Isolation And Functional Analysis Of Stripe Rust Resistance Genes In Common Wheat (Triticum Aestivum L.)

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most devastating diseases worldwide,which is responsible for major damage in wheat and result in both yield losses and downgrading in quality. The use of resistant cultivars is the most economical and environmentally sound method to reduce the damage. Therefore, understanding of the disease-resistant mechanism will be greatly important for genetically improving disease resistance in wheat using molecular genetics and genetic engineering.One hundred and seven uniESTs were obtained and sequenced in cDNA-AFLP assay to identify genes involved in the hypersensitive resistance response of Yr9NIL, Yr10NIL and Avocet background to stripe rust isolates CYR29-1 and 78028-1. ESTs similarity analysis via the BLASTx and BLASTn indicated that 20 ESTs matched the proteins or genes with known function, 30 matched those with unknown function, and 45 did not appear any homologies to the sequences with known ESTs in GenBank. The other ESTs matched the homologous sequences from chloroplast, mitochondrion, and fungus. The 20 uniESTs matching the known genes and proteins were involved in pathogen response, energy metabolism, and signal transduction etc.In the Q-PCR assay, the transcript levels of 10 ESTs were up-regulated in Yr9NIL, Yr10NIL and Avocet inoculated with CYR29-1 or 78028-1. PTaRtL, TaSBL and TaHLRG with complete coding sequences were cloned in PCR amplification of cDNA and genomic DNA. The amino acid sequence identity between the putative PTaRtL and retrotransposon proteins (GenBank accession ABA95227) was 64%. The putative TaSBL protein exhibited 67% sequence identity to Saposin B protein in barrel medic(GenBank accession ABE83256). TaHLRG (GenBank accession EU364815) encodes a protein of 337 amino acid residues with a predicted 66-amino-acid homeobox domain HOMEOBOX₂, containing a helix-turn-helix structure with the first helix at the position 11-43aa, a turn at the position 44-55aa and the second helix at the position 56-76aa.Transient expression of PTaRtL-LPC-GFP and TaSBL-LPC-GFP fusion proteins was mainly found in the membrane and nuclear of onion epidermis cells. A functional marker, THR1, based on the cDNA sequence of TaHLRG was developed and located on chromosome 6A using a set of Chinese Spring nulli-tetrasomic lines. PTaRtL was mapped between Xgwm333 and Xwg180 on the 7B, with 11.5cM and 8.0cM respectively, using one DH population derived from the cross Fukuho-komugi/Oligoculm. Statistical analyses indicated that the TaHLRG gene was significantly associated with APR to stripe rust and powdery mildew, explaining 9.3-9.7% and 11.9-20.5% of the phenotypic variance for powdery mildew resistance in populations Bainong 64/Jingshuang 16 and Lumai 21/Jingshuang 16, respectively, and accounting for 11.8-22.5% of the phenotypic variance for stripe rust reaction in Strampelli/ Huixianhong across different environments. VIGS assay for PTaRtL, TaSBL and TaHLRG resulted in none of Yr10-induced HR.