Ascobolus immersus as a test tube to unravel truffle sexual reproduction

The growth of trees in forests is promoted by root colonization by ectomycorrhizal fungi, which assist the trees in nutrient uptake. Most ectomycorrhizal fungi belong to Basidiomycota and Ascomyceta and some of them provide also non-wood products of forest such as Tuber spp.. These are ecomycorrhizal Ascomycota producing edible fructifications, called truffles upon fertilization between strains of opposite mating type. Truffle formation faces important bottlenecks: the initiation of the sexual reproduction and the growth of ascocarps during a period of several months. It is therefore critical to better understand the mechanisms leading to sexual reproduction such as recognition between compatible strains. Using genomic resources the genes involved in Tuber melanosporum (the Pe?rigord black truffle) sexual reproduction have been characterized but their role cannot be verified by genetic approach due to the absence of genetic tools. More generally, genetic tools are lacking for many ecologically and economically important Ascomycota for which genomic resources are or will be soon available. Belonging to the Pezizomycete class, the saprophytic Ascomycete Ascobolus immersus is closely related to T. melanosporum, also a Pezizomycete. A. immersus has been used as model organism for decades: it is easy to grow in vitro and its sexual cycle can be completed within two weeks in lab conditions. To propagate itself, as truffle species do, this heterothallic fungus relies on sexual reproduction only. Thus, when two strains of opposite mating type (MAT) encounter each other, female gametes are fertilized by male gametes of the opposite MAT, which leads to the building of the fruiting body. Germination of the produced ascospores gives rise to new generations of fungal individuals. In addition, A. immersus can be efficiently transformed and using reverse genetic strategy, several of its genes have already been knocked-out. The aim of our project is to use A. immersus as a test tube to address fundamental questions for other Ascomycete species such as truffles. To start with, we will focus on deciphering the truffle genetic mechanisms involved in strains recognition for sexual reproduction. Recently the first genetic transformation of A. immersus with promoters of mating type genes from T. melanosporum that drive the expression of the GFP gene was realized to gain functional evidence on these regulatory sequences. Overall, the use of A. immersus as test tube should lead to the development of new tools to address fundamental issues on life cycle and reproductive biology of un-culturable ascomycetes or ascomycetes that cannot be efficiently transformed.