Isolation And Functional Analysis Of Disease-Resistance Related Genes To Wheat Leaf Rust From TcLr24

Wheat leaf rust caused by Puccinia triticina is one of the most devastating diseases worldwide, which is resposible for major damage in wheat and results 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. So, discovery and utilization of favorable genes in wild source and prolonging resistant cultiver the life of becomes an important task to reduce the damage caused by wheat leaf rust. The study was carried out to investigate resistance related genes from TcLr24 by homology-based method and complementary DNA amplified fragments length polymorphism (cDNA-AFLP) analysis. The expression patterns of the test fragments were conducted through real- time PCR. At the same time the genes were selected for further functional analysis using BSMV VIGS technique. The main results were discrived as follows:1. One through reading fragment was obtained and sequenced from cDNA of TcLr24 according to the conserved sequence of resistance gene analogs (RGA). The deduced amino acids of protein RGA1 contained a NB-ARC conserved domain which were identical to the NBS conserved domains (P-Loop, kinase-2, kinase-3a) of many plant resistance genes.2. The full length sequence of disease resistance homology gene in the cDNA from TcLr24 was 2822bp named as RGA1 which was obtained by the rapid amplification cDNA ends (RACE) based on RGA1 fragment. The predicated RGA1 protein encode 799 amino acids. BLASTp analysis showed that the deduced amino acids of protein contained NBS conserved domain and many leucine-rich repeats (LRR) domains, which were identical to the conserved domains of many plant resistance genes. These sequences appeared to be induced by Puccinia triticina and were genes in the wheat leaf tissue . There is one copy in DNA of TcLr24 by Southern hybridazation. Expression patterns revealed by real-time PCR analyses indicated that RGA1 gene was constitutive genes in the wheat leaf tissue in the incompatible interaction between host and pathogen. Silencing RGA1 using BSMV VIGS reduced the transcript level of gene through real time PCR analysis, of which the gene showed no changed phenotype.3. Eighty-four pairs of primers could amplify differential expression bands among TcLr24 and Thatcher by cDNA-AFLP analysis. Polymorphic bands were grouped into nine different types, and four types of them were supposed to correspond to disease- resistant gene.4. One hundreds and twenty-one differential expression bands were cloned and sequenced. Blastx analysis showed that the thirty-two transcript derived fragments (TDFs) accounted for 27% of all the TDFs showed high homology with the function known gene. Thirty-seven TDFs accounted for 30% were homology with unknown function genes. Fouty-five sequences accounted for 37% were no match with any sequence in the database. All TDFs were classified, energy and metabolism, transporters, disease/defense, transcription, energy gene, cell structure, signal transduction, protein storage were supposed to correspond to the expression of the disease-resistant genes.5. Based on their functional analysis, nine genes were selected for further real time PCR analysis. The results indicated that expression patterns revealed by real time PCR analysis were basically in accord with the cDNA-AFLP analysis. These results demonstrated that the TDFs were most likely pathogen-induced genes.6. Three kinase-encoding genes were obtained using the rapid amplification cDNA ends (RACE) method, including TaSTK (1475bp), TaRLPK (2398 bp), TaLRRK (2038 bp), which respectively encoded Ser/Thr protein kinase, Receptor protein kinase, LRR-receptor protein kinase. InterProScan analysis revealed that three genes were deduced to proteins with protein kinase activity, thus characterizing as kinase-encoding genes. ATP-dependent phosphorylation feature were observed for TaSTK,TaRLPK,TaLRRK. Psort analysis indicated TaSTK had 85.4% probability of target in chloroplast stroma. TaRLPK,TaLRRK had 51.4%, 46% probability of target in membrane. The analysis of multiple alignment and phylogenetic tree demonstrated that TaSTK, TaRLPK, TaLRRK were highly similar to barley, rice, wheat kinase.7. Three kinase genes were carried out for further functional analysis using BSMV VIGS technique. Positive silencing for each gene could be detected using real time PCR analysis, of which wheat plants of three silenced genes each showed changed phenotype. Further histological observation indicated the same results. Plants of silenced gene TaSTK showed larger area of hypersensitive cell death with slight amount sporulation showing infection type of"1". Wheat plants with silenced TaRLPK, TaLRRK gene, each showed sporulation around with relatively small area of hypersensitive cell death, indicating resistant infection type of"2". The results implied three kinase genes might play an important role in the defense response.