Detection of chitin synthase class I and II type sequences in six different arbuscular mycorrhizal fungi and gene expression in Glomus intraradices

The arbuscular mycorrhizal (AM) fungus Glomus intraradices was grown in vit ro with RIT-DNA transformed Daucus carota roots on plates divided into two compartments. One side of the plates contained only fungal hyphae and spore s while the other contained mycorrhizal roots. Using this technique, the li mitations of insufficient biological material available for molecular analy sis of this obligate symbiont were overcome. Fungal material from the first compartment was used as a pure source of G. intraradices for genomic DNA e xtraction. PCR amplification as well as southern hybridization were conduct ed using this DNA. Sufficient pure genomic DNA and mRNA was obtained to car ry out Southern analysis, achieve optimum PCR results with 25-35 cycles and conduct RT-PCR. Differential expression of chitin synthase class I and II was detected in G. intraradices. Using degenerate primers specific to funga l chitin synthase sequences, a single amplification product obtained after 25 cycles of PCR was cloned. Sequencing of this fragment revealed similarit y to other fungal chitin synthase genes. PCR with these primers and additio nal primers allowed the amplification of chitin synthase fragments from spo res of different isolates of G. intraradices as well as from G. mosseae, Gi gaspora margarita, Acaulaspora scrobiculata, Scutellospora calospora and En trophosphora colombiana. A total of 21 chitin synthase sequences from diffe rent species and isolates of various AM fungi were successfully amplified S equencing of these fragments permitted their classification into class I an d II of the chitin synthase groups.