Home > Browse Issues > Vol.40 No.7
Lighting the Cellular Structures of Sexual Generation in Magnaporthe oryzae with Fluorescent Proteins and Fluorescent Dyes
Guo Xiaoyu1,2, Li Ling2,3, Dong Bo4, Wang Jiaoyu2*, Chai Rongyao2, Zhang Zhen2, Mao Xueqin2, Qiu Haiping2, Hao Zhongna2, Wang Yanli2, Sun Guochang2*
1College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China; 2State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 3School of Agricultural and Food Sciences, Zhejiang Agriculture and Forest University, Lin’an 311300, China; 4Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Abstract: Magnaporthe oryzae causes rice blast disease， the most devastating disease on rice， and also is a model organism for the study on plant pathogenic fungi. M. oryzae is a heterogeneous fungus， but its sexual generation rarely occurs in nature. The studies are thus limited on cytological process， morphological structure and molecular mechanism of sexual generation， and on the contribution of sexual reproduction to the pathogenicity variation of the fungus. The methods for such studies are still required. In the present work， the sexual structures of M. oryzae were investigated microscopically by using fluorescent protein labeling combined with fluorescent staining. The green fluorescent protein (GFP) and the red fluorescent protein (mCherry) were firstly introduced respectively into the M. oryzae strains with opposite mating types， Guy11 (MAT1-2) and 70-15 (MAT1-1). The transformants， together with the wild types were cross-cultured on OMA medium basing their mating types to induce the sexual generation， which were then detected fluorescent-microscopically. The results showed that the promoters of histone H3， ribosomal protein RP27 and hydrophobin MPG1 could be highly expressed in ascospores of M. oryzae. Subsequently， we labeled and detected the nuclei and peroxisomes in asci and ascospores of M. oryzae via the three promoters and the two fluorescent proteins. The mCherry fused with H2B and the GFP fused with a nuclear localization signal (NLS) can be well distributed on the nuclei in ascospores. The GFP fused with peroxisomal localization signal 1 (GFP-PTS1) can effectively label the peroxisomes in the cells of ascospores. The presence of peroxisomes in sexual structures may suggest that the metabolism in peroxisomes is involved in the sexual reproduction of the fungus. We also used fluorescent dyes Nile red， BODIPY and Calcofluor white to stain the asci and ascospores of the fungus. The data indicated that these fluorescent dyes did not interfere with the fluorescent proteins and brighten clearly the cellular and subcellular structures of the asci and ascospores. Our work provides a method for future study on sexual reproduction of M. oryzae.
CN
EN