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

端粒是形成真核生物染色体末端的特殊DNA序列，在染色体复制过程中防止末端DNA序列的逐渐丢失。在大多数真核生物中，端粒由简单序列重复的串联阵列组成；端粒酶是一种特殊的逆转录酶，它通过在现有的端粒上周期性地添加新的重复序列来解决末端维持问题。一些真核生物缺乏端粒酶，有不同的策略来解决末端复制问题。研究得最好的例子是果蝇，它的端粒由逆转录转座子TART和HeT A组成，它们也通过逆转录添加到染色体末端。除了保护染色体末端外，端粒还参与染色体配对和运动，可以使邻近基因沉默，并且具有高度的可塑性。本实验室正在研究植物病原真菌米大孔菌（Magnaporthe oryzae）的端粒可塑性，因为这与致病变异性有关。感染多年生黑麦草的M. oryzae分离株具有异常不稳定的端粒，在培养物和植物中都经历不断的重排。相比之下，这种真菌的大多数其他宿主特定形式的端粒是相当稳定的。测序显示，黑麦草病原体的染色体末端与具有稳定端粒的菌株的染色体末端组织非常不同。具体来说，黑麦草病原体端粒包含由短TTAGGG基序分隔的反转录转座子样序列串联阵列。这些阵列在染色体末端由一个“正常”端粒序列覆盖，在m.o ryzae中是（TTAGGG）n。这种排列让人想起家蚕（Bombyx mori）中插入端粒的TRAS和SART反转录转座子。然而，m.o ryzae元件，我们称之为MoTERs （m.o ryzae端粒排他重复序列），其组织方式与TRAS和SART完全不同。最重要的是，它们缺乏插入DNA所需的核酸内切酶基因。相反，染色体末端的MoTERs的特殊组织表明它们以与果蝇中的TART和HeT A相同的方式被添加到染色体末端。这些观察结果导致了一种假设，即m.o ryzae的端粒不稳定性是由于两种端粒维持机制的活性所致——一种是利用端粒酶延长TTAGGG重复序列，另一种是通过端粒降解将MoTERs序列反转录转位到自由DNA末端。实验的目的是验证这一假设，并为未来的机制研究开发资源。具体目的是：1)确定端粒不稳定性的分子基础；2)研究MoTER转位的遗传学。智力价值：实验是一个高度新颖的端粒维持系统在一个完善的实验模型表征的重要的第一步。它将为影响端粒稳定性的因素和端粒维持的动力学提供重要的见解。此外，该研究将为未来研究一些尚不清楚的领域打开大门，如末端转位的机制和调控、两种端粒维持策略之间的进化关系以及端粒动力学对邻近基因表达的影响。更广泛的影响：该项目将为博士后学者和本科生提供一个刺激的研究经验。此外，每年夏天将提供一个本科生独立研究项目。少数族裔学生参与项目将通过与肯塔基青年科学家暑期研究项目的联系来促进。博士后将参加计算机技能等研讨会，并强烈鼓励他们参加英国校区提供的各种专业发展研讨会，以帮助他们为未来的职业生涯做好更好的准备。

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.