Identification of fungal genes involved in the preinfection events between ectomycorrhizal association (Pisolithus tinctorius and Pinus massoniana)

Abstract

The preinfection stage of establishment of the mycorrhiza before physical contact between symbionts is crucial, as changes that occur throughout mycorrhiza formation are set in motion at this time. To improve the understanding of the molecular mechanisms involved in the fungus Pisolithus tinctorius during its interaction with its symbiotic partner Pinus massoniana, we set up a cellophane-separated culture system avoiding physical contact between the symbionts. A cDNA subtraction library representing the differentially expressed genes of P.tinctorius during the preinfection stage was constructed using suppression subtractive hybridization combined with mirror orientation selection (SSH/MOS). Differential screening of the SSH library allowed us to identify 133 unique cDNA clones. Sequences analysis of the expressed sequence tags (ESTs) showed that they represent 23 differentially expressed genes, of which 10 are involved in metabolism and energy, transcription and protein fate, membrane proteins and signaling, and cell rescue and defense, and 13 encoding hypothetical proteins with unknown function. Some fungal genes have not been previously identified in other ectomycorrhizal associations. Expression of the genes that code for ubiquitin-conjugating enzyme E2, 40S ribosomal protein, maternal g10 transcript, ADP ribosylation factor 6, and cytochrome P450 were evaluated by RT-qPCR, confirming the activation of these genes during the preinfection stage.