Microstructural Analysis Of Rice-free Stamen Mutants Infested With Rice Smut And Preliminary Study On Activation Of Genes Related To Rice Filling

Rice false smut （RFS）, one of widely distributed rice panicle diseases, is caused by the fungal pathogen Ustilaginoidea virens （Cke） Tak. The RFS not only reduces grain production, but also produces mycotoxins which are harmful to human and animal health. In recent years, the occurrence of RFS has greatly increased,and become one of the three major rice diseases nowadays. Although more and more research has been conducted on RFS, the underlying mechanism of its pathogenesis is still elusive.It has recently been reported that U. virens specifically infects rice flower filaments to form false smut balls; while some reports also showed that anthers and stigma may also be infected. Whether the filament is the only or essential infection site for U. virens needs to be clarified. In addition, we have previously found that U. virens infection interrupted rice seed formation, but activated grain-filling-related genes, suggesting that U. virens may hijack rice nutrient reservoir for growth and formation of false smut balls.In this thesis, a stamenless rice mutant was employed for anatomical analysis upon U. virens infection. The molecular mechanism of how U. virens activates rice grain-filling-related genes was also investigated. The main results are as follows:（1） U. virens can infect a stamenless rice mutant and form false smut balls, indicating that filament is not the only infection site for U. virens.（2） Anatomical analysis revealed that U. virens can infect the ovary and stigma in the stamenless rice mutant.（3） Transmission electron microscopy analysis revealed that vesicles were detected in ovary cells of the stamenless rice mutant upon U. virens infection, similar to those in ovary cells after pollination, supporting that U. virens infection may activates rice grain filling.（4） To investigate whether the activation of rice grain-filling-related genes is an important part of the infection process, we collected some rice mutants with grain filling deficiency, including gifl, risbzl and pdil-1. Next, we will inoculate U. virens and analyze the infection process and changes in the formation of false smut balls. To monitor the expression pattern of rice grain filling genes upon infection, transgenic plants expressing grain filling genes fused with reporter genes were obtained.（5） U. virens candidate effectors were screened for rice grain filling activators. Interestingly enough, three Ustilaginoidea virens grain filling activators （UvGFAs） were identified to induce the expression of OsRISBZ1 by dual-luciferase reporter assay. These three effectors are currently underwent function confirmation by transgenic approach.（6） We studied the subcellular localization of Uv effectors by transient expression in Nicotiana benthamiana. The results showed that Uv₁214 and other fifteen effectors were localized in both nuclei and plasma membrane; Uv₁114 and other six effectors were localized in plastids and plasma membrane; Uv₁324 and other seven effectors were localized in plasma membrane; UvGFA3 displayed spot-like localization in the cell.In summary, we found that Ustilaginoidea virens can infect rice and form false smut ball in the absence of filaments by using a stamenless mutant, indicating that the filament is not the only site for U. virens infection. U. virens infection not only activates the expression of rice grain-filling-related genes, but also induces the formation of vesicles in ovary cells. U. virens may secrecte effectors UvGFA1, UvGFA2, and UvGFA3 to activate the expression of OsRISBZ1, thus hijacking the nutrition reservoir of rice. This study reveals a new infection rounte of U. virens in a stamenless rice mutant, and proposes a special mechanism of U. virens infecting rice and forming false smut ball, i.e. hijacking rice grain filling system. Our work should be important for further understanding and controlling rice false smut disease.