Chlorimuron ethyl

For the fungal transformation, we used an AtMT protocol that was previously described [21] (link) with slight modifications. The hygromycin-resistant transformants were selected on the PDA medium containing 100 µg/ml of hygromycin B (Wako Chemicals, Osaka, Japan), 50 µg/ml of cefotaxim (Wako Chemicals, Osaka, Japan), and 50 µg/ml of spectinomycin (Wako Chemicals, Osaka, Japan). The bialaphos-resistant transformants were selected on an SD medium containing 10 µg/mL of bialaphos (Meiji Seika Kaisha, Ltd., Tokyo, Japan), 100 µg/ml of cefotaxim, and 100 µg/ml of spectinomycin. The sulfonylurea-resistant transformants were selected on an SD medium containing 4 µg/ml of chlorimuron-ethyl (Chem Service West Chester, PA, USA.), 100 µg/ml of cefotaxim, and 100 µg/ml of spectinomycin.

The wild-type strain (EV-MIL31) of A. alternata was cultured from diseased leaves of Minneola tangelo and has been previously characterized [14 (link),18 (link)]. Fungal strains defective for a Yap1 regulator (Δyap1), a Skn7 response regulator (Δskn7), a high osmolarity glycerol (Δhog1) mitogen-activated protein kinase (MAPK), a cell wall integrity MAPK (Δslt2), a Fus3 MAPK (Δfus3), and a “two component” histidine kinase (Δhsk1) were generated from the EV-MIL31 strain in previous studies [11 (link),12 (link),15 (link),38 (link),39 (link)]. YCp1 strain was previously created by expressing a functional copy of Yap1 in a Δyap1 mutant [11 (link)]. Fungal strains were cultured on potato dextrose agar (PDA; Difco, Sparks, MD) at 28°C with constant fluorescent light. For DNA and RNA purification, Alternaria strains were cultured on PDA covered with a sterile cellophane membrane. For protoplast isolation, fungi were cultured in potato dextrose broth (PDB) on a shaker for 3 to 4 days. Fungal transformants were recovered from a regeneration medium [40 ] amended with hygromycin (250 μg/ml, Roche Applied Science, Indianapolis, IN) or sulfonylurea (5 μg/ml, chlorimuron ethyl; Chem Service, West Chester, PA).