We then carried out a multiple alignment of EGF-like repeats of TaWAK5 and WAKs from wheat, barley, rice, and Arabidopsis, in which each EGF-like repeat contained six conserved cysteine residues ( Fig. 3-B). The positions of the six cysteine residues are conserved in TaWAK5 and the other tested WAKs, although

the amino acid sequences between the cysteine residues varied. To study the subcellular Selumetinib datasheet localization of TaWAK5, the p35S:TaWAK5-GFP and p35S:GFP constructs were separately introduced into onion epidermal cells. As presented in Fig. 4-A, the TaWAK5-GFP fusion proteins were localized on the cell periphery, whereas the fluorescence of GFP alone as a control was distributed throughout the cell. To verify the nature of the subcellular localization

of TaWAK5, a plasmolysis experiment was performed. When onion cells expressing TaWAK5-GFP were plasmolyzed in a 0.8 mol L− 1 sucrose solution, TaWAK5-directed GFP fluorescence signal was observed on the plasma membrane ( Fig. 4-B). Thus, TaWAK5 may be a plasma membrane-localized protein. To determine if TaWAK5 was responsive to various phyto-hormone (SA, ABA, ethylene, or MeJA) treatments, we used qRT-PCR to monitor the transcriptional patterns of TaWAK5 in wheat following treatment for 0, 1, 3, 6, 12, and 24 h with exogenous SA, ABA, ethylene or MeJA. As shown in learn more Fig. 5, the expression of TaWAK5 was significantly induced by SA, ABA, or MeJA treatment. The greatest induction effect was observed with the SA treatment. Upon SA treatment, the expression of TaWAK5 was induced at 1–12 h post-treatment (hpt), reached a peak at 6 hpt (about 33-fold over that of 0 hpt), and Methane monooxygenase then decreased

to a normal level by 24 hpt ( Fig. 5-A). The expression pattern of TaWAK5 after treatment with ABA was similar to that induced by SA; the induction reached a peak (about 17-fold over that of 0 hpt) at 6 hpt ( Fig. 5-B). Upon MeJA treatment, the transcript of TaWAK5 was induced from 1 to 24 hpt, and peaked at 12 hpt (more than 11-fold over that of 0 hpt) ( Fig. 5-C). Upon ethylene treatment, the transcriptional level of TaWAK5 decreased from 1 to 24 hpt ( Fig. 5-D). These results suggested that TaWAK5 may be responsive to the SA, ABA, and MeJA signals. Transcriptional regulation is important in mediating the responses of plants to external stimuli. To study which stimuli TaWAK5 may respond to, we analyzed cis-acting elements in the TaWAK5 promoter region using the PLACE database. Many important transcriptional motifs were identified in the promoter of TaWAK5, including a TATA box (at position 956), basal transcription, transcription factor binding site, hormone (ABA, SA, gibberellins, cytokinins, and auxin) responsiveness sites, and sites for responsiveness to elicitors and other processes ( Table S3). To investigate whether TaWAK5 plays a critical role in wheat resistance response to R.