| Ⅰ. Knockout of Eghl6 family genesMagnaporthe oryzae is a well-known ascomycete that causes rice blast disease. A better understanding of the molecular basis of rice blast benefits its control. As a model fungal pathogen, M. oryzae has typical infection processes, including conidiation, germination and appressorium differentiation. Isolation of functional genes of M. oryzaeis is helpful to the understanding of other fungal-plant interactions. Despite the pre-penetration processes of M. oryzae, especially appressorium differentiation and formation, has been well studied and many genes involved have been characterized, many mechanisms unclear in infection cycles still left to be understood, such as hypha differentiation and colonization, establish of parasite, nutrition acquirement, and the roles played by each organelles in these processes.Egh16 gene was firstly found in Blumeria graminis, an obligate parasite of wheat, by using subtractive hybridization and differential display methods, They are expressed specifically during appressorium formation in the wild-type strains. The homologues genes were found in a variety of plant pathogenic fungi, including important pathogenicity genes GAS1 and GAS2 of M. oryzae. In addition, the homologous genes are specifically expressed after the invasion in Fusarium graminearum.Therefore, we presumed that Egh16 is a class of important gene family in plant pathogenic fungi,8 Eghl6 genes were abtained in M. oryzae genome by homologous search (including GAS1 and GAS2).To understand the functions of Egh16 gene family in M. oryzae pathogenicity, including appressorium formation, invasive and the role of invasive growth process, in this study,3 Egh16 family genes from M. oryzae, MG02253, MG00703 and MG09875, were cloned and disrupted by gene replacement strategy. The mutants of each gene were phenotypic analyzed. The results are showed as follows:Eight homologues (MG02253,MG00703,MG09875,MG03504,MG00992,MG11595,GAS1,GAS2) were found by using the amino acid sequence of Egh16 gene of Blumeria graminis to search form M. oryzae genome databases. Sequence analysis to the genomic DNA of MG02253 revealed an open reading frame of 1067 bps, including 1 intron,2 exons, and a coding sequence of 843 bps, coding a 280 amino acid peptide. The open reading frame of MG00703 is 1053 bps long, including 1 intron,2 exons, a coding sequence of 969 bps, coding a 322 amino acid peptide. The open reading frame of MG09875 is 925 bps, including 1 introns,2 exons, and a 828 bps coding sequence, coding a 275 amino acid peptide.Seventy-five hygromycin-resistance transformants of MG02253, seventy-two of MG00703 and seventy-three of MG09875 were selected via twice agrobacterium mediated transformations. Six knockout mutants for each gene were obtained and confirmed by PCR and Southern blot. At least 90% coding regain of the corresponding gene was replaced by HPH in each mutant. Two mutants for each gene were chosen randomly for phenotypes analysis.One copy of MG02253,MG00703,MG09875 was detected by genomic Southern blot in the genome of Guy11.The colony morphology, vegetative growth rate and conidiation of mutants on different media were not changed compared to Guy11. Under the condition of N or C starvation or high hyperosmotic stress, the colony modality and growth rates of mutants kept unchanged.Conidia of mutants were allowed to germinate and form appressoria in water drops on the surface of Terylene. All the spores of strains germinated, and the germination rate of mutants and wild-type show no difference, appressoria formation rate up to 95%. All mutants can penetrate into onion epidermal cells and form infection hyphae.The mutants produced the typical brown lesions, have no significant difference in the pathogenicity to wild-type and random insertion strains.Cross-cultured with M. oryzae 2539 strain on OMA media, all mutants generate perithecium normally, indicting no changes in the fertility of the fungus.Ⅱ.Pathogenicity research of M. oryzae on Brachypodium distachyonThe study of interactions of M. oryzae to the different hosts out of rice is also helpful to understand the mechanism of plant-fungi interation and to find new source of resistance genes. But up to now, the research of M.oryzae interactions with other host is relatively limited.Brachypodium distachyon is a new model of Poaceae plants, sensitive to several important phytopathogens. To investgate whether B. distachyon can be used as a candidate for studying fungal plant interactions and as a probable source of disease resistance, we developed inoculation methods of rice blast fungus M. oryzae to B. distachyon in this report, observed the infection processes and symptom development, and compared with those on rice (Oryza sativa) and barley (Hordeum Vulgare).The results are as follows:All the leaves, sheathes, stems and panicles of B. distachyon could be infected by M. oryzae and form blast disease. Spraying conidia suspension on either whole seedlings or leaf segments led to typical symptoms on B. distachyon.In the whole seedling inoculation, the symptoms on B. distachyon leaves developed accordantly to those on rice; but the lesions on B. distachyon had better uniformity in shapes and sizes than those on rice or barley. In the leaf segments inoculation, rice only produced initiate and low-developed lesions, while B. distachyon and barley could form normal symptoms.Inoculated with low-virulent mutants of M. oryzae, B. distachyon produced low-level symptoms. And the symptom levels of each mutant showed on B. distachyon corresponded well to those on rice.In addition, M. oryzae performed the typical infection processes on B. distachyon leaves:formed melanized appressoria, penetrated into host epiderm and then formed hyphae in epidermal cells. |
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