Isolation and preliminary characterization of 5‐fluoroindole‐resistant and IAA‐overproducer mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi

Abstract

summaryIAA‐overproducer mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi were isolated in an attempt to clarify the role of fungal IAA in ectomycorrhiza formation. By contrast to the wild type, mutants are able to metabolize endogenous Trp to IAA. They were isolated using a two‐step method. One hundred and forty‐four mutants with altered regulation of the Trp branch of the shikimate pathway were isolated from two compatible monokaryons by selecting for resistance to the indole analogue, 5‐fluoroindole. These mutants are Trp‐overproducers and most of them accumulate free Trp. Seventeen mutants able to synthesize IAA in the absence of exogenous precursor (tryptophan or intermediates of the tryptophan biosynthetic pathway) were identified by screening for the ability to metabolize endogenous Trp into IAA. By studying the activity of key enzymes of the Trp biosynthetic pathway (i.e. anthranilate synthase and Trp synthase), it was evident that different mechanisms led to IAA‐overproduction. A correlation was detected between the level of intracellular Trp accumulation and the ability of the mutants to metabolize endogenous Trp to IAA. This indicates that, irrespective of the mutation, intracellular Trp accumulation is a prerequisite for IAA overproduction. A positive correlation was also detected between, on the one hand, resistance to fluoroindole and, on the other hand, intracellular Trp accumulation and, subsequently, IAA‐overproduction. Thus, resistance to high fluoroindole concentrations appeared to provide a method for the direct selection of IAA‐overproducer mutants. In order to be able to compare these mutants with the wild parental dikaryon and to perform genetic analyses, the mutants were fused with a compatible wild monokaryon. The resulting dikaryons were able to form mycorrhizas with the normal host plant, Pinus pinaster, and to fruit under controlled culture conditions, thus allowing stabilization and purification of the mutations. IAA‐overproducer monokaryons were detected within the progeny of all the mutants. They are now being used to study the role of fungal IAA in ectomycorrhizas.