Isolation and analysis of a symbiosis-regulated and Ras-interacting vesicular assembly protein gene from the ectomycorrhizal fungus Laccaria bicolor

A yeast two-hybrid library prepared from Laccaria bicolor x Pinus resinosa mycorrhizas was screened using a LbRAS clone, previously characterized, as a bait to isolate LbRAS interacting signaling-related genes from L. bicolor. Using this method, a novel line of Ras-interacting yeast two-hybrid mycorrhizal (Rythm) clones were isolated and analysed for their symbiosis-regulation. One such clone identified (RythmA) had homology to Ap180-like vesicular proteins. Sequence homology and parsimony-based phylogenetic analysis showed its relatedness to Ap180-like proteins from other systems. DNA analysis suggested that L. bicolor had one or two copies of the RythmA gene. An RNA analysis showed that the expression of RythmA could be detected 36 h after interaction with the host, which follows the expression of Lbras. Immunolocalization of LbRAS near dolipore septum of the fungal cells in the Hartig net area suggests that RythmA protein may be involved in the transport of signaling proteins such as LbRAS.