Cultivation of the edible white button mushroom Agaricus bisporus is a complicated process. The change from the vegetative to reproductive stage (edible fruit bodies) is induced by the manipulation of several environmental conditions. The molecular mechanisms underlying this process are unknown and the phase change does not happen uniformly (e.g. a thick layer of colonised compost covered with a humid, peaty casing layer prevents an uniform change of environmental conditions throughout this substrate), leading to variations in yield and quality and requiring manual harvest.
1) Morphological changes during mushroom initiation and development --> high resolution photography/microscopy
2) Development of a A. bisporus gene database and annotation of genes driving the initiation process --> comparative bioinformatics
3) Construction of gene expression profiles of the phase change under normal and modified growth conditions --> custom microarray technology
4) Identification of key genes involved in the initiation process --> combination of the first 3 points and use of transformation technology and gene silencing
- A. bisporus is a non-sequenced organism: which genes are involved in the development of A. bisporus and need to be present on the microarray?
- A. bisporus cultivated in vitro does not produce fruiting bodies. Therefore it needs to be studied in vivo creating a number of technical difficulties.
- RNA required for microarray hybridisations will be extracted from the normal growth substrate of this fungus, a mixture of casing and compost, known to contain high amounts of RNA degrading enzymes. Moreover, peaty casing contains high concentrations of substances (e.g. brown humic and fulvic acids) which inhibit subsequent processing required to prepare the RNA for microarray experiments very efficiently. Therefore a method to extract intact RNA, free of inhibitory substances will have to be developed.
The information acquired from these studies will be combined to get an understanding in the molecular processes during initiation and fruit body development. This knowledge is likely to give an insight on the function of multiple environmental parameters and might lead to optimised This knowledge is likely to lead to optimised growth conditions, improved quality and automated/more efficient harvesting.
project and the developed methodology is an example of creating a gene expression profile of a non-sequenced organism which cannot be cultivated properly in vitro using novel microarray technology whereby up to eight different samples can be hybridised and tested on one microarray slide, reducing the costs involved with this kind of experiments significantly.