Cloning And Functional Analysis Of The Effectors Containing LXAR Motif And Molecular Markers Of Avirulence Genes Of Magnaporthe Oryzae

Rice blast is one of the most destructive diseases caused by Magnaorpthe oryzae, which posed a tremendous threat to the production and safety of rice. When invades rice,Magnaorpthe oryzae deliver effectors inside the rice cells via characterized mechanism, interfering and suppressing innate immunity of rice, which in further successfully invades rice.Bioinformatics assay and tobacco syringe experiments discovered 17 effectors containing LXAR motif could significantly suppress the BAX-mediated cell death in tobacco leaves. The functional analysis of these 17 genes contributes greatly to the molecular mechanism of interactions between Magnaorpthe oryzae and rice. The present research work is divided into four parts:1 Construct and transform over-expressed vectors of effectors into the Guy11 strain. All the over-expressed vectors of 17 effectors containing LXAR motif are finished.2. Sequence polymorphism assays of effectors containing LXAR motifWe collect 60 various strains from different regions of the world, and confirm 11 typical strains after identifying the pathogenicity. We select 10 out of 17 effectors to assay the sequence polymorphism. The sequence polymorphism is more frequent in promoter than in coding regions shows that effectors are under positive selection.3. Partial effectors containing LXAR motif deletion and morphogenesis of related transformantsFive gene replacement vectors have been constructed (P1₃119, P2₂484, P1₂310, P2₆480, P2₃959), and two typical genes, P1₃119 and P2₂484 are deleted by homologous recombination and get transformants, respectively. Compared with wild type strain Guy11, P2₂484 over-expressed transformants grow a little faster and have a few spores of abnormality, while the P2₂484 deleted transformants make no changes, which show that P2₂484 might be minor gene related with development. The P1₃119 transformants have no obvious morphological changes.4. Moleculart markers of AvrPiSH-SWe work on the molecular markers of avirulence genes from the cross P131 and S1528 of Magnaporthe oryzae. Firstly, we screened IRBLSH-S out of 28 isogenic lines showed avirulence to S1528 while virulence to P131. Based on IRBLSH-S, the pathogenicities of all the 97 ascospores are tested, and the pathogypes ratio is about 1:1, which demonstrated the single control of avirulence in S1528 strain. Out of 75 PATE primers, P7 could geneate a specific band with similar length of 800bp in avirulent strains but absent in virulent strains, which might link with the avirulence gene.