Characterization of an NBS-LRR resistance gene homologue from soybean

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Abstract

Conserved motifs such as the nucleotide-binding site (NBS) were found in many characterized plant disease resistance genes. Based on the NBS domain, resistance gene analogs have been isolated in our previous study and were used as probes to screen a soybean (Glycine max) cDNA library. A full-length cDNA, KR4, was isolated by screening the library and rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA was 3818 bp in length and the open reading frame coded for a polypeptide of 1211 amino acids with an NBS and five leucine-rich repeats domains, which were identified by Pfam protein analysis. Sequence alignment showed that KR4 was similar to I2 protein of tomato. Southern analysis indicated that the KR4 gene had low copies in soybean genome and it was mapped on the molecular linkage group E. Its expression was also investigated and it was found that KR4 was induced by exogenous salicylic acid and responded upon infection of soybean mosaic virus strain N3.

Introduction

To ward off the invading of microbial pathogens, nematodes and insects, plants have evolved elegant and complex mechanisms to protect themselves. One of the major mechanisms was characterized by a gene-for-gene interaction that required a specific plant resistance (R) gene and a cognate pathogen avirulence (Avr) gene (Flor, 1971). More than 30 R genes have been cloned from various plant species that provide resistance to a variety of different pathogen and pest species by map-based cloning or transposon-tagging strategies (Hulbert et al., 2001). Five main classes of R genes have been defined according to the structural characteristics of their predicted protein products (Dangl and Jones, 2001). The majority of functionally described R genes are the nucleotide-binding site (NBS)-leucine-rich repeat (LRR) type. The NBS domains are characteristic of various proteins with ATP/GTP binding activity and comprise the P-loop, kinase 2a, kinase 3a and GLPL motifs (Traut, 1994), while LRR domains play roles in the protein–protein interaction (Kobe and Deisenhofer, 1994).

Conserved motifs in NBS domain have been used to isolate resistance gene analogs (RGAs) from soybean (Kanazin et al., 1996; Yu et al., 1996; He et al., 2001; Peñuela et al., 2002), common bean (Creusot et al., 1999; Rivkin et al., 1999), Arabidopsis thaliana (Aarts et al., 1998; Speulman et al., 1998), potato (Leister et al., 1996), lettuce (Meyers et al., 1998), maize (Collins et al., 1998), and cereals (Leister et al., 1999). These RGAs are useful in physical mapping and as gene candidates in positional cloning.

Soybean mosaic virus (SMV) is one of the common diseases in soybean production, often significantly reducing soybean yields in many regions. To date, three SMV R gene loci (Rsv1, Rsv3, Rsv4) have been reported. Rsv1 has been mapped on the soybean MLG F, Rsv3 on the MLG B2 and Rsv4 on the MLG D1b (Yu et al., 1994; Hayes et al., 2000a; Jeong et al., 2002). Previously, we have found a molecular marker that was linked to the SMV R gene (Zhang et al., 1999) and isolated a defense gene SbPRP and an R gene candidate KR1 from Kefeng1 (He et al (2002), He et al (2003)). In the present study, RGAs of soybean (He et al., 2001) were used as probes to isolate more R gene candidates from Kefeng1, a SMV-resistant soybean cultivar. An R gene candidate KR4 was obtained and its genomic organization and expression were also analyzed.

Section snippets

Plant growth condition and nucleic acid isolation

SMV-resistant and susceptible soybean (Glycine max) cultivars, Kefeng1 and Nannong1138-2, were grown in the greenhouse under natural light condition. Leaves of 2-week-old soybean plants were harvested by amputation into liquid nitrogen and stored at 70°C until DNA isolation. Total DNA extraction was performed as previously described (Chen et al., 1991). For salicylic acid (SA) treatment, SA was dissolved in water and adjusted to pH 7.0 with NaOH. Two-week old seedlings were then sprayed with

Isolation and structural analysis of R gene homologue KR4

To isolate R gene candidates, RGA-specific probes were used to screen a cDNA library constructed with the mRNA from SA-treated Kefeng1. A total of 54 postulated positive clones were obtained after screening approximately 2.7 million plaques. Sequence analysis of the clones with longer inserts suggested that they had conserved domains related to R genes, but some were partial in 5′ cDNA sequences (data not shown). 5′-RACE-PCR was then carried out for a cDNA, KR4-1, and a product of 285 bp which

Discussion

Several full-length R gene candidates have been isolated from soybean (Graham et al., 2000; Hayes et al., 2000b; He et al., 2003; Wang et al., 2003). In the present study, KR4 belonged to the class of plant R gene products that contain NBS and LRR domains. NBS domain was necessary for the function of ATP or GTP binding and LRR domain was assumed to be Avr protein recognition site (Whitham et al., 1994), and the domains were common to most cloned plant R proteins. KR4 was most similar to I2,

Acknowledgements

This work was supported by State Key Basic Research and Development Plan of China (2002CB111303).

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