Cloning and characterization of a symbiosis-related gene from an ectomycorrhizal fungus Laccaria bicolor.

An in vitro system for a Laccaria bicolorxPinus resinosa interaction was used to identify and clone a symbiosis-regulated gene from L. bicolor employing the mRNA differential display technique (DDRT-PCR). The DDRT-PCR identified several cDNAs that are differentially expressed as early as 6h into the interaction. One such cDNA was used to screen a L. bicolor cDNA library enriched for mRNAs expressed during early interaction with red pine seedlings. Characterization of a cDNA clone, PF6.2, showed that it contained a 1551 bp insert coding for a protein of 433 amino acids. Sequence analysis of the PF6.2 cDNA revealed the presence of several evolving repeats in the protein. To confirm this, the gene corresponding to PF6.2 was isolated and sequenced. The PF6.2 gene consisted of seven exons interrupted by six relatively small introns. Although the amino-acid sequence of the PF6.2 did not show significant overall similarity to any previously characterized proteins, of several direct repeats it contained a feature similar to other proteins involved in signal transduction through protein-protein interaction. Northern analysis showed that the PF6.2 mRNA was detectable in the fungus 6h after interaction and continued to be expressed in established ectomycorrhizas, suggesting that it plays an important role in the formation and maintenance of the symbiosis.