Elsevier

Gene

Volume 407, Issues 1–2, 15 January 2008, Pages 193-198
Gene

Cloning of a putative hypersensitive induced reaction gene from wheat infected by stripe rust fungus

https://doi.org/10.1016/j.gene.2007.10.010Get rights and content

Abstract

The hypersensitive response (HR) is one of the most efficient forms of plant defense against biotrophic pathogens and results in localized cell death and the formation of necrotic lesions. In this study, a novel putative hypersensitive induced reaction (HIR) gene from wheat leaves infected by incompatible stripe rust pathogen CY23, designated as Ta-hir1, was identified by using rapid amplification of cDNA ends (RACE). Ta-hir1 encodes 284 amino acids, with a predicted molecular mass of 31.31 KDa. A phylogenetic analysis showed that Ta-hir1 was highly homologous to Hv-hir1 from barley at both cDNA and deduced amino-acid levels. Amino-acid sequence analysis of the wheat HIR protein indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain typical for stomatins which served as a negative regulator of univalent cation permeability, especially for potassium. The expression profile of the Ta-hir1 transcript detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (real time-PCR), respectively, showed that the highest expression occurred 48 h post inoculation (hpi), which is consistent with our previous histopathology observations during the stripe rust fungus–wheat incompatible reaction.

Introduction

Hypersensitive response (HR), one of the most efficient forms of plant defense against biotrophic pathogens, is usually defined as ‘the rapid death of plant cells in association with the restriction of pathogen growth’ (Goodman and Novacky, 1994). It occurs in resistant plant cultivars in response to incompatible viruses, fungi, bacteria (Lam et al., 2001, Vleeshouwers et al., 2000, Baker et al., 1993), and causes a series of biochemical processes that result in cell death in the adjacent living cells thus causing an adverse environment for the pathogens. Elicitors of HR cause intracellular ion influx, membrane dysfunction, and increase in the generation of reactive oxygen species and salicylic acid (Heath, 1998, Heath, 2000). A cascade of events triggered within the signaling pathway results from the interaction of ligands originating from pathogens with intra or extra-cellular plant receptors in an incompatible reaction which will further lead to modifications of plant cell walls, accumulation of pathogenesis-related (PR) proteins or phytoalexins, and accumulation of oxidized phenolic compounds that can cause browning of the dead cells. In addition, the HR typically induces systemic changes throughout the plant, including induced resistance to a variety of previously compatible pathogens (Kombrink and Sossich, 1995).

Karrer et al. (1998) used a functional screening method to isolate several genes whose products elicited the HR in tobacco challenged by the tobacco mosaic virus. One of the isolated cDNAs, NG1, was able to induce the formation of lesions and expression of PR-2 protein, an acid β-glucanase. Three maize hypersensitive induced reaction (HIR) genes, Zm-hir1, Zm-hir2 and Zm-hir3, have been isolated based on their amino-acid homology to the tobacco NG1 sequence (Nadimpalli et al., 2000). Studies on the regulation of the Zm-hir3 transcript indicated higher level of expression of this gene in the maize disease lesion mimic mutant Les9 when compared to the wild type, and reduced expression in maize line which suppressed the Les9 lesion mimic phenotype. Many other data also suggested participation of the maize HIR genes in HR. In addition to three Zm-hir genes, Nadimpalli et al. (2000) also isolated and identified four genes closely related to prohibitins (Zm-phb1, Zm-phb2, Zm-phb3, and Zm-phb4) and one to stomatins (Zm-stm1). They further used PHI-BLAST searches combined with multiple sequence alignments and neighbor-joining tree construction to show that maize HIR proteins, along with prohibitins and stomatins, formed the superfamily PID (proliferation, ion, and death). Prohibitins are involved in proliferation and cell cycle control, stomatins in ion channel regulation, and HIR genes are involved in cell death. Although members of this superfamily are involved in diverse functions, their structural similarity suggested a conserved molecular mechanism, which Nadimpalli et al. (2000) postulated to be involved in ion channel regulation.

Recently, four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hv-hir3, Hv-hir4 were identified (Rostoks et al., 2003). Sequence analyses of the barley HIR proteins indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain (Tavernarakis et al., 1999). Barley HIR genes were expressed in all organs and development stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression thus implicating HIR genes in the induction of HR (Rostoks et al., 2003).

In a previous study, we isolated from an incompatible suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis, a cDNA fragment, WSRP1878, homologous to the barley Hv-hir1 gene (Yu et al., 2007). In the present study, we cloned a full length wheat HIR gene using rapid amplification of cDNA ends (RACE). Based on the sequence of the WSRP1878 fragment and designated it as Ta-hir1. The characteristics of Ta-hir1 cDNA and deduced amino-acid sequences were identified by a series of bioinformatic softwares. In addition, the expression profile of Ta-hir1 at different post-inoculation time points was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and real time-polymerase chain reaction (real time-PCR).

Section snippets

Plant materials and inoculation

Wheat (Triticum aestivum L.) cultivar Suwon11 and stripe rust race CY23 were the biological materials used for the full length cloning of the wheat HIR1 gene and its expression analysis. Suwon11 was presumed to contain a stripe rust resistant gene YrSu (Cao et al., 2003, Li et al., 2006). Plants were grown and maintained as described in Kang and Li (1984). Freshly collected urediospores were applied with a paintbrush to the surface of the primary leaf of seven-day old wheat seedlings. After

Characterization of Ta-hir1 cDNA

Following 3′ and 5′ RACE analyses, two DNA fragments around 600 and 1000 bp, respectively, were detected on 1% agarose gel. Following plasmid construction, transformation and cDNA clones sequencing, 501 bp for 3′RACE and 944 bp for 5′RACE fragments were obtained by removing vector and adapter sequences. Furthermore, CAP3 software was used to combine the two fragments into an 1132 bp consensus sequence including a poly-A signal region in the 3′-UTR. BLAST analysis showed that the 1132 bp

Discussion

In the present study, we initially identified a gene fragment isolated from an incompatible SSH library of wheat leaves infected by the stripe rust fungus which showed a high similarity to the barley Hv-hir1 gene. In order to understand whether it would be involved in HR of wheat, a full-length cDNA sequence of 1132 bp with a complete ORF was isolated from infected wheat leaves using the RACE technique. We designated it as Ta-hir1 because many characteristics of this gene predicted by different

Acknowledgements

This study was supported financially by the National Basic Research Program of China (No. 2006CB101901), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (NO.2005295), the Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education of China (No.200558), the Nature Science Foundation of China (No.30671350), and the 111 Project from Ministry of Education of China (B07049).

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    Present address: College of Life Science, Agriculture University of Hebei, Baoding, Hebei, 071001, PR China.

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