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

在分子水平上，食用菌子实体的形成知之甚少。作为子实体形成的基础，性发育在裂褶菌和灰盖鬼伞中得到了很好的研究。与此相反，食用蘑菇到目前为止还不太容易获得功能遗传学方法。茶树菇是一种商业化栽培的食用菌，具有严格意义上的单核子实体的罕见特征，这意味着它能够在不进行任何有性交配的情况下提前子实体。最近发表的工作探索分子工具的转化和组成型基因表达在A。aegerita现在允许在这种蘑菇中使用基于功能遗传学的方法。在拟议的项目中，到目前为止尚未表征的果实相关基因（FRG）将从四个基因开始进行功能表征，这些基因在果实形成过程中的差异表达已经得到验证，并且预计可能编码转录因子。除了从初步工作中已经建立的用于组成型感兴趣基因表达（“过表达”）的功能遗传学工具之外，基于CRISPR/Cas9的基因缺失将适用于A. aegerita。最后，比较了异单核子实体和常规二核子实体的异同。Aegerita将被定义。为了实现这一点，将进行RNA测序，采用野生型单核子实体阶段与本项目中产生的两种选定突变体表现出的不同单核子实体表型。这种方法也将有助于理解诱变基因在A. aegerita。使用这个模型系统，预期的结果将进一步弥合子实体形成的分子生物学知识的差距，也将提供第一次的见解是什么区别单核子实体从常规的双孢子实体在蘑菇。

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.