Telomere Hypervariability in the Fungus, Magnaporthe Oryzae - A Model Plant Pathogen

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Telomeres are specialized DNA sequences that form the ends of eukaryotic chromosomes and are required to prevent the progressive loss of terminal DNA sequences during chromosome replication. In most eukaryotes, the telomeres consist of tandem arrays of simple sequence repeats; and the end-maintenance problem is solved by the periodic addition of new repeats to existing ends by telomerase, a specialized reverse transcriptase. Some eukaryotes lack telomerase and have different strategies for solving the end replication problem. The best studied example is Drosophila, whose telomeres consist of the retrotransposons TART and HeT A, which also are added to chromosome ends via reverse transcription. In addition to protecting chromosome ends, telomeres are involved in chromosome pairing and movement, can silence neighboring genes and are highly plastic. This laboratory is studying telomere plasticity in the plant pathogenic fungus Magnaporthe oryzae, as this has been associated with pathogenic variability. M. oryzae isolates that infect perennial ryegrass have unusually unstable telomeres that undergo continual rearrangements both in culture and in planta. By comparison, telomeres in most other host specific forms of this fungus are quite stable. Sequencing revealed that the chromosome ends of the ryegrass pathogens are organized very differently from those in a strain with stable telomeres. Specifically, the ryegrass pathogen telomeres contain tandem arrays of retrotransposon-like sequences separated by short TTAGGG motifs. The arrays are capped at the chromosome end by a "normal" telomere sequence, which in M. oryzae is (TTAGGG)n. This arrangement is reminiscent of the TRAS and SART retrotransposons, which insert into telomeres in the silkworm, Bombyx mori. However, the M. oryzae elements, which we call MoTERs (for M. oryzae telomere-exclusive repeats), are organized quite differently to TRAS and SART. Most importantly, they lack the endonuclease gene required for insertion into DNA. Instead, the particular organization of MoTERs at the chromosome ends suggests they are added on to the ends of chromosomes in the same manner as TART and HeT A in Drosophila. These observations lead to the hypothesis that telomere instability in M. oryzae is due to the activities of two telomere maintenance mechanisms - one utilizing telomerase to extend TTAGGG repeats, and the other involving retrotransposition of MoTERs sequences onto free DNA ends that result from end degradation. The goal of the experiments is to test this hypothesis and develop resources for future mechanistic studies. The specific aims are: 1) To determine the molecular basis for telomere instability, and 2) To study the genetics of MoTER transposition.Intellectual merit: The experiments are an important first step in the characterization of a highly novel system of telomere maintenance in a well-developed experimental model. It will provide important insight into factors affecting telomere stability and the dynamics of telomere maintenance. In addition, this study will open the door to future investigations into poorly understood areas, such as mechanisms and regulation of terminal transposition, the evolutionary relationship between the two telomere maintenance strategies, and the influence of telomere dynamics on neighboring gene expression.Broader impacts: This project will provide a stimulating research experience for a postdoctoral scholar and an undergraduate student. In addition, an undergraduate independent research project will be offered each summer. Minority student involvement in the project will be fostered through links with the Kentucky Young Scientist Summer research program. The Postdoc will attend workshops for computer skills, etc. and are strongly encouraged to attend the various professional development workshops offered on the UK campus, to help them become better prepared for their future careers.