Role Of Mating-type Genes In Sexual Reproduction And Pathogenicity Of Magnaporthe Oryzae

Rice is one of the most important grain crops in the world.With the growth of population in the world,the demanding for rice is also on the rise.Rice blast is the main factor affecting the rice yield.It can infect many parts in rice plants,such as rice leaves,stem nodes,branches,stalks,spikes,and grains.Conidium plays a decisive role in the invasion of Magnaporthe oryzae.M.oryzae which belongs to heterothallic fungi also has sexual reproduction,so it needs two parents who carry the opposite mating-type genes to complete the mating process.The mating type of M.oryzae divide into two species:MAT1-1 and MAT1-2.MAT1-1 contains three genes:MAT1-1-1,MAT1-1-2 and MAT1-1-3.MAT1-2 contains two genes:MAT1-2-1 and MAT1-2-2.Up to now,there is no report about the regulatory mechanism of the above genes in the process of sexual reproduction of M.oryzae.In the present work,we investigated the roles of the two mating genes,MAT1-2-1 and MAT1-2-2 in sexual reproduction and pathogenicity of M.oryzae.We constructed two knockout vectors,pko-MAT121 and pko-MAT122.MAT 1-2-1 knockout mutant and MAT1-2-2 knockout mutant were obtained via ATMT transformation,and a further analysis was then made on effects of knocking out these two genes to asexual growth and development(including colony morphology,sporulation,spore germination,appressorium formation and pathogenicity)and sexual generation of the fungus.The results are as follows:after knocking out the MAT1-2-1 gene,the mutant strain(MAT1-2-1)were failed to form ascospores when cross-cultured with Guy 11(MAT1-1),which suggested that the MAT1-2-1 gene has a decisive influence on the generation of sexual generations.The artificial inoculation showed that the pathogenicity of the MAT 1-2-1 mutant was slightly lower than that of the wild type 70-15,but no differences were found in morphology and size of colonies or the germination of conidium and the formation of appressorium.In addition,there was no difference between the mutant and the wild type in the capability of using carbon sources and the tolerance of methyl viologen and Congo red(interfering cell wall).The deletion of MAT1-2-2 gene had no significant effect on the sexual generation of the strain.Under microscopes,asci and ascospores produced by MAT1-2-2 knockout mutant and wild-type Guyll had no significant difference in the number and morphology compared with the asci and ascospores produced by wild-type Guyl 1 and 70-15.On barley leaves,the pathogenicity of MAT1-2-2 mutants decreased.MAT1-2-2 mutant was reduced in the anti-interference ability to Calcofluor white compared with 70-15.The conidial germination and appressorium formation of MAT 1-2-2 mutant were similar to those of 70-15.There was no difference in morphology and size of colonies and the capability of using carbon sources.The tolerance to methyl viologen and anti-interference ability to Congo red of the mutant were similar to those of 70-15.Analysis of the expression of the two genes revealed that the expression of MAT1-2-2 in MAT1-2-1 mutants increased dramatically,while the expression of MAT1-2-1 gene in MAT 1-2-2 mutant increased slightly,which suggests that the two genes have direct or indirect regulation.In order to clearly detect the sexual structures and provide a method for the study of sexual generation of M.oryzae,we used GFP and mCherry fluorescent proteins to label 70-15 and Guy11 respectively,and then we observed the fluorescent expression in asci and ascospores under fluorescence microscopy.In the resulting stains,the fluorescent proteins could clearly label the cell structures and organelles of the sexual structrues.We also observed the sexual structures by staining lipid and cell wall of the asci and ascospores.