, 1984). In this study, we identified three PDCs from the G. zeae genome and performed functional analyses of the genes. Although the PDC2 and PDC3 deletions had no observable defect, PDC1 deletion mutants exhibited defected perithecia maturation. These defects in perithecia Afatinib datasheet maturation could be attributed to the reduced production of lipids in the aerial mycelia and reduced growth of embedded mycelia. These results, taken together with results from our previous study on ACSs, suggest that the PAA pathway plays a crucial role in the production of lipids in the aerial mycelia and growth of embedded mycelia in G. zeae. All strains used in this study are listed in Supporting information, Table S1. Minimal

medium containing 5 mM agmatine (MMA) was used to induce the production of trichothecenes (Gardiner et al., 2009). Conidia were induced using carboxymethyl cellulose (CMC) medium (Cappellini & Peterson, 1965). Standard laboratory methods and culture conditions for Fusarium

species were used (Leslie & Summerell, 2006). Fungal transformations were conducted as previously described (Han et al., 2007). Genomic Ibrutinib DNA extraction and Southern analysis using 32P-labeled probes were performed using standard protocols (Sambrook & Russell, 2001; Leslie & Summerell, 2006). PCR primers used in this study were synthesized by an oligonucleotide synthesis facility (Bionics, Seoul, Korea; Table S2). Constructs for gene deletion and complementation assays were generated using the double-joint (DJ) PCR method (Yu et al., 2004). Geneticin resistance cassettes (gen) were amplified with Gen-F/Gen-R primers and fused to the 5′ and 3′ flanking region of the genes targeted for deletion and amplified with the appropriate Etofibrate primer pairs (Table S2). To generate the complementing construct for the PDC1 deletion mutant, the promoter and open reading frame (ORF) of the PDC1 gene were fused to GFP-hyg amplified using pIGPAPA-sGFP F/HYG-F1 primers from the pIGPAPA vector (Horwitz et al., 1999) and 3′ flanking region of PDC1 gene. To generate the strain containing both the PDC1 deletion and the ACS1-GFP fusion (HK60),

the ∆mat2 mutant was outcrossed with the ∆pdc1 strain, and progeny from this cross (∆mat2 ∆pdc1, strain HK59) were then cross-fertilized with the strain containing the ACS1-GFP fusion (HK23). The strain containing both the ACS1 deletion and the PDC1-GFP fusion (HK65) was generated by outcrossing (∆mat1; PDC1-GFP) × HK22 (∆acs1), and the ∆pdc1 ∆acs1 strain (HK61) was created by outcrossing HK59 × HK22. For every cross, progeny with the desired genetic characteristics were selected using antibiotic resistance, and genotypes were verified by PCR (Table S2). For self-fertilization, mycelia grown on carrot agar for 5 days were removed with a glass spreader or with the back of the surgical blade (surgical blade #11; Feather Safety Razor, Osaka, Japan) with 2.