ORIGINAL PAPER Nils Rostoks Deric Schmierer Sharon Mudie Thomas Drader Robert Brueggeman David G. Caldwell Robbie Waugh Andris Kleinhofs Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1 Received: 8 September 2005 / Accepted: 2 November 2005 / Published online: 9 December 2005 Ó Springer-Verlag 2005 Abstract Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic lo- cus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive dele- tions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and b-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Keywords NEC1 HLM1 Cyclic nucleotide-gated ion channel Necrotic mutant Disease lesion mimic mutant Introduction Plant lesion mimic or necrotic mutants are a broad category of morphological mutants that display a dis- ease lesion phenotype in the absence of a pathogen. A large number of dominant and recessive mutants have been identified in maize (Johal et al. 1995) and Arabid- opsis (Lorrain et al. 2003). Morphological studies iden- tified similarities in the mutant phenotypes with the hypersensitive response (HR), a form of programmed cell death (PCD). The increased resistance of some necrotic mutants to pathogens, as well as constitutive expression of some defense molecules, e.g., pathogene- sis-related (PR) proteins, collaborated the hypothesis that necrotic mutants may help to identify the compo- nents of plant defense pathways (Johal et al. 1995; Lorrain et al. 2003). The availability of genomic sequence and functional genomics tools, such as knock- out and mutant collections, and highly efficient transformation procedures enabled cloning and func- tional studies of many necrotic mutants in Arabidopsis (Lorrain et al. 2003). The emerging picture of the rela- tionship among the mutants enabled visualization of the pathways leading to PCD; however, a clear picture of the interactions between the genes and gene products in pathways is still missing. Recently, a necrotic mutant gene hlm1 (HR-like lesion mimic) was cloned from Arabidopsis encoding a cyclic nucleotide-gated ion channel (CNGC) 4 (Balague et al. 2003). A different member of the Arabidopsis CNGC family, a CNGC2, is encoded by dnd1 (defense, no death) gene which failed to produce HR in response to avirulent pathogens, but could exhibit a rare, condi- tional lesion phenotype (Clough et al. 2000). Both genes have been implicated in the regulation of PCD (Kohler et al. 2001; Lorrain et al. 2003), although the molecular Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00438-005- 0073-9 and is accessible for authorized users. Communicated by R. Hagemann N. Rostoks (&) S. Mudie D. G. Caldwell R. Waugh Scottish Crop Research Institute, Genome Dynamics, Invergowrie, DD2 5DA, Dundee, Scotland, UK E-mail: nils.rostoks@scri.ac.uk Tel.: +44-1382-562731 Fax: +44-1382-568587 D. Schmierer T. Drader R. Brueggeman A. Kleinhofs Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA A. Kleinhofs School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA Mol Gen Genomics (2006) 275: 159–168 DOI 10.1007/s00438-005-0073-9