In this study, we addressed
the roles of areA in virulence, secondary metabolism, vegetative growth, and sexual development. A functional study of areA can increase our understanding of the relationships between nitrogen metabolism and fungal development in Ivacaftor G. zeae. The wild-type strain GZ3639 of G. zeae (Bowden & Leslie, 1999) and transgenic strains derived from this strain were used in this study (Table 1). All strains were stored as mycelia and conidia in a 20% glycerol solution at −70 °C. Standard laboratory methods and culture media for the Fusarium species were used (Leslie & Summerell, 2006). The growth rate of wild-type and transgenic strains was measured in complete medium (CM) and minimal medium (MM) (Leslie & Summerell, 2006) supplemented with 20 mM sodium nitrate, urea, ammonium tartrate, or l-glutamine as the sole nitrogen source. Fungal genomic DNA was isolated from freeze-dried mycelia powder as previously described (Leslie & Summerell, 2006). Standard procedures were used for agarose gel electrophoresis, restriction endonuclease digestion, and Southern blot analysis using 32P-labeled probes (Sambrook & Russell, 2001). Primers used in this study were synthesized by an oligonucleotide synthesis facility (Bionics, Seoul, Korea; Supporting
Information, Table S1). General PCR was performed following the manufacturer’s instructions (Takara Bio Inc., Otsu, Japan). DNA cassettes for targeted gene deletion and complementation were constructed using a slightly modified double-joint (DJ) PCR procedure (Yu et al., 2004). For deletion PARP activity of areA, geneticin resistance gene cassette (gen) was amplified from pII99 (Namiki et al., 2001). The 5′ and 3′ regions of the target gene were amplified and the three amplicons (5′ region, gen, and 3′ region) were fused by a second round of PCR. The fusion constructs were amplified with nested primers to generate split markers (Son et al., 2011a ,b). To complement the deletion mutant, the areA gene region including the 5′ region and open reading frame (ORF) was amplified and fused with hygromycin resistance cassette (hyg) from pBCATPH (Gritz & Davies, 1983), generating
Epothilone B (EPO906, Patupilone) areA-hyg construct. The GFP-areA-hyg construct was generated to visualize the localization and expression level of AreA. The 5′ flanking region of areA and ORF of green fluorescent protein (GFP) was fused with the areA-hyg construct. Fungal transformation was performed as previously described (Kim et al., 2005a,b). The virulence of G. zeae strains was determined using a susceptible wheat cultivar, Eunpamil, as previously described (Lee et al., ,b). A 10-μL aliquot of conidial suspension (1 × 105 conidia mL−1) was injected into a center spikelet of the wheat head at midanthesis. The inoculated plants were incubated in a humidity chamber for 3 days and then transferred to a greenhouse. At 14 days’ post-inoculation, the spikelets with head blight symptoms were counted. Cultures were grown on carrot agar plates for 5 days.