Knockout Analysis Of The Genes Involved In Mycelium Growth And Sporulation In Magnaporthe Grisea

Rice blast disease is one of the most severe diseases of rice worldwide caused by Magnaporthe oryzae. The fungus is also an important model organism for investigating pathogenicity of filamentous fungi.Identification of genes involved in mycelial growth of M. oryzae contribute to reveal their functions in mycelial growth, and also to establish the foundation for the future construction of the gene network for regulation of the mycelial growth in M. oryzae.A populated insertional mutagensis library and co-segregated mutants provides us necessary research materials and it is a fundamental platform to get mutants as well previously. The author selected 12 co-segregated mutants from the library, deducing that the mutant phenotypes were disrupted by the insertion of T-DNA. By using Tail-PCR technique, the flanking sequences of the insertion sites in 20 mutants were amplified. In order to verify the roles of these genes in mycelium growth and sporulation,15 genes were selected for gene knockout experiments.Gene knockout vector construction and transformation of vector into the wild-type strain P131, and the phenotype observation of transformants and PCR identification was performed. At least two or more knockout transformants of each gene were obtained. After cultivated on tomato oatmeal medium for 5 days, the colony diameters of the knockout transformants were measured. The results showed that?Mg12709,?Mg07336,?Mg03074,?Mg04145,?Mg05482 and?Mg12553 mycelium growth was much slower than the wild-type strain P131, and the sporulation also reduced respectively. MGG₁2709.5 encoding proteasome regulatory subunit Nas2p, MGG₀7336.5 encoding BCAS2 domain protein, MGG₀3074.5 encoding nucleoporin Nup44, MGG₀4145.5 encoding chitin synthase?, MGG₀5482.5 and MGG₁2553.5 were hypothetical protein. MGG₀6075.5 was predicted to encode Fislp, which was involved in mitochondrial membrane fission. MGG₀5135.5 encoding mitochondrial distribution and morphology protein Mdm31p. The colony diameter of its konchout transformants was reduced and no difference in sporulation efficacy as compared to wild-type strain P131. The other seven genes could not affect the mycelial growth of M. oryzae, which were MGG₀4401.5 encoding F-box and JmjC domain protein, MGG₀8302.5 encoding rRNA-processing protein Fcflp, MGG₀7162.5 encoding dihydrosphingosine phosphate lyase, MGG₀9953.5 which may be proteasome regulatory subunit Nas2p, MGG₀6473.5, MGG₀0555.5 and MGG₀4540.5 were hypothetical protein. The sporulation efficacy of?Mg06473 and?Mg04540 was significantly decreased as compared to wild-type strain P131.Identification of the genes involved in mycelial growth of M. oryzae mentioned above will lead towards further study on the control of mycelial growth, reveal the pathogenicity mechanism of M. oryzae and control the rice blast disease.