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

土耳其9604856丝状真菌通常通过有性和无性过程繁殖。性子囊菌可以是异性体(交配时需要配偶)或同性体(交配时没有伴侣)。丝状真菌的术语同源同源是指单个个体能够完成有性周期，通常但不总是如此，因为每个细胞核都携带两种交配型(MAT)基因。在酵母菌中，同系化是指在以前统一交配型的培养中，一些细胞的交配类型发生了变化，然后“切换”的细胞与“未切换”的细胞进行交配。丝状子囊菌的某些属同时含有异株和同株。其中，玉米的病原菌Cochliobolus被选为本研究的对象，因为它生活方式的自然多样性(无性和有性、异性关系和同性关系、腐生和寄生)，从各种自然栖息地和地理位置随时可以获得田间分离株，以及易于进行分子遗传操作。与其他子囊菌一样，模式种(异源异型侧耳)有一个交配型基因座(MAT)，具有不同的形式(称为自形，命名为MAT-1和MAT-2)，每个基因大小约为千碱基，在核苷酸水平上无关，编码不同的转录调节因子。在所有Cochliobolus物种中，自形基因高度保守；在异源物种中，每个菌株携带一个MAT独特型，而在同源物种中，每个菌株都有两个自形，彼此之间的碱基相差约一千个碱基。同种交配型之间的自形两侧的序列几乎完全相同，并且在种间保存良好。然而，最近在Neuropora物种中发现，独特型的一个侧翼携带一个可变区，在同源物种中(7kb)大约是异源物种(3-5kb)的两倍。此外，可变区毗邻一个短的(57-59bp)恒定区，该恒定区在交配型和所有物种中都高度保守。虽然MAT自形的功能很清楚，但可变区和恒定区的意义尚不清楚，也不容易在神经孢子虫中进行测试，因为该属不容易接受有效的基因靶向。初步证据表明，Cochliobolus的可变区和恒定区与脉孢子虫的相似。该项目的一个目标是利用Cochiobolus高效的基因打靶能力来研究这些区域的功能：它们在交配、同种或异种生活方式、物种形成中是重要的，还是对所有这些过程都是可有可无的？这些分析将通过序列分析、在不同遗传背景下的异源表达以及特定的基因缺失、替换或核苷酸替换来执行。另一个目标是根据大量异源和同源菌株的MAT独形和侧翼区域序列构建系统发育树。其目的是追踪上述各种Cochliobolus生活方式的进化史。该项目的一个实际好处是，如果成功，将证明通过对MAT基因的差异破坏，同源物种可以转化为异源物种。这将通过强迫杂交和消除自体来促进同源植物病原真菌物种的遗传分析。

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.