Expression profiling of in vitro Poplar roots during early interaction with the ectomycorrhizal fungus Laccaria bicolor

The early phase of the interaction between tree roots and ectomycorrhizal (ECM) fungi, prior to symbiosis establishment, is accompanied by a stimulation of lateral root (LR) development. We set out to identify gene networks that regulate LR development during the early signal exchanges between Populus tremula x Populus alba (hereafter called poplar) and the ECM fungus Laccaria bicolor. A sandwich culture system was developed in order to bring plant and fungus into an indirect contact, which permits signal molecule exchange and LR stimulation (emergence after 4-5 days of contact) but prohibits root colonization. A NimbleGen full genome poplar oligo-array was used to investigate transcript profiles at three days of indirect poplar/L. bicolor contact, referring to the time point of LR initiation in response to the fungus. The Populus whole-genome expression array version 2.0 (S. DiFazio, A. Brunner, P. Dharmawardhana, and K. Munn, unpublished data) manufactured by NimbleGen Systems Limited (Madison, WI) contains in duplicates three independent, non-identical, 60-mer probes per whole gene model plus control probes and labeling controls. Included in the microarray are 65,965 probe sets corresponding to 55,970 gene models predicted on the P. trichocarpa genome sequence version 1.0 and 9,995 aspen cDNA sequences (Populus tremula, Populus tremuloides, and P. tremula x P. tremuloides). NimbleGen whole genome microarray analyses were performed in triplicate as per manufacturers instructions. We carried out six hybridizations (NimbleGen) with samples derived from each six pooled, entire in vitro grown root systems on MES-buffered medium. Three samples (biological replicates) originated from control roots in the absence of L. bicolor (GSM415421, GSM417830, GSM417831) and another serie of three biological replicates consisted of roots from a three days indirect contact with L. bicolor (GSM417832, GSM417833, GSM417856). cDNA was synthesized using CLONTECH Smart cDNA Synthesis kit containing an amplification step on the cDNA level. All samples were labeled with Cy3.