Quantitative analysis was performed LDK378 molecular weight using the GeneAmp®7000 Sequence Detection System (PE Applied Biosystems) with PCR conditions of 95 °C for 15 s and 60 °C for 1 min for 40 cycles. Three independent experiments were carried out. Each sample was examined in triplicate, using relative quantification analysis. The plasmid pSilent1 (Nakayashiki et al., 2005) was obtained from the Fungal Genetics Stock Center (McCluskey, 2003). A 340-bp fragment

from the Tas-acdS encoding region was cloned into pSilent1 in sense and reverse/complementary orientations on both sides (XhoI/HindIII sites and StuI/ApaI sites) of the 147-bp intron 2 of the cutinase gene from Microdochium oryzae driven by the PtrpC promoter. The coding region, in the sense orientation, was amplified by PCR with the primers ACCXhoI (5′-CCGCTCGAGCACAAGCCCACGCTGGCAAACC-3′) and ACCHindIII (5′-CCAAGCTTTGGCAGCAGTGAATTTAGC-3′). The coding region in the

antisense orientation was amplified by PCR with the primers ACCApaI (5′-AAAGGGCCCCACAAGCCCACGCTGGCAAACC-3′) and ACCStuI (5′-AAGGCCTTGGCAGCAGTGAATTTAGC-3′). Microprojectile bombardment of intact T. asperellum T203 conidia with the pSilent1-Tas-acdS/RNAi plasmid was performed as described in Viterbo et al.(2002). Silencing of Tas-acdS in ACC-induced cultures was analyzed by comparing the relative gene expression of Tas-acdS/RNAi XL765 order lines to the wild type by real-time RT-PCR using the same primer sets as described above. Intron-free cDNA was obtained from total RNA extracted from T. asperellum cultures grown in the presence of ACC (3 mM) as the sole nitrogen source. The coding region was amplified by PCR (5′-ATGGCTACCCTCAACATCC-3′, 5′-TCAGTCTAAAAGAGAGGAATACGC-3′), Unoprostone subcloned in pGEM-T Easy vector (Promega) and cloned in the pALTER-EX1 (Promega) vector in NdeI/NcoI sites under the control of the tac promoter. The hybrid plasmid was then transformed into JM109 cells and ACCD activity

was tested as described in the next section. For ACCD activity determination in recombinant E. coli and Pseudomonas putida UW4, bacteria were grown as described in Penrose & Glick (2003). For determination of ACCD activity in Trichoderma, a 20-μL spore suspension was inoculated in 10-mL synthetic medium (SM; Yedidia et al., 1999) and the culture was grown for 48 h. The washed mycelia were then transferred to 5 mL of SM without ammonium and with 0.3–3 mM ACC. At the end of the induction period, the cultures were resuspended in half volume of Tris buffer 0.1 M (pH 8.5) and homogenized using an ULTRA-TURRAX apparatus (Janke & Kunkel, Staufen, Germany). Toluene (25 μL) was added to a 200-μL aliquot and vortexed vigorously for 30 s. ACC (20 μL of 0.5 M solution) was added, and after an incubation period of 15 min at 30 °C, 1 mL of 0.56 N HCl was added. The lysates were centrifuged (10 000 g, 10 min) and 1 mL of the supernatant was mixed with 800 μL of 0.