GrainGenes

Mapping and sequencing of the barley putative hypersensitive induced reaction genes

Nils Rostoks¹, David Kudrna¹ and Andris Kleinhofs^1,2

¹ Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164

² School of Molecular Biosciences, Washington State University, Pullman, WA 99164

Hard-copy edition pages 34 - 37.

Abstract

Hypersensitive induced reaction (HIR) genes encode proteins involved in plant hypersensitive response (HR) against infection. HR is one of the most efficient forms of the plant defense against a pathogen and results in a localized cell death and formation of necrotic lesions. Three maize HIR genes have been identified and their products were shown to belong to a protein superfamily involved in regulation of cell cycle, ion channels and cell death (Nadimpalli et al. 2000). Tobacco cDNA NG1 (GenBank accession U66271) which is homologous to HIR genes has been shown to cause formation of HR-like lesions and to induce a pathogenesis-related acidic beta-glucanase in transient assays in tobacco (Karrer et al. 1998). We screened for the barley (Hordeum vulgare L.) cDNAs homologous to maize HIR genes, positioned them on the barley genetic map and isolated corresponding genomic DNA sequences. We detected four barley putative HIR genes (Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4) in four distinct chromosomal regions. They did not co-segregate with previously mapped barley morphological "necrotic" (nec) mutant loci (reviewed by Lundqvist et al. 1997). Analysis of the genes Hv-hir1, Hv-hir2 and Hv-hir3 revealed high homology at both DNA and deduced amino acid level. Hv-hir4 gene, however, had little DNA homology to other genes, although it exhibited significant conservation to the others at the amino acid sequence level.

Isolation of the cDNAS for the barley putative HIR genes

mRNA sequences of the maize HIR genes (Zm-hir1 (AF236373), Zm-hir2 (AF2363743), Zm-hir3 (AF236375); Nadimpalli et al. 2000) were used in BLASTN search to screen barley EST database at http://www.ncbi.nlm.nih.gov/BLAST/. EST sequences with BLASTN score higher than 100 were identified, aligned using Clustal_X 1.8 multiple sequence alignment program (Thompson et al. 1997) and divided by manual inspection into three groups: Hv-hir1, Hv-hir2 and Hv-hir3. This division was supported by EST clustering done by Dr. Close and Mr. Wanamaker (HarvEST 0.73; http://harvest.ucr.edu/). The most complete cDNA clone for each group was isolated from the Clemson University Genomics Institute (CUGI) EST libraries (http://www.genome.clemson.edu/projects/barley/) and complete sequence of the cDNA was obtained.

cDNA sequences from each group were used in the second search of the barley EST database at the NCBI using the TBLASTX procedure. This yielded the fourth group of barley putative HIR genes, Hv-hir4, with high amino acid homology, but little DNA homology to the previous three groups.

Isolation of the genomic clones for the barley putative HIR genes

Representative cDNAs of Hv-hir1, Hv-hir2 and Hv-hir3 genes were used as probes to screen the barley cv. Morex 6.3x genome coverage BAC clone library by Southern hybridization. BAC clones for each cDNA were recovered and reconfirmed. One BAC clone for each cDNA probe was subcloned in the plasmid vector pBluescript II KS+. Plasmid subclones hybridizing with the appropriate cDNA probe were isolated and sequenced yielding the genomic sequence covering the full length of the gene. Genomic sequence of the Hv-hir4 gene was obtained from a PCR product amplified from the Morex genomic DNA using primers designed from the cDNA sequence.

Genetic mapping of the barley putative HIR genes

cDNAs for Hv-hir1 and Hv-hir2 genes were mapped genetically in the Steptoe x Morex "minimapper" population consisting of 35 doubled haploid lines (DHL) selected from the original 150 DHL of the NABGMP cross. "Minimapper" population allows positioning of the marker within approximately 10 cM chromosome bin (Kleinhofs and Graner 2001). cDNA for Hv-hir3 gene was mapped in Oregon Wolf Barley Dominant x Recessive 94 DHL population (Costa el al. 2001). cDNA for Hv-hir4 gene was mapped in Harrington x Morex "minimapper" population consisting of 35 DHL selected from the original 140 DHL (Hayes et al. 1997).

Since different mapping populations were used, the genetic map location of the barley putative HIR genes could only be determined to a chromosome bin. Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4 genes mapped to the chromosomes 4(4H) bin10, 7(5H) bin07, 7(5H) bin04 and 1(7H) bin03, respectively (Fig. 1). None of the four barley Hv-hir genes mapped in the vicinity of the previously mapped necrotic (nec) barley mutations (reviewed by Lundqvist et al. 1997).

Sequence analysis of the barley putative HIR genes

Conceptual translation of the cDNAs for the Hv-hir1, Hv-hir2 and Hv-hir4 genes, isolated from the CUGI EST libraries suggested that they contain full-length coding sequences because the predicted amino acid sequences aligned with the maize HIR gene products over their entire length. In addition, complete cDNA sequence was obtained for the Hv-hir2 group by the 5'-RACE technique and it did not extend the coding part of the cDNA in the 5' direction. The only barley cDNA for the Hv-hir3 group found in the CUGI EST libraries was 5'-truncated. The full length coding sequence was reconstructed using a combination of FGENESH gene prediction program (http://www.softberry.com/) and alignment with cDNAs from the other barley HIR groups.

A pairwise similarity matrix of the deduced amino acid sequences of the barley and maize HIR genes is given in the Table 1. Deduced amino acid sequences of the Hv-hir1, Hv-hir2 and Hv-hir3 genes were highly homologous among themselves and to the sequences of maize HIR genes (>90% amino acid sequence similarity). Hv-hir1, Hv-hir2 and Hv-hir3 genes were also highly homologous at the DNA sequence level (data not shown). In contrast, Hv-hir4 gene had little homology to other barley and maize HIR genes at the DNA level, although there was a notable conservation at the amino acid level. Comparison of the Hv-hir4 gene to the maize EST database identified several EST sequences with very low E value (<10^-80) indicating that maize has a transcribed homologue of the barley Hv-hir4 gene.

Table 1 Pairwise similarity matrix of the deduced amino acid sequences of the barley and maize HIR genes. The values in % (%) indicate sequence identity and similarity, respectively. They were obtained from pairwise BLASTP alignment at the NCBI BLAST Web page run at default setting with low-complexity filter "off".

Figure 1 Chromosome locations of the barley putative hypersensitive induced reaction genes (Hv-hir) on the barley consensus bin map.

References

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