpose of identifying the HUT gene(s), a physical map of the PDA1-CD chromosome was constructed. This map, in combination with synteny analysis, and Southern hybridizations led to the identification of a region of 365Kb that is likely to contain the HUT gene. By searching the publicly available genome of N. haematococca several candidates for HUT were identified.The synteny evaluation between the PDA1-CD chromosome and a different CD chromosome that carries the MAK1 gene, for chickpea pathogenicity, revealed a region (> 463Kb) of synteny, which advocates for a common ancestor for these CD chromosomes. However a large region (~ 1 Mb) in each of the CD chromosomes was found to carry unique DNA, therefore we proposed that individual isolates of this fungus contain large regions of unique DNA located on the CD chromosomes. The localization of syntenic regions also suggests that breakage points previous identified in the MAK1-CD chromosome could potentially be "hot spots" for recombination between both CD chromosomes. Furthermore, the anchoring of the PDA1-CD map to the genome of N. haematococca allowed the identification of additional putative habitat colonization genes present on both CD chromosomes, and niche-defining genes on the PDA1-CD chromosome.

Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...

Soilborne plant pathogens are responsible for many of the major crop diseases worldwide. However, plant root tips are generally resistant to pathogen infections. The goal of this dissertation research is to understand the mechanism of this natural resistance by testing the hypothesis that root caps and root border cells control the rhizosphere community through the biological products which they deliver to the soil. Specific objectives of this dissertation project are 1) identifying, isolating, and characterizing the genes important for border cell development and for root exudates delivery, and 2) analyzing the function of extracellular macromolecules in root exudates in root tip-fungal pathogen interaction. The expression of a primary cell wall synthesis gene, PsFut1, encoding Pisum sativum fucosyltransferase, was characterized during border cell production, and the impact of silencing this gene on border cell development was examined. Another gene, BRDgal1, encoding β-galactosidase, was identified and characterized in Pisum sativum during this study. It was shown that this β-galactosidase is specifically produced in and secreted from root border cells. The microarray transcriptional profiling in M. truncatula and mRNA differential display analysis in pea plants were carried out following the induction of border cell production to gain a broader understanding of the genes which potentially influence border cell development. In order to study the commonality of border cell production across different plant species, the expression of rcpme1, the marker gene for border cell production, was compared between the garden pea and a gymnosperm species, the Norway spruce (Picea abies). To accomplish the second objective, the focus of this study was shifted from border cell development to mucilaginous root exudates excreted by border cells and root cap cells. This resulted in a breakthrough in the understanding of the mechanisms of root tip resistance. The presence of extracellular DNA in the root mucilage was discovered and its requirement for root tip resistance to fungal infection was demonstrated. Extracellular proteins in the root mucilage were identified and they were shown to be also required for the root tip resistance to fungal infection. This work provided new insights into understanding plant defense mechanisms.