Molecular Analysis of Mating Type Loci in Heterothallic and Homothallic Cochliobolus spp

丝状真菌通常通过有性和无性过程进行繁殖。有性子囊菌可以是异宗配合的（需要配偶进行交配）或同宗配合的（没有配偶进行交配）。用于丝状真菌的术语同宗配合意味着单个个体能够完成性周期，通常但不总是，因为每个细胞核携带两个交配型（MAT）基因。在酵母中，同宗配合是指在先前统一交配型的培养物内的一些细胞中交配型的改变，随后是“转换”细胞与“未转换”细胞的交配。某些属的丝状子囊菌同时含有异宗配合和同宗配合的种类。其中之一，玉米的病原体Cochliobolus，已被选择用于本研究，因为它的自然多样性的生活方式（无性与有性，异宗配合与同宗配合，寄生与寄生），现成的现场分离物从各种自然栖息地和地理位置，并服从分子遗传操作。与其他子囊菌一样，模式种（异宗配合的C. heterostrophus）具有单一交配型基因座（MAT），其具有交替形式（称为独特型，并命名为MAT-1和MAT-2），其大小各自约为一个核糖核酸酶，在核苷酸水平上不相关，并编码不同的转录调节因子。所有旋孢霉属物种的独特型是高度保守的;在异宗配合物种中，每个菌株携带一个单一的MAT独特型，而在同宗配合中，每个菌株都有两个独特型，彼此之间的距离约为1个碱基。同一物种的交配型个体两侧的序列几乎相同，并且在物种间具有很好的保守性。然而，最近在脉孔菌属物种中发现，自异形体的一侧携带一个可变区，该可变区在同宗配合物种中约为异宗配合物种（3-5 kb）的两倍（7 kb）。此外，可变区与一个短的（57- 59 bp）恒定区相邻，该恒定区在两种交配型和所有物种中高度保守。虽然MAT独特型的功能是明确的，可变区和恒定区的意义是未知的，不容易测试，在脉孔菌属，因为该属是不容易服从有效的基因靶向。初步证据表明，Cochliobolus的可变区和恒定区类似于脉孢菌。该项目的一个目标是利用Cochilobolus的高效基因靶向能力来研究这些区域的功能：它们在交配中，在同宗配合与异宗配合的生活方式中，在物种形成中是否重要，或者它们是否对所有这些过程都很重要？将通过序列分析、各种遗传背景中的异源表达和特定基因缺失、替换或核苷酸取代的组合进行分析。另一个目标是基于来自大量异宗配合和同宗配合菌株的MAT独特型和侧翼区的序列构建同源基因。目的是跟踪上述各种旋孢霉生活方式的进化历史。该项目的一个实际好处是，如果成功的话，将证明一个同宗配合的物种可以通过其MAT基因的差异破坏转化为异宗配合的物种。这将有助于同宗配合植物病原真菌物种的遗传分析，通过强迫杂交和消除自交。

Turgeon 9604856 Filamentous fungi typically reproduce by both sexual and asexual processes. Sexual ascomycetes may be either heterothallic (require a partner for mating) or homothallic (mate without a partner). The term homothallic as used for filamentous fungi means that a single individual is capable of completing the sexual cycle, usually, but not always, because each nucleus carries both mating type (MAT) genes. In the yeasts, homothallism refers to a change in mating type in some of the cells within a culture of a formerly uniform mating type, followed by mating of "switched" cells with "unswitched" cells. Certain genera of filamentous ascomycetes contain both heterothallic and homothallic species. One of these, Cochliobolus, a pathogen of maize, has been chosen for this study because of its natural diversity of life styles (asexual vs. sexual, heterothallic vs. homothallic, saprophytic vs. parasitic), ready availability of field isolates from a variety of natural habitats and geographical locations, and amenability to molecular genetic manipulation. Like other ascomycetes, the type species (heterothallic C. heterostrophus) has a single mating type locus (MAT) with alternate forms (called idiomorphs and designated MAT-1 and MAT-2) which are each about a kilobase in size, unrelated at the nucleotide level, and encode different transcriptional regulators. The idiomorphs are highly conserved among all Cochliobolus species; in heterothallic species each strain carries a single MAT idiomorph, whereas in homothallics each strain has both idiomorphs within about a kilobase of each other. The sequences on both flanks of the idiomorphs are nearly identical between mating types of the same species and well conserved among species. However, it has recently been found in Neuropora species that one flank of the idiomorph carries a variable region which is about twice as large (7 kb) in homothallic species as in heterothallic (3-5 kb). In addition, the variable region is adjacent to a short ( 57-59bp) constant region which is highly conserved in both mating types and among all species. Although the function of the MAT idiomorphs is clear, the significance of the variable and constant regions is unknown and not easily tested in Neuropora because the genus is not readily amenable to efficient gene targeting. Preliminary evidence suggests variable and constant regions in Cochliobolus analogous to those in Neurospora. One goal of this project is to utilize the efficient gene-targeting ability of Cochilobolus to investigate the functions of these regions: are they important in mating, in the homothallic vs. heterothallic life style, in speciation, or are they dispensable for all of these processes? The analyses will be performed by a combination of sequence analyses, heterologous expression in various genetic backgrounds and by specific gene deletions, replacements, or nucleotide substitutions. Another goal is to construct a phylogeny based on sequences of MAT idiomorphs and flanking regions from a large number of heterothallic and homothallic strains. The intention is to track the evolutionary histories of the various Cochliobolus life styles mentioned above. A practical benefit of the project will be the demonstration, if successful, that a homothallic species can be converted to heterothallic by differential disruption of its MAT genes. This would facilitate genetic analysis of homothallic plant-pathogenic fungal species by forcing crosses and eliminating selfs.