Molecular characterization and deployment of the high-temperature adult plant stripe rust resistance gene Yr36 from wheat

Abstract

Stripe rust, caused by Puccinia striiformis f. sp. tritici is one of the most destructive fungal diseases of wheat. Virulent races that appeared within the last decade caused drastic cuts in yields. The incorporation of genetic resistance against this pathogen is the most cost-effective and environmentally friendly solution to this problem. However, race specific seedling resistance genes provide only a temporary solution because fungal populations rapidly evolve to overcome this type of resistance. In contrast, high temperature adult plant (HTAP) resistance genes provide a broad spectrum resistance that is partial and more durable. The cloning of the first wheat HTAP stripe rust resistance gene Yr36 (Science 2009, 323:1357), funded by our previous (2007-2010) BARD grant, provided us for the first time with an entry point for understanding the mechanism of broad spectrum resistance. Two paralogous copies of this gene are tightly linked at the Yr36 locus (WKS1 and WKS2). The main objectives of the current study were to characterize the Yr36 (WKS) resistance mechanism and to identify and characterize alternative WKSgenes in wheat and wild relatives. We report here that the protein coded by Yr36, designated WKS1, that has a novel architecture with a functional kinase and a lipid binding START domain, is localized to chloroplast. Our results suggest that the presence of the START domain may affect the kinase activity. We have found that the WKS1 was over-expressed on leaf necrosis in wheat transgenic plants. When the isolated WKS1.1 splice variant transcript was transformed into susceptible wheat it conferred resistance to stripe rust, but the truncated variant WKS1.2 did not confer resistance. WKS1.1 and WKS1.2 showed different lipid binding profiling. WKS1.1 enters the chloroplast membrane, while WKS1.2 is only attached outside of the chloroplast membrane. The ascorbate peroxidase (APX) activity of the recombinant protein of TmtAPXwas found to be reduced by WKS1.1 protein in vitro. The WKS1.1 mature protein in the chloroplast is able to phosphorylate TmtAPXprotein in vivo. WKS1.1 induced cell death by suppressing APX activity and reducing the ability of the cell to detoxify reactive oxygen. The decrease of APX activity reduces the ability of the plant to detoxify the reactive H2O2 and is the possible mechanism underlying the accelerated cell death observed in the transgenic plants overexpressing WKS1.1 and in the regions surrounding a stripe rust infection in the wheat plants carrying the natural WKS1.1 gene. WKS2 is a nonfunctional paralog of WKS1 in wild emmer wheat, probably due to a retrotransposon insertion close to the alternative splicing site. In some other wild relatives of wheat, such as Aegilops comosa, there is only one copy of this gene, highly similar to WKS2, which is lucking the retrotransposon insertion. WKS2 gene present in wheat and WKS2-Ae from A. showed a different pattern of alternative splice variants, regardless of the presence of the retrotransposon insertion. Susceptible Bobwhite transformed with WKS2-Ae (without retrotansposon insertion in intron10), which derived from Aegilops comosaconferred resistance to stripe rust in wheat. The expression of WKS2-Ae in transgenic plants is up-regulated by temperature and pathogen infection. Combination of WKS1 and WKS2-Ae shows improved stripe rust resistance in WKS1×WKS2-Ae F1 hybrid plants. The obtained results show that WKS1 protein is accelerating programmed cell death observed in the regions surrounding a stripe rust infection in the wheat plants carrying the natural or transgenic WKS1 gene. Furthermore, characterization of the epistatic interactions of Yr36 and Yr18 demonstrated that these two genes have additive effects and can therefore be combined to increase partial resistance to this devastating pathogen of wheat. These achievements may have a broad impact on wheat breeding efforts attempting to protect wheat yields against one of the most devastating wheat pathogen.