Functional characterisation of genes putatively involved in fruiting body formation of the cultivated edible mushroom Agrocybe aegerita

在分子水平上，人们对食用菌的子实体形成知之甚少。作为担子菌子实体形成的基础，裂鳃裂殖酵母和墨帽平菇的性发育得到了广泛的研究。相比之下，在功能遗传学方法方面，食用菌到目前为止还不太容易获得。茶树菇(Agrocybe Aegerita)是一种商品栽培的食用菌，它具有单核结实的罕见特征，这意味着它可以在没有任何性交的情况下结实。最近发表的一项研究探索了山羊草转化的分子工具和构成基因的表达，现在允许在这种蘑菇中采用基于功能遗传学的方法。在拟议的项目中，到目前为止，尚未鉴定的果实相关基因(FRG)将从四个基因开始，它们在果实期间的差异表达已经得到验证，并被预测可能编码转录因子。除了已从初步工作中建立的用于结构性目的基因表达(过表达)的功能遗传学工具外，基于CRISPR/Cas9的基因缺失将适用于山羊草。最终，将确定异常的单核和正常的双核结实之间的共性和差异。为了实现这一点，需要进行RNA测序，利用野生型单核果期与本项目中产生的两个选定突变体所表现的不同单核果型进行比较。这一方法也将有助于理解突变基因在预测的Aegerita中FRGS的复杂遗传网络中的作用。利用这个模型系统，预期的结果将进一步弥合在子实体形成的分子生物学知识方面的差距，并将提供关于蘑菇单核果与常规双核果的区别的初步见解。

On the molecular level, fruiting body formation by edible mushrooms is poorly understood. As a basis of fruiting body formation in basidiomycetes, sexual development is well studied in the split gill mushroom Schizophyllum commune and the ink cap Coprinopsis cinerea. In contrast to that, edible mushrooms are up to now less accessible with regard to functional genetics approaches. Agrocybe aegerita is a commercially cultivated edible mushroom which exhibits the rare feature of monokaryotic fruiting sensu stricto which means that it is able to fruit without any sexual mating beforehand. A recently published work exploring molecular tools for transformation and constitutive gene expression in A. aegerita now permits functional genetics-based approaches in this mushroom. Within the proposed project, up to now uncharacterised fruiting-related genes (FRGs) will be functionally characterised, starting with four genes, the differential expression of which during fruiting has already been validated and which are predicted to likely encode transcription factors. In addition to the already set up functional-genetics tools for constitutive gene of interest expression (‘overexpression’) from preliminary work, CRISPR/Cas9-based gene deletion is to be adapted for A. aegerita. Ultimately, commonalities and differences between exceptional monokaryotic and regular dikaryotic fruiting of A. aegerita will be defined. To achieve this, RNA-sequencing is to be conducted, employing wild type monokaryotic fruiting stages versus different monokaryotic fruiting phenotypes exhibited by two selected mutants generated within this project. This approach will also help to understand the mutagenised genes’ roles within the complex genetic network of predicted FRGs in A. aegerita. Using this model system, the expected results will further bridge gaps in the knowledge on molecular biology of fruiting body formation and will also provide first insights on what differentiates monokaryotic fruiting from regular dikaryotic fruiting in mushrooms.