With Ponceau S (0.1 in 5 acetic acid). Alternatively, unspecific bands reacting using the antisera employed are marked for demonstration of equal gel loading.YEAST TWO-HYBRID AND THREE-HYBRID ASSAYSYeast two-hybrid and yeast three-hybrid analyses in absence and presence of SA have been conducted as reported earlier (Weigel et al., 2001; Maier et al., 2011). LacZ reporter gene activities are given in Miller units. Most plasmids applied in the protein?protein interaction assays have been described (Weigel et al., 2001). pGAD10/NIMIN1 35/142, pGAD10/NIMIN2 20/122, and pGAD10/NIMIN3 13/112 encode NIMIN proteins truncated at their N-terminus. The plasmids were isolated within a Y2H screen with all the At NPR1 bait (Weigel et al., 2001).ACKNOWLEDGMENTSWe would prefer to thank Sylvia Zwicker for communication of Y2H data, Xinnian Dong, Duke University, USA, and Jane Glazebrook, University of Minnesota, USA, for npr1 mutant seeds, Jim Haseloff, University of Cambridge, England, for the generous gift of pBIN 35S-mGFP4, and Klaus Harter, Universit T ingen, Germany, for providing the Agrobacterium strain with the p19 silencing suppressor. Thanks also to David Neeley for tips around the English language. Ashir Masroor was supported by a fellowship from Katholischer Akademischer Ausl der-Dienst (KAAD).M., et al. (1996). A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant J. ten, 61?0. Fu, Z. Q., Yan, S., Saleh, A., Wang, W., Ruble, J., Oka, N., et al. (2012). NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486, 228?32. Glazebrook, J., Rogers, E. E., and Ausubel, F. M. (1996). Isolation of Arabidopsis mutants with enhanced illness susceptibility by direct screening. Genetics 143, 973?82. Glocova, I., Thor, K., Roth, B., Babbick, M., Pfitzner, A. J. P., and Pfitzner, U. M. (2005). Salicylic acid (SA)-dependent gene activation can be uncoupled from cell deathmediated gene activation: the SAinducible NIMIN-1 and NIMIN-2 promoters, as opposed to the PR-1a promoter, usually do not respond to cell death signals in tobacco.223556-14-7 Chemscene Mol.Formula of 364794-69-4 Plant Pathol.PMID:28322188 six, 299?14. Gr er, R., and Pfitzner, U. M. (1994). The upstream area in the gene for the pathogenesis-related protein 1a from tobacco responds to environmental also as to developmental signals in transgenic plants. Eur. J. Biochem. 220, 247?55. Gr er, R., Strompen, G., Pfitzner, A. J. P., and Pfitzner, U. M. (2003). Salicylic acid along with the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element in the PR-1a promoter. Eur. J. Biochem. 270, 4876?886. Haseloff, J., Siemering, K. R., Prasher, D. C., and Hodge, S. (1997). Removal of a cryptic intron and subcellular localization of green fluorescent protein are needed to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. U.S.A. 94, 2122?127. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989). Site-directed mutagenesis by overlap extension making use of the polymerase chain reaction. Gene 77, 51?9. Horvath, D. M., Huang, D. J., and Chua, N.-H. (1998). 4 classes of salicylate-induced tobacco genes. Mol. Plant Microbe Interact. 11, 895?05. Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in larger plants. EMBO J. 6, 3901?907. Jones, J. D. G., and Dangl, J. L. (2006). The plant immune technique. Nature 444, 323?29. Lawton, K. A., Friedrich, L., Hunt, M.,.