The contribution of horizontal gene transfer (HGT) to the evolution of prokaryotic genomes has been substantial. However, the existence of this phenomenon and its relative role in shaping eukaryotic genomes is controversial. This research aims to elucidate how fungi become pathogens of plants. The specific fungus and disease that serve as the primary model are Nectria haematococca and the disease it causes on pea (Pisum sativum). Past research has established that pathogenic isolates of this fungus have a specific enzyme, a cytochrome P450, that demethylates and thus detoxifies the antimicrobial chemical pisatin produced by the pea. The gene (PDA) encoding pisatin demethylase (pda) has been shown to serve as a virulence factor in N. haematococca. Research funded by NSF has lead to the discovery that PDA is clustered with three other genes that also function independently as virulence traits on pea. This cluster of pea pathogenicity genes (the PEP cluster) is on a "conditionally dispensable" chromosome in N. haematococca. Recent results have shown that a complete PEP cluster is found only in pea-pathogenic isolates of this fungus and that the PEP genes are distinguished from the genes located in the other regions of the genome by their G+C content and codon usage. Although the PEP cluster is present in two other related pea pathogens (Neocosmospora and Fusarium oxysporum), it is not present in Nectria species that are more closely related to N. haematococca. The dispensability, uneven distribution within a species, difference in codon usage and a discontinuous phylogenetic distribution are traits that have been associated with HGT in prokaryotic organisms. Biochemical studies indicate that other more distantly related pea pathogens lack PDA homologs but have paralogous cytochrome P450s with pda activity. These pea pathogens may have convergently evolved their pisatin-detoxifying ability by gene recruitment. The distribution of this enzymatic capacity is also restricted to the pea pathogens and not found in intervening lineages. Thus, two parallel systems are available to assess the role of gene recruitment and the relative roles of HGT versus vertical gene evolution in the evolution of pathogenicity in these eukaryotes. The role of gene recruitment will be evaluated by determining the evolutionary history of the PDA P450 genes that have evolved to detoxify pisatin. This research addresses basic questions on the evolution of eukaryotic genomes. In addition, the identification of pathogenicity traits and knowledge about how fungi acquire them will directly affect the current methods for the control of pathogenic fungi and aid in the design of new strategies to prevent plant diseases. The research will also allow undergraduates from the University of Arizona and from the Dept. of Plant Science's Winter Internship Program to continue to participate in research. This internship program involves ten liberal arts colleges and funds from this grant will expand the collaboration between the University of Arizona and one of these colleges, Luther College, thus providing undergraduates at Luther College with hands-on research experience along with training in contemporary technologies, all at their home institution.
