Finally, it is worth noting that those non-identified transcripts that were detected in this study as up-regulated in T. harzianum by the presence of tomato plants (non-annotated sequences BTSA1 from additional file 3) are also an additional resource for future research on Trichoderma-plant interactions, especially those that did not respond significantly to other culture conditions assessed. Conclusion The Trichoderma HDO microarray presented here has enabled us to define a gene set probably involved
in the transcriptional response of the fungus T. harzianum CECT 2413 within the first hours of contact with tomato plant roots. Many of the genes identified had not been previously related to Trichoderma-plant interactions, including those respsonsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, micoparasitic activities, mycelium development, or those related to the formation of infection structures in plant tissues, which can provide
new insight into the mechanisms and roles of this fungus in the Trichoderma-plant interaction. The effectiveness of the Trichoderma HDO microarray in the detection of different gene responses in T. harzianum under different growth conditions strongly indicates that this tool should be useful for further assays addressing different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Methods Fungal and plant growth Cilengitide chemical structure conditions Trichoderma harzianum CECT 2413 (Spanish Type Culture Collection, Valencia, Spain) was grown on potato dextrose agar (Sigma, St. Louis, aminophylline Mo, USA) plates in the dark at 28°C for 10 days. Spores were collected and used as inoculum (107 spores as counted with a hemocytometer) for fungal pre-cultures in 250-ml Erlenmeyer
flasks containing 100 ml of liquid minimal medium [67] supplemented with 2% glucose as carbon source. Flasks were then maintained at 28°C and 150 rpm for 48 h. After this time, fungal biomass was harvested by filtration, washed twice with sterile distilled water, and immediately transferred to the definitive cultures (see below). Tomato seeds (Solanum lycopersicum, formerly Lycopersicon esculentum Mill. var. Manitu) from Ramiro Arnedo S.A. (Calahorra, La Rioja, Spain) were surface-sterilized by vigorous sequential shaking in 70% ethanol and 2% hypochlorite solution, for 5 min each, and then thoroughly washed with sterile distilled water and air-dried on a sterile gauze sheet. Seeds were germinated in multi-cell growing trays containing sterile soil selleck substrate covered with vermiculite in a controlled environment chamber with 75% humidity and a photoperiod of 16 h light at 23°C. Plants were then allowed to grow under these conditions for twelve weeks.