We further analyzed the function of TaWAK5 in wheat defense responses to R. cerealis using virus-induced gene silencing (VIGS) technique. Six wheat (T. aestivum L.) lines/cultivars exhibiting different levels of resistance buy BKM120 and susceptibility to R. cerealis

were used in this study. They included CI12633 and Shanhongmai (resistant to R. cerealis); Navit 14, and Shannong 0431 (moderately resistant to R. cerealis); Wenmai 6 (susceptible to R. cerealis); and Yangmai 158 (moderately susceptible to R. cerealis) [28]. A major Jiangsu virulent isolate strain of pathogen fungus R. cerealis causing the sharp eyespot disease, R0301, was provided by Profs. Huaigu Chen and Shibin Cai from Jiangsu Academy of Agricultural Sciences, China. Wheat plants were grown in a 14 h light/10 h dark (22 °C/10 °C) regime. At the tillering stage, the 2nd base sheath of each wheat plant was inoculated with small toothpick fragments harboring well-developed learn more mycelia of the pathogen R. cerealis following Chen [27]. Mock treatment (control) plants were inoculated with small toothpick fragments soaked in liquid potato dextrose agar (PDA). Inoculated plants were grown at 90% relative humidity for 4 days. The inoculated stems were sampled at 0, 4, 7, 10, 14, and 21 days post inoculation,

quickly frozen in liquid nitrogen, and stored at − 80 °C prior to total RNA extraction. At 4 dpi, the roots, sheaths, stems, and leaves of the inoculated CI12633 plants were collected, respectively. At 45 dpi, the

roots, stems, leaves, and young either spikes of the inoculated CI12633 plants were separately sampled and used for RNA extraction and the tissue expression profiles of TaWAK5. In additional experiments, the seedlings at the three-leaf stage of the resistant line CI12633 were treated with phyto-hormones, including 1.0 mmol L− 1 SA, 0.1 mmol L− 1 MeJA (JA analog), ethylene released from 0.2 mmol L− 1 ethephon, and 0.2 mmol L− 1ABA, following Zhang et al. [29]. Leaves were collected for RNA extraction at 0, 1, 3, 6, 12, and 24 h after treatment with these hormones. Total RNA was extracted using TRIzol reagent (Qiagen, China) according to the manufacturer’s instructions. DNase I treatment was used to remove genomic DNA. First-strand cDNA was synthesized using 2 μg purified RNA, AMV reverse transcriptase, and oligo (dT15) primers (TaKaRa Inc., Tokyo, Japan) according to the manufacturer’s instructions for the cDNA synthesis kit. Based on microarray analysis results, a partial cDNA fragment (GenBank accession number CA642360), which was differentially expressed between the resistant wheat genotype CI12633 and the susceptible wheat Wenmai 6, was identified. Based on the sequence of CA642360 and using a 3′-Full RACE Core Set kit v.2.0 from TaKaRa Inc., the sequence of the 3′ untranslated region (UTR) was amplified from cDNA of CI12633 stems that had been challenged with the pathogen R. cerealis for 21 days.